Ankle-brachial index as a marker of cognitive impairment and

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Volume 12, issue 1, June 2011
ISSN 1567-5688
atheros clerosis
supplements
ABSTRACTS OF THE 79TH EAS CONGRESS
26–29 JUNE 2011, GOTHENBURG, SWEDEN
Society of
Atherosclerosis
Imaging and
Prevention
Official Journal of the European Atherosclerosis Society
Affiliated with the International Atherosclerosis Society and
the Society of Atherosclerosis Imaging and Prevention
SUPPLEMENTS
Official Journal of the European Atherosclerosis Society
Affiliated with the International Atherosclerosis Society and
Abstracts of the
79th EAS Congress
26−29 June, 2011, Gothenburg, Sweden
Vol. 12 No. 1 (June 2011)
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doi:10.1016/S0021-9150(11)00402-3
Contents
Abstracts of the
79th EAS Congress
26−29 June, 2011, Gothenburg, Sweden
Vol. 12 No. 1 (June 2011)
Cited in: Biological Abstracts – Chemical Abstracts –
Current Contents/Life Sciences – Current Contents/Clinical Medicine – Elsevier BIOBASE/Current Awareness in Biological
Sciences – EMBASE/Excerpta Medica Abstract Service – Index Internacional de Cardiologia – Medline/Index Medicus –
Informedicus – PASCAL M – Sociedad Iberoamericana de Información Cientifica (SIIC)
Oral presentations
1
Poster presentations
Author Index
13
185
The Table of Contents, Instructions to Authors and other information on Atherosclerosis, can be
accessed on the World Wide Web at the following URL addresses:
http://www.elsevier.nl/locate/atherosclerosis or http://www.elsevier.com/locate/atherosclerosis
Atherosclerosis Supplements 12 (2011) 1–11
Contents lists available at ScienceDirect
Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis
79th EAS Congress, 26−29 June, 2011, Gothenburg, Sweden
Oral presentations
1 HUMAN BIGLYCAN EXPRESSION IN A MOUSE MODEL OF
ATHEROSCLEROSIS TRIGGERS LIPID RETENTION
P. Fogelstrand, C. Mogensen, K. Skålén, J. Borén. Wallenberg Laboratory/
CMR, Gothenburg University, Gothenburg, Sweden
Objective: Accumulation of vascular smooth muscle cells (VSMCs) within the
intimal vessel wall layer (intimal hyperplasia) has been identified as an early
step in atherogenesis. Proteoglycans secreted by intimal VSMCs are believed
to promote arterial retention of lipoproteins through an electrostatic binding
between negatively charged proteoglycans and positively charged sites on
lipoproteins. Mice are widely used to study atherosclerosis, but unlike humans,
mice do not naturally form intimal hyperplasia. The aim of this study is to answer
whether proteoglycans secreted by VSMCs is a significant contributor to lipid
retention and consequently to the development of atherosclerosis after inducing
intimal hyperplasia in the mouse carotid artery.
Methods: Atherosclerotic prone mice expressing inducible SMC-specific human
biglycan underwent operation to induce intimal hyperplasia in the left carotid
artery. The mice were fed an atherogenic diet for two weeks and subsequently
sacrificed. Both carotid arteries were collected (operated and non-operated) and
analyzed for lipid accumulation, human biglycan expression and macrophage
infiltration.
Results: Lipid accumulation was seen in vessels with intimal hyperplasia, but
not in non-operated vessels. Biglycan expression dramatically increased the
lipid accumulation in vessels with intimal hyperplasia, but not in non-operated
vessels. No macrophages were observed at this early time point.
Conclusion: Biglycan derived from VSMCs activated by vascular injury
contributes to lipid retention independently of macrophage infiltration. Hence,
biglycan is a probable key player in the initial phase of atherosclerosis.
2 SMOOTH MUSCLE CELL APOPTOSIS PROMOTES VESSEL
REMODELING, VIA ACTIVATION OF CELL MIGRATION,
PROLIFERATION AND COLLAGEN SYNTHESIS
H. Yu, M. Clarke, N. Figg, T.D. Littlewood, M.R. Bennett. Cardiovascular
Medicine, University of Cambridge, Cambridge, UK
Rationale: Although vascular smooth muscle cell (VSMC) apoptosis occurs
during remodeling and atherosclerosis, the direct role of VSMC death in
determining final vessel structure and the regulation of remodeling are unclear.
Objective: To determine the role of VSMC apoptosis in vessel remodeling,
medial repair and neointima formation, and the mediators responsible for the
effects of VSMC apoptosis.
Methods and Results: Vessel remodeling was studied after surgical ligation
of the left common carotid artery in SM22a-human diphtheria toxin receptor
mice, in which VSMC apoptosis can be selectively induced by DT treatment.
Acute apoptosis induced from Day 7−14 after ligation significantly increased
intimal and medial areas, cell proliferation, and vessel size. In contrast, chronic
apoptosis prior to ligation significantly reduced intimal area and cell counts,
but augmented the vessel compliance of RCCA for 1 fold in comparison with
the saline control. To examine the mechanism of action of apoptotic cells on
‘bystander’ VSMCs, we studied conditioned media from apoptotic VSMCs which
promoted cell migration, proliferation and collagen synthesis. The chemotactic
effects of apoptotic supernatants could be prevented by the pan-caspase
inhibitor z-VAD or by siRNA to caspase-3. IL-6 secretion increased 5-fold and
IL-1a 1.5-fold after apoptosis; IL-6 inhibition negated the effect of apoptotic
VSMC supernatants on VSMC migration, proliferation and matrix synthesis.
Conclusions: Signaling from apoptotic VSMCs directly promotes vessel
remodeling, medial repair and neointima formation after flow reduction. Whilst
lumen size appears to depend upon flow, VSMC apoptosis is a major
determinant of vessel, medial and intimal size after flow reduction.
1567-5688/ $ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
3 MAJOR ROLE OF PERICYTES IN THE CALCIFICATION OF
ATHEROMATOUS ARTERIES
J. Davaine1 , R. Brion1 , M.F. Heymann1 , F. Hérisson1 , D. Heymann1 ,
Y. Goueffic2 , G. Lambert1,3 . 1 Inserm U957, 2 Nantes University Hospital,
3
Faculté de Médecine, Nantes, France
Background: Atheroma lesions display a great heterogeneity in terms of
recurrence after surgical or endovascular treatment. There is mounting evidence
that vascular calcifications, a highly regulated pathophysiological process, play
a key role in this process.
Methods and Results: We have harvested atheromatous plaques following
femoral (n = 45) or carotid endarterectomy (n = 45). Histological, morphological
and biochemical analysis showed more fibrolipidic plaques in carotid arteries
(72%) and fibrocalcic plaques in femoral arteries (93%), p < 0.01, with a
high prevalence of osteoid metaplasia and calcium content in femorals vs.
carotids (p < 0.01). NG2 positive pericytes were found around neovessels
in carotid plaques but they were distributed within fibrocalcic plaques in
femorals. We also showed that primary human pericytes are able to calcify
in vitro, a process accompanied by a 2−4 fold increased mRNA and protein
expression of the bone differentiation (osteoblastic) markers cbf1a and alkaline
phosphatase. In addition, we found that pericytes secrete the bone resorption
(osteoclastic) inhibitor osteoprotegerin (OPG) and that pericytes conditioned
medium dose dependently reduces the osteoclastic differentiation of CD14+
monocytes, a process that persists under inflammatory conditions. Conversely,
pericytes conditioned medium dose dependently reduced the osteoblastic
differentiation of mesenchymal stem cells, but this process was inhibited under
pro-inflammatory conditions induced with TNFa or IL6.
Conclusion: Our results indicate that pericytes play a major regulatory role
in the calcification of atheromatous arteries and that variable distribution of
pericytes within the arterial wall explains, at least in part, why calcified plaques
occur more often in femoral vs. carotid arteries.
4 REGRESSION AND STABILIZATION OF ADVANCED MURINE
ATHEROSCLEROTIC LESIONS: A COMPARISON OF LDL LOWERING
AND HDL RAISING GENE TRANSFER STRATEGIES
E. Van Craeyveld, S.C. Gordts, E. Nefyodova, F. Jacobs, B. De Geest. Center
for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
Both reductions in atherogenic lipoproteins and increases in high density
lipoprotein (HDL) levels may affect atherosclerosis regression. Here, the relative
potential of low density lipoprotein (LDL) lowering and HDL raising gene transfer
to induce regression of complex murine atherosclerotic lesions was directly
compared.
Male C57BL/6 LDL receptor (LDLr)−/− mice were fed an atherogenic diet
(1.25% cholesterol, 10% coconut oil) to induce advanced atherosclerotic
lesions. A baseline group was sacrificed after 6 months and remaining mice
were randomized into a control progression (Adnull or saline), an apolipoprotein
(apo) A-I (AdA-I), an LDLr (AdLDLr), or a combined apo A-I/LDLr (AdAI/AdLDLr) adenoviral gene transfer group and followed-up for another 12 weeks
with continuation of the atherogenic diet.
Transfer with AdLDLr decreased non-HDL cholesterol levels persistently by
95% (p < 0.001) compared to baseline. This induced lesion regression by 28%
(p < 0.001) in the aortic root and by 25% (p < 0.05) in the brachiocephalic artery
at 12 weeks after transfer. Change in lesion size was accompanied by enhanced
plaque stability, as evidenced by increased collagen content, reduced lesional
macrophage content, a drastic reduction of necrotic core area, decreased
expression of inflammatory genes, and a shift of macrophages toward a more
favourable M2 phenotype. Elevated HDL cholesterol following AdA-I transfer
increased collagen content in lesions, but did not induce regression. AdA-I
transfer on top of AdLDLr transfer resulted in additive effects, particularly on
inflammatory gene expression.
In conclusion, drastic lipid lowering following AdLDLr gene transfer leads to
pronounced regression and stabilization of advanced murine atherosclerosis.
2
Atherosclerosis Supplements 12, no. 1 (2011) 1–11
5 THE GLUCAGON-LIKE PEPTIDE-1 RECEPTOR AGONIST EXENDIN-4
INHIBITS VLDL BIOSYNTHESIS AND SECRETION IN APOE*3-LEIDEN
MICE
Y. Wang1 , E.T. Parlevliet1 , J.J. Geerling1 , J.P. Schröder-Van der Elst1 ,
J.W.A. Smit1 , L.M. Havekes1,2 , J.A. Romijn1 , H. Pijl1 , P.C.N. Rensen1 . 1 Leiden
University Medical Center, 2 Netherlands Organization for Applied Scientific
Research, Leiden, The Netherlands
Introduction: The glucagon-like peptide 1 (GLP-1) receptor agonist exendin-4
improves glucose intolerance and is currently being used for the treatment of
type 2 diabetes mellitus. Since recent studies suggest that GLP-1 may also
ameliorate plasma triglyceride (TG) metabolism, the aim of this study was to
evaluate the effect of chronic infusion of exendin-4 on VLDL metabolism by
using APOE*3-Leiden (E3L) mouse, a unique animal model for human-like
lipoprotein metabolism.
Methods and Results: Male mice were fed a high-fat diet (44 energy%
fat derived from bovine fat) for 8 weeks to induce insulin resistance.
Subsequently, mice were treated for 4 weeks with exendin-4 by a
subcutaneous osmotic minipump. Chronic administration of exendin-4 (15 and
50 mg/kg/day) reduced plasma glucose levels (−38%; P < 0.001 and −42%;
P < 0.001, respectively) compared to vehicle (11.6±1.2 mmol/L). Exendin-4
also decreased insulin levels (−50%; P = 0.06 and −70%; P < 0.01) compared
to vehicle (1.0±0.3 ng/mL), confirming that exendin-4 ameliorates glucose
intolerance. In addition, exendin-4 markedly reduced the production rate of
VLDL-TG (−24% and −56%; P < 0.05) compared to vehicle (12.4±1.2 mmol/h)
as well as that of VLDL-apoB (−8% and −46%, P < 0.05), without affecting VLDL
composition. This indicates that exendin-4 reduces the hepatic production of
VLDL particles rather than reducing the intrahepatic lipidation of apoB. Hepatic
mRNA analysis showed that this effect of exendin-4 was accompanied by
downregulation of Apob as well as Dgat1 expression.
Conclusion: These data demonstrate that the GLP-1 receptor agonist
exendin-4 not only improves glucose intolerance but also reduces VLDL
biosynthesis and secretion.
6 POSTPRANDIAL TRIGLYCERIDE METABOLISM IS IMPAIRED IN
STATIN-TREATED TYPE 2 DIABETIC MEN
H. Barrett, J. Pang, V. Tenneti, D. Chan, G.F. Watts. Medicine & Pharmacology,
University of Western Australia, Perth, WA, Australia
Aim: To investigate postprandial triglyceride metabolism in type 2 diabetic men
treated to an LDL target of less than 2.5 mmol/L.
Design: Seven statin-treated type 2 diabetic men (mean±SD, BMI > 32.2±5.7
kg/m2 ), treated to LDL target <2.5 mmol/L, and fourteen normolipidemic
non-diabetics (BMI > 23.2±2.2) were studied. Postprandial (PP) triglyceride
(TG) was measured following a standardized meal (1305 kcal, 87% fat, 7%
carbohydrate, 6% protein) and 100,000 IU vitamin A. Plasma was assayed for
cholesterol, TG, apoB, glucose, insulin and retinyl palmitate (RP). TG and RP
area under the curve (AUC0−10 ) and incremental AUC (iAUC) were calculated.
Results: Compared with controls, plasma cholesterol, LDLc, HDLc, nonHDLc and apoB were significantly lower in diabetics (all p < 0.0001). Plasma
insulin and glucose concentrations were 4.7× and 1.3× higher, respectively
(p < 0.0001) compared with controls. PP TG AUC and iAUC were significantly
higher in diabetic subjects (34.5±2.4 vs. 13.3±1.1 and 20.1±2.2 vs.
6.4±0.7 mmol/L.h, respectively, p < 0.0001). The high AUC and iAUC in
diabetics, compared with controls, was independent of differences in BMI. RP
AUC was also significantly higher in diabetic subjects (p < 0.05).
Conclusion: Despite achieving an LDL cholesterol target of less than
2.5 mmol/L, TG metabolism in diabetic subjects was significantly impaired
compared with controls. This finding was independent of differences in BMI
between subject groups. Even in the presence of low plasma apoB and
cholesterol, residual risk, due to elevated fasting and PP TG, remains in
diabetic men. Future studies to investigate therapies that improve postprandial
dyslipidemia may help reduce residual risk in this population.
7 ORP10, A NOVEL REGULATOR OF LIPOGENESIS AND APOB100
SECRETION
E. Nissilä1,2 , G. Liebisch3 , Y. Zhou2,4 , J. Perttilä1,2 , G. Schmitz3 ,
V.M. Olkkonen1,2 . 1 Institute of Biomedicine/Anatomy, University of Helsinki,
2
Minerva Foundation Institute for Medical Research, Helsinki, Finland,
3
Institute of Clinical Chemistry and Laboratory Medicine, University of
Regensburg, Regensburg, Germany, 4 Faculty of Medicine, University of
Helsinki, Helsinki, Finland
Oxysterol binding protein (OSBP) related proteins (ORPs) constitute a family
of homologous proteins that share a unique domain capable of binding
hydrophobic ligands. Previous studies have shown that ORPs play different
roles in various cellular processes including intracellular lipid transport and
membrane trafficking. We recently showed that polymorphisms in the OSBPL10
gene show linkage and association with extremely high serum triglyceride (TG)
levels in dyslipidemic subjects.
Oral presentations
We have created Huh7 hepatoma cell lines in which ORP10 is stably silenced
using shRNA encoding lentiviruses, for detailed functional analysis. Silencing of
ORP10 resulted in increased TG synthesis and apoB100 secretion. Lipidome
analysis revealed increased cellular content of numerous phospholipids species
and of free cholesterol. This suggests that the function of endogenous ORP10
involves negative regulation of hepatic lipogenesis and VLDL secretion.
We have also studied the localization of full length ORP10, C-terminal region,
N-terminal ligand binding domain, and the pleckstrin homology (PH) domain
of ORP10 in Huh7 cells by generating EGFP or Cherry fusion constructs. Full
length EGFP-ORP10 mostly and EGFP-C-terminal ORP10 partly associates
with microtubules, whereas Cherry-full length ORP10 distributed between
microtubules and Golgi-complex. EGFP-N-terminal region is localized to the
Golgi complex. We measured the binding of phosphoinositides (PIPs) by the PH
domain of ORP10. The result shows that the PHD specifically binds PI4P, a PIP
characteristic of Golgi membranes. The microtubule and Golgi association of
ORP10 indicate a function that involves microtubule-dependent membrane/lipid
trafficking in the endoplasmic reticulum – Golgi region, a location where VLDL
are assembled.
8 ALIPOGENE TIPARVOVEC GENE THERAPY ENHANCES
POST-PRANDIAL CLEARANCE OF CHYLOMICRONS IN LIPOPROTEIN
LIPASE DEFICIENT PATIENTS
A.C. Carpentier1 , F. Frisch1 , S.M. Labbé1 , J. Méthot2,3 , C. Gagné4 , S. Déry3 ,
K. Tremblay3 , J. de Wal5 , J. Twisk5 , S. Greentree5 , N. van den Bulk5 ,
D. Brisson3 , D. Gaudet2,3 . 1 Centre Hospitalier Universitaire de Sherbrooke,
Centre de Recherche Clinique Étienne-Le Bel, Sherbrooke, 2 Department of
Medicine, Université de Montréal, Montreal, 3 Ecogene-21 and Lipid Clinic,
Chicoutimi, 4 Clinique des Maladies Lipidiques de Québec, Quebec City, QC,
Canada, 5 Amsterdam Molecular Therapeutics (AMT) B.V., Amsterdam, The
Netherlands
Introduction: Alipogene tiparvovec (Glybera® ) gene therapy for lipoprotein
lipase deficiency (LPLD) constitutes DNA encoding gene variant LPLS447X
in an adeno-associated virus derived vector. It is administered by one-time
intramuscular injection at a dose of 1×1012 gc per kg BW. To establish a marker
for long term efficacy of alipogene tiparvovec, the post-prandial response to a
fat-labeled meal, both before and after therapy, was studied.
Methods: Fasted patients were given a 13% fat meal containing tritium-labeled
palmitate 2 weeks before (n = 5) and 14 (n = 5) and 52 weeks (n = 3) after
therapy and blood was drawn over 24 hours. A chylomicron (CM) enriched
fraction was isolated by ultracentrifugation. NEFA and glycerol appearance rates
were determined using IV U-13 C-palmitate and d5-glycerol tracers.
Results: Before therapy, the post-prandial response in LPLD was grossly
abnormal with no or only a limited decline of CM fraction-tracer until 24 hours.
After therapy, 3 H-tracer amount in plasma and CM fraction was greatly reduced:
93% and 68% reduction in AUC0−24hrs in the CM fraction (p = 0.03, n = 5 and
p = 0.18, n = 3, vs week-2, at week 14 and 52 respectively). The LPLD results at
14 weeks after alipogene tiparvovec were similar to those of 5 healthy subjects
given a similar calorie, 33% fat meal. The enhanced post-prandial CM clearance
was independent of fasting plasma triglyceride level. No statistically significant
change in NEFA and glycerol appearance rates was observed.
Conclusion: One-time alipogene tiparvovec resulted in (near) normalized postprandial CM clearance after 14 and 52 weeks, without NEFA spillover.
9 LACK OF ANTIGEN PRESENTATION ON MHC CLASS II AGGRAVATES
ATHEROSCLEROSIS IN APOE−/− MICE
M. Wigren1 , H. Björkbacka1 , G.N. Fredrikson1,2 , J. Nilsson1 . 1 Clinical Sciences
Malmö, Lund University, Skane University Hospital, 2 Faculty of Health and
Society, Malmö University, Malmö, Sweden
Hypercholesterolemic mice lacking factors required for activation of CD4+
effector T cells are characterized by reduced development of atherosclerosis.
Against this background it has been assumed that atherosclerosis involves
a loss of tolerance against modified self-antigens generated in response to
hypercholesterolemia and that presentation of such antigens on MHC class II
molecules lead to activation of pro-inflammatory Th1 cells. To test this possibility
we investigated atherosclerosis development in ApoE−/− mice deficient for
MHC class II (ApoE−/−MHCII−/−). As expected ApoE−/−MHCII−/− mice had
reduced levels of CD4+ T cells, low levels of IgG and IgM, as well as
of Th1 and Th2 cytokines in plasma. CD115+ monocytes were reduced in
spleen as well as the plasma levels of TNF-a, IL-1b and IL-6 indicating
reduced systemic inflammation. In spite of this, ApoE−/−MHCII−/− mice had
significantly more atherosclerosis as assessed both by en face Oil Red O
staining of the aorta (4.7±2.9% versus 1.9±1.3%; P < 0.01) and cross-sectional
area of subvalvular lesions (7.7±2.2x105 versus 4.6±2.8x105 mm2 ; P < 0.05).
Moreover, macrophage accumulation in lesions was significantly increased
(44.8±8.0% versus 24.8±7.8% MOMA-2 stained area; P < 0.001). The present
observations unexpectedly show that the net effect of MHC class II-dependent
antigen presentation in atherosclerosis is athero-protective and suggest that
this effect occurs locally in the arterial wall rather than at the systemic level.
79th EAS Congress
10 CD4+ NKT CELLS CONTRIBUTE TO ATHEROSCLEROSIS
VIA PERFORIN- AND GRANZYME B-DEPENDENT CYTOTOXIC
MECHANISMS
K. To1 , A. Bobik2 , P. Tipping3 , B.-H. Toh3 . 1 Vascular Biology and
Atherosclerosis, Baker IDI Heart & Diabetes Institute, 2 Vascular Biology and
Atherosclerosis, BakerIDI Heart & Diabetes Institute, Melbourne, 3 Department
of Medicine, Monash University, Clayton, VIC, Australia
The proatherogenic activity of NKT cells resides with the CD4+ NKT cell
subtype. However, the mechanisms by which they augment atherosclerosis
are unknown. We investigated potential mechanisms in ApoE−/− mice −
the role of bystander immune T, B and NK cell activation and the roles of
cytokines IFN-gamma, IL-4 and IL-21 and cytotoxins perforin and granzyme B.
Adoptive transfer of CD4+ NKT cells into ApoE−/−Rag2−/− mice augmented
atherosclerosis assessed by Oil Red O staining, by 88%; macrophage
accumulation increased 250%. Similarly, adoptive transfer of these cells into
ApoE−/−Rag2−/−gamma C−/− mice also augmented atherosclerosis, by 60%
and macrophage accumulation by 200%, indicating that CD4+ NKT cell
proatherogenic activity is independent of T, B or NK cells. To investigate the role
of cytokines and cytotoxins CD4+ NKT cells were isolated from mice deficient
in these substances and transferred into ApoE−/−Jalpha18−/− mice. CD4+
NKT cells deficient in IL-4, IFN-gamma and IL-21 all augmented development
of atherosclerosis in ApoE−/−Jalpha18−/− mice, increasing lesion size by 80%,
82% and 70% respectively. However adoptive transfer of CD4+ NKT cells
deficient in either perforin or granzyme B failed to augment atherosclerosis.
To determine whether post apoptotic necrosis associated inflammation is
responsible for the proatherogenic effect of CD4+ NKT cells we also assessed
necrotic core formation and proinflammatory mediators VCAM-1 and MCP-1.
These were reduced by 50% and 30% respectively in mice receiving perforin
deficient NKT cells. Thus CD4+ NKT cells augment atherosclerosis via cytotoxic
mechanisms, increasing post apoptotic necrosis and associated inflammation,
possibly via an inflammasome dependent mechanism.
11 THE EXPRESSION OF TLR3 IS INCREASED IN ATHEROSCLEROSIS
AND MEDIATES PROTECTION AGAINST LESION DEVELOPMENT
AND WEIGHT GAIN IN APOE−/− MICE
J.E. Cole1 , T. Navin1 , A.J. Cross1 , M.E. Goddard1 , L. Alexopoulou2 ,
A.T. Mitra1 , A.H. Davies3 , R.A. Flavell2 , M. Feldmann1 , C. Monaco1 . 1 Kennedy
Institute of Rheumatology, Imperial College London, London, UK , 2 Department
of Immunobiology, Yale University School of Medicine, New Haven, CT, USA,
3
Imperial Vascular Unit, Division of Surgery and Cancer, Imperial College
London, London, UK
Introduction: Previous studies have assigned detrimental roles to toll-like
receptors (TLR) in cardiovascular disease. Recently we described a novel
protective role for TLR3 in vivo in intimal hyperplasia.
Aim: Using human and murine systems we investigated the consequence of
TLR3 signaling in atherosclerosis.
Methods and Results: We compared the responses of human atheromaderived smooth muscle cells (AthSMC) and control aortic smooth muscle
cells (AoSMC) to various TLR ligands. AthSMC exhibited a specific increase
in TLR3 expression and TLR3-dependent functional responses. Interestingly,
exposure to the TLR3 ligand poly(I:C) induced both pro- and anti-inflammatory
gene expression in vitro in AthSMC and in vivo in vascular tissues.
We examined the role of TLR3 signaling in atherosclerosis in vivo using
15- and 30-week ApoE−/− and ApoE−/−TLR3−/− mice fed a chow diet.
Aortic root lesion area was significantly increased in ApoE−/−TLR3−/− mice
compared to ApoE−/− mice at 15- (p = 0.035) but not 30-weeks of age
(p > 0.05) suggesting that TLR3 is protective in early but not more advanced
stages of lesion development. The absence of an exogenous viral stimulus
implicates an endogenous vasculoprotective TLR3 ligand. TLR3 deficiency
did not affect lesional macrophage or collagen content nor cholesterol levels.
Intriguingly, high-fat feeding for 15-weeks induced increased weight gain in
ApoE−/−TLR3−/− mice compared to ApoE−/− mice (p = 0.047).
Conclusions: Collectively, our data describe for the first time a protective role
for TLR signaling in atherosclerosis and suggest that an endogenous TLR3
ligand may mediate the observed protection. Moreover, genetic deletion of TLR3
leads to susceptibility to High-fat diet induced obesity.
3
12 IDENTIFICATION OF A DANGER-ASSOCIATED PEPTIDE FROM
APOLIPOPROTEIN B100 (APOBDS-1) THAT TRIGGERS INNATE
PRO-ATHEROGENIC RESPONSES
D.F.J. Ketelhuth1 , F.J. Rios2 , H. Liu3 , Y. Wang1,4 , M.E. Johansson5 ,
G.N. Fredrikson6 , M. Gidlund2 , J. Nilsson6 , G.K. Hansson1 , Z.-Q. Yan1 .
1
Department of Medicine, Karolinska University Hospital, Stockholm, Sweden,
2
Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade
de São Paulo, São Paulo, Brazil, 3 Department of Pharmacology, Shandong
University College of Medicine, 4 Department of Geriatrics, Qilu Hospital,
Shandong University, Jinan, China, 5 Department of Physiology, Institutes
of Neuroscience and Physiology, University of Gothenburg, Gothenburg,
6
Department of Clinical Sciences, Skåne University Hospital, Lund University,
Malmö, Sweden
Background and Aims: Compelling evidence suggests that low density lipoprotein (LDL) deposition and subsequent vascular inflammation are associated
to atherosclerosis. Oxidation of LDL phospholipids and crystallization of LDLcholesterol in the subendothelial space of the arterial wall have been shown to
confer new properties on LDL, enabling it to initiate and propagate inflammatory
responses. As the chief protein component of LDL, apolipoprotein B-100
(ApoB100) undergoes biochemical modifications and fragmentation in the
vessel. Whether ApoB100-derived fragments contribute to inflammation has
been unclear.
Methods and Results: By screening a peptide library of ApoB100, we identified
a distinct native peptide, referred as ApoB100 danger-associated signal 1
(ApoBDS-1), which has sequence-specific bioactivity. ApoBDS-1 activates
MAPK and calcium signaling in innate immune cells, effecting the production
of IL-6 and a specific subset of chemokines, including IL-8 and CCL2. Ex
vivo stimulation of carotid plaques with ApoBDS-1 enhances IL-8 and PGE2
release.
Conclusions: Our data suggest that ApoBDS-1 is a previously unrecognized
peptide with strong proinflammatory activity that contributes to causal roles of
LDL in the pathogenesis of atherosclerosis.
13 HOW BROWN ADIPOSE TISSUE CORRECTS HYPERLIPIDEMIA
AND OBESITY: A ROLE FOR LIPOPROTEINS
A. Bartelt1,2 , O.T. Bruns3 , H. Ittrich4 , A. Niemeier1,2 , M. Merkel1,5 ,
J. Heeren1 . 1 IBMII: Molecular Cell Biology, N30, 2 Department of Orthopedics,
University Medical Center Hamburg-Eppendorf, 3 Electron Microscopy and
Micro-Technology, Heinrich-Pette-Institut, 4 Department of Radiology, University
Medical Center Hamburg-Eppendorf, 5 I. Department of Internal Medicine,
Asklepios Clinic St. George, Hamburg, Germany
Objective: Brown adipose tissue (BAT) burns fatty acids in order to defend
the body against cold. Recently, we demonstrated that BAT activation can
correct hyperlipidemia and obesity. Here we show that activation of BAT leads
to massive increase in triglyceride-rich lipoproteins (TRL) turnover and that
lipoprotein lipase (LPL) in BAT and lipid transporter CD36 are master regulators
of this process.
Methods: C57BL/6J mice were kept for 24 hours in a cold room (4ºC) or at room
temperature. Plasma clearance and organ uptake of radiolabeled TRL were
determined in control and cold-adapted wild-type and CD36-KO mice and in
mice treated with heparin or a specific LPL inhibitor to manipulate LPL function.
TRL organ uptake was visualized by magnetic resonance imaging (MRI) and
intravital imaging using nanocrystals embedded into the TRL core.
Results: We observed a massive acceleration of TRL turnover and a
concomitant 10-fold increase in organ uptake of TRL components into BAT
of cold-adapted mice. Using in vivo intravital imaging we visualized initial
heparin-sensitive binding of TRL to the endothelium which was followed by TRL
internalization. Heparin and THL pre-treatment abolished uptake of TRL and
fatty acids; CD36-KO mice lost body temperature rapidly during cold exposure
due to impaired TRL uptake.
Conclusion: Local LPL and CD36 in BAT drive lipolysis and organ uptake of
TRL into BAT. Increasing LPL activity in BAT might a novel approach to combat
hyperlipidemia and obesity efficiently.
14 ROLE OF HUMAN ANGIOPOIETIN-LIKE PROTEIN 3 (ANGPTL3)
GLYCOSYLATION IN THE PROTEIN SECRETION AND STABILITY
M.A. Burza1,2 , C. Pirazzi3 , C. Maglio3 , A. Molinaro2 , S. Ginanni Corradini2 ,
S. Romeo1,3 . 1 University of Cambridge, Cambridge, UK , 2 Clinical Medicine
Department, Division of Gastroenterology, Sapienza University of Rome,
Rome, Italy, 3 Department of Molecular and Clinical Medicine and Center
for Cardiovascular and Metabolic Research, the Sahlgrenska Academy,
Gothenburg University, Gothenburg, Sweden
Introduction: Angiopoietin-like protein 3 (ANGPTL3) is a secreted protein
with a natural inhibitory activity on the lipoprotein lipase, one of the key
enzymes in regulating extracellular triglyceride metabolism. Rare loss of
function nonsynonymous human mutations have been found to be associated
with lower circulating triglyceride levels by determining cellular protein retention.
N-glycosylation is a post-translational modification involved in protein secretion
4
and susceptibility to degradation. Aim of the study is to evaluate the role of
N-glycosylation in ANGPTL3 trafficking and stability.
Methods: By using a bioinformatic approach we identified 4 putative human
ANGPTL3 N-glycosylation sites at aminoacid position 23, 115, 296, 357. Wild
type ANGPTL3 was cloned into a pcDNA 3.1 6xhys and V5 tag vector. Mutants
were generated at these sites by changing the asparagine into a glutamine
after in situ mutagenesis. Human embryonic kidney cells (HEK 293A) were
transiently transfected with recombinant wild type and mutants. ANGPTL3
protein processing, secretion and stability was evaluated in cell lysate and
medium by enzymatic digestion, western blotting and protein purification.
Results: We found ANGPTL3 to be N-glycosylated at position 115, 296 and
357. Defect in N-glycosylation at position 296 determined a severe reduction
in protein secretion. Extracellular protein stability was not affected by lack of
glycosylation at any site.
Conclusions: N-Glycosylation of the aminoacid 296 but not 115 and 357 is
required for complete ANGPTL3 secretion and may be contributing to determine
lower lipoprotein levels in humans.
15 THE VLDL RECEPTOR PROMOTES LIPOTOXICITY AND INCREASES
MORTALITY IN MICE FOLLOWING AN ACUTE MYOCARDIAL
INFARCTION
J. Perman, J. Borén. Department of Molecular and Clinical Medicine, University
of Gothenburg, Gothenburg, Sweden
Myocardial triglyceride accumulation is associated with impaired cardiac
function, but it is not clear how the lipids accumulate and if this accumulation
is detrimental. Here we showed that hypoxia/ischemia-induced accumulation
of lipids in HL-1 cardiomyocytes/mouse hearts was dependent on expression
of the VLDL receptor (VLDLr), which promoted endocytosis of lipoproteins.
Furthermore, VLDLr expression was higher in ischemic compared with nonischemic left ventricles from human hearts, and correlated with the total lipid
droplet area in the cardiomyocytes. Hypoxia-induced VLDLr expression in HL-1
cells was dependent on hypoxia-inducible factor-1a through its interaction with
a hypoxia-responsive element in the VLDLr promoter. Endoplasmic reticulum
(ER) stress, which leads to apoptosis, is known to be involved in ischemic heart
disease, and we showed that ischemia-induced ER stress in mouse hearts
was reduced in VLDLr−/− mice and in mice treated with antibodies against
VLDLr. Furthermore, ischemia-promoted apoptosis was significantly decreased
in VLDLr−/− mouse hearts and showed a strong tendency to decrease in hearts
from mice treated with antibodies against VLDLr. Importantly, VLDLr−/− mice
showed improved survival and decreased infarct area following an induced
myocardial infarction. These findings suggest that the VLDLr-induced lipid
accumulation in the ischemic heart worsens survival by increasing ER stress
and apoptosis.
16 SKELETAL-MUSCLE LIPOPROTEIN EXPRESSION IS REGULATED
BY TRAINING AND TYPE 2 DIABETES IN HUMANS AND IMPROVES
INSULIN SENSITIVITY IN OBESE MICE
E. Bartels1 , T. Ploug2 , J. Størling3,4 , T. Mandrup-Poulsen2,3,5 , F. Dela2,6 ,
L.B. Nielsen1,2 . 1 Department of Clinical Biochemistry, Copenhagen University
Hospital, Rigshospitalet, 2 Department of Biomedical Sciences, University
of Copenhagen, Copenhagen, 3 Steno Diabetes Center and Hagedorn
Research Institute, Gentofte, 4 Glostrup Research Institute, Glostrup, Denmark,
5
Karolinska Institute, Stockholm, Sweden, 6 Center for Healthy Aging,
University of Copenhagen, Copenhagen, Denmark
Lipid accumulation in skeletal muscle is associated with impaired insulin
sensitivity in type 2 diabetes. In cardiac myocytes, lipoprotein secretion
controlled by apolipoproteinB (apoB) and microsomal triglyceride transfer
protein (MTP) affects lipid homeostasis. In this study, we investigated whether
skeletal muscle lipoprotein formation
1. affects triglyceride accumulation in myocytes and insulin sensitivity in obese
mice and
2. is regulated by type 2 diabetes and training in humans.
Both murine and human skeletal muscle biopsies expressed apoB and
MTP mRNA. Transgenic mice with increased skeletal muscle human apoB
expression accumulated 28% less triglycerides in skeletal myocytes after one
year of fat-feeding as compared with WT mice (32±5, n = 10 vs. 44±4 nmol/mg
ww, n = 13, P = 0.04). Moreover, overexpression of apoB in fat-fed mice was
associated with 32% (P = 0.02) and 37% (P = 0.01) lower plasma insulin levels
after 9 and 12 months respectively, improved i.p. glucose tolerance after 6
months, and increased insulin-stimulated glucose uptake in isolated skeletal
muscle. On average, the MTP mRNA content was 35% lower (P < 0.05) in
individuals with type 2 diabetes (n = 16) than in healthy controls (n = 15), and
physical training reduced skeletal muscle MTP mRNA expression by 25−40%
in healthy subjects (P = 0.03, n = 15). The data suggests that local lipoprotein
secretion is a novel pathway for exporting lipids from skeletal muscle, which
attenuates peripheral insulin resistance in obese mice and may affect skeletal
muscle lipid accumulation associated with type 2 diabetes and physical training
in humans.
Oral presentations
17 THE PLASMA CONCENTRATION OF HDL-ASSOCIATED APOM IS
CONTROLLED BY THE ACTIVITY OF THE LDL-RECEPTOR
C. Christoffersen1 , M. Benn2 , R. Frikke-Schmidt1 , A. Tybjaerg-Hansen1 ,
B. Dahlbäck3 , L.B. Nielsen1 . 1 Clinical Biochemistry, Rigshospitalet, University
of Copenhagen, Copenhagen, 2 Department of Clinical Biochemistry, Herlev
Hospital, Herlev, Denmark, 3 Division of Clinical Chemistry, Department of
Laboratory Medicine, University of Lund, University Hospital Malmö, Malmö,
Sweden
Apolipoprotein (apo) M is mainly associated with HDL and has antiatherogenic effects. Nevertheless, we have consistently observed close positive
correlations of apoM with plasma LDL cholesterol in humans. Moreover, in
mice LDL-receptor deficiency is associated with increased plasma apoM. Here,
we have tested the idea that plasma apoM is affected by the activity of the
LDL-clearance pathway. First, we injected human apoM-containing HDL into wt
or LDL-receptor deficient mice and saw that the removal of HDL-associated
human apoM was delayed in the LDL-receptor deficient mice: after 2 hrs 48%
versus 90% (p < 0.005) of the initial amounts of human apoM remained in
plasma of wt and LDL-receptor deficient mice, respectively. Next, we measured
apoM in human patients with 3 different LDL-receptor mutations (n = 9) or
carriers of the apoB3500 mutation (n = 12). These patients had increased
plasma apoM (41%, p = 0.006 and 37%, p = 0.0002, respectively) as compared
with healthy individuals. Finally, we compared the turnover of radio-iodinated
LDL and plasma apoM concentrations in 51 human individuals. There was a
negative correlation between plasma apoM and the fractional catabolic rate of
LDL (r = −0.36, p = 0.01, n = 51). These data suggest that the plasma clearance
of apoM, despite apoM primarily being associated with HDL, is affected by the
activity of the LDL receptor.
18 ROLE OF ENDOTHELIAL LIPASE IN HDL METABOLISM AND
ATHEROSCLEROSIS IN HUMANS
T. Ishida, T. Yasuda, L. Sun, K.-I. Hirata. Cardiovascular Medicine, Kobe
University Graduate School of Medicine, Kobe, Japan
Aims: Endothelial lipase (EL) regulates metabolism of high-density lipoprotein
cholesterol (HDL-C). However, the role of EL in HDL-C levels and
atherosclerosis in humans has not been fully elucidated. In this study, we
investigated the role of plasma concentration and activity of EL in HDL-C levels
in human subjects.
Methods: Plasma EL mass was evaluated by a sandwich ELISA. After plasma
EL protein was immunopricipited, the EL phospholipase activity was measured
using a fluorogenic phospholipid as a substrate. The anti-inflammatory functions
of HDL after complete inactivation of EL were evaluated in vitro using HDL
isolated from EL−/− mice.
Results: The plasma EL mass and activity were inversely correlated
with HDL-C levels in humans. The EL mass and activity in patients
with cardiovascular diseases were significantly higher than those without
cardiovascular diseases. The EL activity in smokers was higher than that in
healthy volunteers, which is concomitant with the low HDL-C levels. In addition,
the plasma EL activity was significantly correlated with coronary risk scores
in the Framingham Study. Next, we compared anti-inflammatory functions of
HDL in terms of LPS-neutralizing capacity, inhibition of VCAM-1 expression,
inhibition of LDL-oxidation, and cholesterol efflux activity, between EL−/− and
EL+/+ mice, to estimate the functional quality of HDL after EL inactivation.
These anti-inflammatory functions were preserved in HDL of EL−/− mice.
Conclusions: Plasma EL mass and activity have a significant inverse
association with HDL-C levels, and the activity was high in cardiovascular
disease patients. Moreover, inhibition of plasma EL activity may increase antiinflammatory HDL particles.
19 KRP-206, A SELECTIVE S1P(1) AGONIST INHIBITS DEVELOPMENT
OF ATHEROSCLEROSIS IN LOW-DENSITY LIPOPROTEIN
RECEPTOR-DEFICIENT MICE
S. Costa1 , F. Poti1 , V. Bergonzini1 , M. Galletti1 , H. Freise2 , G. Varga3 ,
M. Simoni1 , J.-R. Nofer1,4 . 1 Dept. of Medicine, Endocrinology, Metabolism
and Geriatrics, University of Modena and Reggio Emilia, Modena, Italy,
2
Department of Anesthesiology and Intensive Care Medicine, University
Hospital Münster, 3 Institute of Immunology, University of Münster, 4 Center for
Laboratory Medicine, University Hospital Münster, Münster, Germany
Background: Sphingosine 1-phosphate (S1P), a bioactive lysosphingolipid
associated with high density lipoprotein (HDL), at least partly accounts for
anti-atherogenic properties of this lipoprotein. We previously demonstrated that
FTY720 − a synthetic S1P analog targeting all S1P receptors but S1P(2) inhibits
development of atherosclerosis in a murine model of disease. The present study
addressed the identity of S1P receptor mediating atheroprotective effects of
S1P.
Methods and Results: LDL receptor-deficient mice on a cholesterol-rich
diet were given KRP-203, a selective S1P(1) agonist, at a dose of
3.0 mg/kg/day for 14 weeks. KRP-203 substantially reduced atherosclerotic
lesion formation both in aortic root and arteria thoracica. Plasma lipids
79th EAS Congress
remained unchanged in course of KRP-203 treatment. However, KRP-203
induced marked peripheral blood lymphopenia, reduced total (CD4+ , CD8+ ) and
activated (CD69+ /CD8+ , CD69+ /CD4+ ) T-cells in peripheral lymphoid organs,
and interfered with lymphocyte function, as evidenced by decreased splenocyte
proliferation and IL-2 and IFN-gamma production in response concanavalin
A or phytohaemagglutinin as well as reduced RANTES levels in plasma.
Plasma concentrations of macrophage-derived cytokines TNF-alpha and IL-6
were reduced by KRP-206 administration. Moreover, peritoneal macrophages
from KRP-206 treated mice showed reduced surface expression of activation
markers MCH-II and CD86 as well as LPS-elicited production of TNF-alpha
and IL-6. In vitro experiments demonstrated reduced production of TNF-alpha
and IL-6 in LPS-stimulated and IP-10 in INF-gamma stimulated bone marrow
macrophages.
Conclusions: Present results demonstrate that activation of S1P signaling
pathways inhibit atherosclerosis by modulating lymphocyte and macrophage
function and suggest that S1P(1) at least partly mediates anti-atherogenic
effects of S1P.
20 LCAT , HDL CHOLESTEROL AND MYOCARDIAL INFARCTION −
A MENDELIAN RANDOMIZATION STUDY OF HDL CHOLESTEROL
IN 54,500 INDIVIDUALS
C.L. Haase1 , A. Tybjærg-Hansen1,2,3 , A.A. Qayyum1 , J. Schou1 ,
B.G. Nordestgaard2,3,4 , R. Frikke-Schmidt1,3 . 1 Dept. Clinical Biochemistry,
Rigshospitalet, Copenhagen University Hospital, Copenhagen, 2 The
Copenhagen City Heart Study, Bispebjerg Hospital, Copenhagen, 3 The
Copenhagen General Population Study, Herlev Hospital, Herlev, 4 Dept. Clinical
Biochemistry, Herlev Hospital, Herlev, Denmark
Background: Epidemiologically, high-density lipoprotein cholesterol (HDL-C)
levels relate inversely to risk of ischemic cardiovascular disease. We tested
whether genetic variation in lecithin:cholesterol acyltransferase (LCAT), a key
enzyme in HDL metabolism, contributes to HDL-C levels and risk of myocardial
infarction (MI) in the general population and whether HDL-C levels associate
causally with risk of MI.
Methods: We resequenced the regulatory and coding regions of LCAT in 380
individuals from the Copenhagen City Heart Study (CCHS) with the lowest 2%
and highest 2% HDL-C levels. All six identified variants were genotyped in the
CCHS (n = 10,281), and a common genetic variant (S208T, rs4986970), which
affected HDL-C levels in the CCHS, was further genotyped in the Copenhagen
General Population Study (CGPS, n = 50,523). Instrumental variable analysis
with MI as endpoint and S208T as the genetic instrument was performed in the
combined study (n = 54,500).
Results: S208T associated with decreased HDL-C levels of up to 0.2 mmol/L
resulting in a predicted increase in risk of 18% for MI. However, S208T did
not associate with increased risk of MI. The estimated causal increase in
risk of MI by instrumental variable analysis for a 50% reduction in genetically
reduced HDL-C levels was −51% (−89–116%) compared to an observational
increase of 111% (70–162%) for a similar reduction in HDL-C on Cox regression
(P-comparison = 0.03).
Conclusion: Common genetic variation in LCAT associated with decreased
HDL-C levels did not associate with increased risk of MI. Low HDL-C levels
robustly associated with increased risk of MI, however genetically decreased
HDL-C did not. This suggests that HDL-C levels are not causally related to risk
of MI.
21 COMPREHENSIVE METABOLIC PROFILING IMPROVES PREDICTION
OF SUBCLINICAL ATHEROSCLEROSIS IN YOUNG ADULTS
P. Würtz1,2,3 , J. Raiko2 , C.G. Magnussen2,4,5 , P. Soininen1,6 , A.J. Kangas1 ,
T. Tynkkynen1,6 , R. Thomson4 , R. Laatikainen6 , M.J. Savolainen1 , A. Jula7 ,
J.S. Viikari8 , M. Kähönen9 , T. Lehtimäki9 , M. Juonala2,8 , M. Ala-Korpela1,6 ,
O.T. Raitakari2 . 1 Computational Medicine, University of Oulu, Oulu,
2
Cardiovascular Research Centre, University of Turku, Turku, 3 Institute of
Molecular Medicine Finland, University of Helsinki, Helsinki, Finland, 4 Menzies
Research Institute Tasmania, University of Tasmania, Hobart, TAS, 5 Murdoch
Childrens Research Institute, Melbourne, VIC, Australia, 6 NMR Metabonomics
Laboratory, University of Eastern Finland, Kuopio, 7 National Institute for Health
and Welfare, Helsinki, 8 Department of Medicine, Turku University Hospital,
Turku, 9 Tampere University Hospital and University of Tampere, Tampere,
Finland
Background: Metabolic profiling holds potential in cardiovascular risk assessment. We evaluated whether high-throughput metabolic profiling improves
prediction of subclinical atherosclerosis as compared with conventional lipid
testing.
Methods: Serum lipids, lipoprotein subclasses, and small molecule metabolites
were analyzed by nuclear magnetic resonance (NMR) for 1570 individuals
aged 24−39 years in the population-based Cardiovascular Risk in Young Finns
Study. Carotid intima-media thickness, a marker of subclinical atherosclerosis,
was measured in 2001 and 2007. Baseline conventional risk factors and
circulating metabolites were used to predict 6-year incidence of high intimamedia thickness (90th percentile) or plaque.
5
Results: The best prediction of high intima-media thickness was achieved when
NMR-based lipoprotein lipids, and two novel biomarkers replaced conventional
LDL-C and HDL-C in models including conventional risk factors. This model
improved risk stratification beyond established risk factors alone; area under
the ROC curve 0.77 vs. 0.74, P = 0.03; net reclassification improvement 15%,
P = 0.003. The novel biomarkers had odds ratios for incident high intima-media
thickness of similar magnitude as cholesterol measures but were independent
of these.
Conclusions: Metabolic profiling, with new systemic biomarkers, improved risk
stratification for subclinical atherosclerosis over and above conventional lipids,
and would be useful for early cardiovascular risk assessment.
22 BIOLOGICAL ROLES OF THE SECRETED SOLUBLE FORM OF
LR11/SORLA
W. Schneider1 , H. Bujo2 , M. Jiang2 , K. Ohwaki2 , Y. Saito2 . 1 Medical
Biochemistry, Medical University Vienna, Vienna, Austria, 2 Genome Research
and Clinical Application, Graduate School of Medicine, Chiba University,
Chiba, Japan
Objectives: The receptor LR11 (also termed SorLA) exists as a membranebound full-length protein as well as a soluble form shed from the cell surface,
sLR11, which is present in the circulation. The aim of our studies is to fully
understand the roles of sLR11 in macrophage-foam cell formation and in smooth
muscle cell (SMC) migration.
Results: LR11−/− mice were protected from macrophage infiltration into cuffinjured arteries, while incubation with sLR11 increased by twofold the migration
activity of THP-1 monocyte-macrophages and their adhesion to extracellular
matrices. Serum concentrations of sLR11 were positively correlated with carotid
intima-media thickness (IMT). Compared to wildtype mice, in LR11 −/− mice
i. angiotensin-II (AngII)-stimulated migration and attachment of SMCs were
largely abolished,
ii. overproduction of sLR11 decreased the sensitivity of AngII-induced
activation pathways to inhibition by an AngII-type1 receptor-blocker, and
iii. femoral artery intimal thickness after cuff-placement decreased.
In isolated wt murine SMCs, exposure to sLR11 caused membrane ruffle
formation via activation of focal adhesion kinase/ERK/Rac1 accompanied by
complex formation between uPAR and integrin avb3, a process accelerated by
AngII. The effects of sLR11 were also inhibited by neutralizing anti-urokinasetype plasminogen activator receptor (uPAR) antibody.
Conclusions: SLR11, originating from intimal SMCs, modulates adhesion,
AngII-stimulated migration, and foam cell formation through activation of uPAR.
Since SLR11 is also required for AngII-induced SMC migration, we propose
that sLR11 may serve a novel role as a bio-marker of IMT.
23 GENETIC POLYMORPHISMS OF THE MAIN TRANSCRIPTION
FACTORS FOR ADIPONECTIN GENE PROMOTER IN REGULATION
OF PLASMA ADIPONECTIN LEVEL
L. Kedenko1 , C. Lamina2 , I. Kedenko1 , F. Kronenberg2 , B. Paulweber1 .
1
University Clinic for Internal Medicine I, Paracelsus Medical University,
Salzburg, 2 Division of Genetic Epidemiology, Innsbruck Medical University,
Innsbruck, Austria
Adiponectin is one of the most promising biomarkers in cardiovascular
epidemiology. It is estimated that 30−70% of the variability in plasma
adiponectin levels is accounted for by genetic factors and variability at
adiponectin gene locus is thought to account for only 2−8% of serum
adiponectin variance.
We hypothesized that genetic polymorphisms in the main transcription factor
genes for adiponectin gene might influence serum adiponectin levels. We
selected 26 SNPs based on their potential ability to modify serum adiponectin
concentration and the haplotype block structure. Genotyping was performed
using TaqMan method. The association analyses on the relation of selected
SNPs at FOXO1, SREBP1c, SIRT1, PPARgamma, PPARdelta, PGC1alpha and
TFAP-2beta genes on adiponectin levels in SAPHIR population (1738 healthy
unrelated subjects, Caucasian origin) was performed using linear regression
models adjusted for age, gender and BMI assuming an additive as well as
recessive genetic model.
Assuming an additive genetic model we found associations between
adiponectin levels and three correlated SNPs in the SREBP1c gene with the
strongest effect for rs1889018, (b = −0.054, SE = 0.016, p = 0.001). Another SNP
(rs2267668) at the PPARdelta gene (b = −0.062, SE = 0.021, p = 0.003) was
borderline significant, if correction for multiple testing was applied. Haplotype
analysis of the FOXO1, SREBP1c, SIRT1, TFAP-2beta genes, stratified anaylsis
for gender and analysis using a recessive model did not give additional
information for the association of plasma adiponectin levels.
The role of genetic variation at SREBP1c and PPARdelta genes in regulation
of adiponectin level has to be further investigated and should be confirmed in
other populations.
6
24 SENSITIVE BIOMARKER FOR STATIN-INDUCED MYOTOXICITY
R. Laaksonen1 , K. Tarasov1 , D. Kauhanen1 , T. Sylvänne1 , M. Jänis1 ,
R. Hurme1 , K. Ekroos1 , G. Mombelli2 , C. Sirtori3 , J.-C. Tardif4 . 1 Zora
Biosciences Oy, Espoo, Finland, 2 Niguarda Hospital, 3 University of Milan,
Milan, Italy, 4 Montreal Heart Institute, Montreal, QC, Canada
Background and Aims: Statins can be associated with a wide range of
muscular side effects, from non-specific or atypical myalgias, to myopathy and
the full-blown rhabdomyolysis syndrome. For the treating physician the serum
CK is not a sensitive biomarker as it becomes diagnostic only after substantial
damage to muscle cells has occurred. Thus, in the vast majority of cases the
CK measurement remains uninformative despite true symptoms. To this end
we aimed to develop new, more sensitive, biomarkers for statin induced muscle
toxicity.
Methods: Plasma samples obtained from subjects (n = 40) with clear muscular
intolerance phenotypes and 40 tolerant patients were analyzed. Direct infusion
coupled to tandem mass spectrometry, i.e. shotgun lipidomics, and two liquid
chromatography tandem mass spectrometry (LC-MS/MS) approaches, i.e.
ceramide and cerebroside lipidomics and ganglioside lipidomics, were used
to identify new lipid biomarkers.
Results: 290 molecular lipids were quantified. Out of those 27 molecular lipids
were differently expressed in the study groups. Logistic models were fitted in
order to find different combinations of lipids that could separate cases and
controls from each other. First, all the lipids were set as possible explanatory
variables and model was selected using a stepwise method. Two significant
lipids were found and with these two lipids in the model AUC value reached
0.81. The model was slightly improved after adding four more lipids to the
model (AUC = 0.88).
Conclusion: Molecular lipid species proved to be excellent biomarker
candidates for statin-induced myotoxicity allowing for more sensitive and early
detection of myopathy.
25 LACTATE AS AN ATHEROSCLEROTIC PLAQUE MARKER FOR
HYPOXIA
S.M. Bovens1,2 , G. Zomer3 , A. Vink4 , G. Pasterkamp1 . 1 Experimental
Cardiology, University Medical Center Utrecht, 2 Interuniversity Cardiology
Institute of the Netherlands (ICIN), Utrecht, 3 National Institute for Public Health
and the Environment (RIVM), Bilthoven, 4 Pathology, University Medical Center
Utrecht, Utrecht, The Netherlands
Background: It is thought that hypoxia and inflammatory cell infiltration promote
neovascularization. In these hypoxic areas new vasculature is leaky and most
likely the primary source of intra-plaque hemorrhage, leading to increased risk
of plaque rupture. One of the metabolites associated with hypoxia is lactate.
A universal technique to detect and quantify metabolites, such as lactate, is
1
H-NMR spectroscopy. We hypothesize that increased levels of lactate are
associated with increased plaque vessel density as well as hypoxia markers
and is consequently a marker for rupture-prone plaques.
Methods and Results: In femoral atherosclerotic plaques (n = 50), obtained
after surgery, 1 H NMR spectra analysis was performed. Frozen samples were
grinded, dissolved in PBS and filtered. The filtered solution was analyzed by
1
H-NMR-spectroscopy at 400 MHz. Spectra were analyzed and lactate peak
integrals (doublet present at 1.32 ppm) used for analysis. Vessel density in the
plaque was determined in histological slides stained for vascular endothelium
(CD34). HIF-1a (hypoxia-inducible factor 1a) staining was graded as no, minor,
moderate or heavy.
We observed that vessel density increased with increasing lactate levels, with
a significant difference (p = 0.01) between the first and fourth quartile (vessel
density: 2.36±0.8 and 7.14±1.3 n/mm2 respectively). Heavy HIF1a staining
was significantly associated with high lactate levels (p = 0.02) and patients
who reached a cardiovascular endpoint had significantly higher lactate levels
(p = 0.03).
In conclusion, high plaque lactate levels are associated with high vessel
density and hypoxia in atherosclerotic plaques and could therefore serve as
a marker for rupture-prone hypoxic plaques.
26 ACIDITY INCREASES THE UPTAKE OF NATIVE LDL BY HUMAN
MONOCYTE-DERIVED MACROPHAGES
K. Öörni, R. Plihtari, P.T. Kovanen. Wihuri Research Institute, Helsinki,
Finland
Due to plaque hypoxia, the extracellular pH is locally decreased in advanced
atherosclerotic lesions. Since accumulation of LDL-derived cholesterol and
formation of foam cells are key processes in atherogenesis, we tested here
the effects of acidic pH on the uptake of native LDL. First, human monocytes
were differentiated into macrophages in the presence of granulocyte-monocytecolony stimulating factor (GM-CSF), after which native LDL was applied to
the monocyte-derived macrophages at pH 5.5, 6.5, or 7.5, and the binding
and uptake of LDL by macrophages were determined. The lower the pH, the
higher was the binding and uptake of LDL by macrophages. Also, acidic pH was
found to increase the production of cell surface proteoglycans by macrophages
Oral presentations
and the binding of LDL to isolated cell-surface proteoglycans. The acidityinduced increase in LDL uptake could be inhibited by pretreating the cells with
heparinase and chondroitinase as well as by interrupting proteoglycan synthesis
by NaClO3 . Thus, the present data indicate that cell surface proteoglycans play
a critical role in acidity-induced LDL uptake by macrophages. By increasing
both the amount of cell surface proteoglycans and the affinity of LDL for them,
acidity increases the effective concentration of LDL on macrophage surfaces
and so promotes LDL uptake and induction of foam cell formation.
27 POSITIVE CROSS-TALK BETWEEN HYPOXIA INDUCIBLE
FACTOR-1a AND LIVER X RECEPTOR a INDUCES FORMATION
OF TRIGLYCERIDE-LOADED FOAM CELLS
T.-Y. Na, M.-O. Lee. College of Pharmacy and Bio-MAX Institute, Seoul
National University, Seoul, Republic of Korea
Background: Atherosclerosis is a chronic and progressive inflammatory
disease of the arteries that is characterized by subendothelial accumulation
of lipid-rich macrophages, called foam cells. Although the mechanism is not
clearly established, hypoxic conditions in the atherosclerotic lesions contribute
to the formation of these lipid-loaded macrophages. The liver X receptor (LXR)
is an orphan nuclear receptor that functions as a regulator of lipid metabolism in
many tissues, however, role of LXRa in the foam cell formation is not known.
Methods and Results: We first observed that expression of LXRa was timedependently induced in human primary macrophages and RAW 264.7 cells
under hypoxia. When hypoxia-inducible factor-1a (HIF-1a) expression was
decreased by transfection of small interfering RNA duplexes that targeted HIF1a, LXRa induction was eliminated. TO901317, an activator of LXRa, enhanced
the expression level and the transcriptional activity of HIF-1a, which was
also decreased by knockdown of LXRa. Second, we demonstrated that LXRa
increased HIF-1a protein stability through a physical interaction between the
ligand binding domain of LXRa and the oxygen-dependent degradation domain
of HIF-1a. Third, we found that the activation of HIF-1a or LXRa synergistically
induced triglyceride accumulation in the macrophages. Finally, we showed that
LXRa and HIF-1a were codistributed in the macrophages of atherosclerotic
arteries obtained from patients.
Conclusions: These results suggest that the positive feedback regulation of
transcriptional induction and protein stability of LXRa and HIF-1a may have an
important impact in foam-cell formation and the development of atherosclerotic
lesions.
28 DURATION OF TYPE 2 DIABETES STRONGLY PREDICTS ALL-CAUSE
MORTALITY, CARDIOVASCULAR MORTALITY AND DEATH FROM
STROKE IN SUBJECTS UNDERGOING CORONARY ANGIOGRAPHY
G. Silbernagel1 , S. Rosinger2 , T.B. Grammer3 , B.R. Winkelmann4 ,
B.O. Boehm2 , W. März3,5,6 . 1 Division of Endocrinology, Diabetology,
Nephrology, Vascular Disease, and Clinical Chemistry, Department of
Internal Medicine, Eberhard-Karls-University Tübingen, Tübingen, 2 Division of
Endocrinology and Diabetes, Ulm University Medical Center, Ulm University,
Ulm, 3 Department of Public Health, Social and Preventive Medicine, Mannheim
Medical Faculty, University of Heidelberg, Mannheim, 4 Cardiology Group
Frankfurt-Sachsenhausen, Frankfurt/Main, 5 Synlab Center of Laboratory
Diagnostics Heidelberg, Heidelberg, Germany, 6 Clinical Institute of Medical
and Chemical Laboratory Diagnostics, Graz, Austria
Aims: Type 2 diabetes is an important cardiovascular risk factor. However,
there is a paucity of data on the impact of the duration of type 2 diabetes on
mortality, especially in subjects at intermediate to high cardiovascular risk. Thus,
we aimed to investigate the predictive value of the diabetes duration for allcause mortality, cardiovascular mortality, and fatal stroke in subjects scheduled
for coronary angiography.
Methods and Results: We studied 2580 participants of the LUdwigshafen
RIsk and Cardiovascular health study, a large cross-sectional and prospective
clinical trial designed to investigate cardiovascular risk factors. Type 2 diabetes
was categorized according to the American Diabetes Association 2010 criteria.
The mean (± standard deviation) duration of the follow-up for all-cause and
cardiovascular mortality and death from stroke was 7.35±2.3 years. 582 deaths
occurred during the follow-up. Among these, 366 were accounted for by
cardiovascular diseases and 27 were due to fatal stroke. The duration of
type 2 diabetes was strongly and positively correlated with all-cause and
cardiovascular mortality and death from stroke (all p < 0.001). The multivariate
adjusted hazard ratios (95% confidence intervals) for cardiovascular mortality
compared to subjects without diabetes were 1.83 (1.40–2.38), 2.91 (2.06–
4.11), 3.03 (1.91–4.80), and 4.91 (3.54–6.80) for subjects with new onset
type 2 diabetes and subjects with known type 2 diabetes (duration 5, >5
and 10, >10 years), respectively.
Conclusion: The data strongly emphasize the need to consider the duration
of type 2 diabetes for the prediction of mortality in subjects at intermediate to
high cardiovascular risk.
79th EAS Congress
29 ASYMPTOMATIC CAROTID ARTERY DISEASE IN VALVULAR HEART
SURGERY
A. Anselmi, M. Morelli, C. Pragliola, N. Pavone, V. Tsiopoulos, F. Glieca,
M. Gaudino. Cardiac Surgery, Caholic University, Rome, Italy
Purpose: We aimed to define the prevalence of asymptomatic carotid artery
disease in patients undergoing valvular surgery.
Methods: In a three-years period, 1198 patients were scheduled for elective
isolated valvular surgery; all of them underwent preoperative echo-Doppler
screening of the epiaortic vessels. Cases who had symptoms of carotid
vasculopathy were excluded from the present analysis. Coexistence, site and
degree of extracardiac vasculopathy were recorded. Operative strategy was
modified according to the results of the carotid screening.
Results: 1012 patients were enrolled; 267 of them (26.4%) had hemodynamically significant carotid vasculopathy. Eighty-two (31%) had bilateral
carotid stenosis, while the remaining 185 had monolateral disease. Thirtyseven patients (3.6%) had carotid stenosis >70% while the remaining 230 had
carotid stenosis between 50% and 70%. In all these 267 cases the planned
surgical approach was modified including either simultaneous or staged carotid
endoarterectomy and/or adoption of hypothermic cardiopulmonary bypass. No
cases of postoperative neurological complication were recorded.
Conclusions: The prevalence of asymptomatic carotid vasculopathy in patients
scheduled for elective isolated valvular surgery is not negligible (26.4% in our
series). Routine echo-Doppler evaluation of the epiaortic vessels in all patients
allows modification of the surgical strategy in order to minimize the neurological
risk and is probably cost-effective.
30 LIPID-LOWERING EFFECTS OF THE THYROID HORMONE
ANALOGUE EPROTIROME REQUIRES FUNCTIONAL LDL
RECEPTORS: STUDIES IN A HOMOZYGOUS FH PATIENT
P. Benedek1 , B. Angelin1 , J. Kristensen2 , M. Rudling1 , M. Eriksson1 .
1
Endocrinology, Metabolism and Diabetes, Karolinska Institutet at Karolinska
University Hospital − Huddinge, Stockholm, 2 KaroBio AB, Huddinge,
Sweden
Introduction: Thyroid hormone stimulates a number of important rate-limiting
steps in lipid metabolism, including bile acid synthesis, cholesterol excretion
and absorption, hepatic lipase and LDL receptor expression. The liver-specific
thyroid hormone analogue Eprotirome elicits pronounced lowering of plasma
total and LDL cholesterol, triglycerides, apoB and Lp(a) when given to
hypercholesterolemic patients. Its detailed mechanism of action in humans is
not known.
Aim: To explore the importance of functioning LDL receptors for the lipidlowering effects of Eprotirome.
Patients and Methods: A 20 yr old female patient with homozygous FH (LDL
receptor gene mutation W556R) with lack of functioning LDL recptors was
studied during continued weekly LDL apheresis therapy. Eprotirome was given
over a 3 month period, with increases in dose every 2 weeks starting from 50 mg
to a maximum dose of 200 mg daily. Blood samples were collected every week
before and after apheresis, and assayed for safety parameters, hormone levels,
lipoprotein pattern and markers of cholesterol metabolism.
Results: Despite showing the expected dose-dependent stimulation of sexhormone binding globulin (SHBG) and bile acid synthesis (measured as 7alphahydroxy-4-cholestene-3-dione, C4), there were no significant decreases in total
or LDL cholesterol, triglycerides or apoB. There was no clinical evidence
of disturbances of thyroid, heart or muscle function, and the hypothalamicpituitary-thyroid hormone axis remained intact.
Conclusion: The results show that the lipid-lowering effects of the liverselective thyroid hormone analogue Eprotirome is dependent on its potential
to increase the expression of LDL receptors.
31 NONFASTING GLUCOSE AND ISCHEMIC HEART DISEASE −
A MENDELIAN RANDOMIZATION APPROACH
M. Benn1 , A. Tybjærg-Hansen2 , M.I. McCarthy3 , G.B. Jensen4 , P. Grande2 ,
B.G. Nordestgaard1 . 1 Copenhagen University Hospital, Herlev Hospital,
2
Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark,
3
Oxford Centre for Diabetes, Endocrinology and Metabolism, University of
Oxford, Oxford, UK, 4 Copenhagen University Hospital, Hvidovre Hospital,
Copenhagen, Denmark
Background: Elevated plasma glucose levels associate with increased risk
of ischemic heart disease (IHD), but whether this is a causal relationship is
unknown. We tested whether elevated nonfasting glucose levels cause IHD.
Methods: Using a Mendelian randomization approach, we studied 80,522
white persons from Copenhagen, Denmark of which 13,824 developed IHD:
the Copenhagen City Heart Study, a prospective general population study
with 34 years follow-up (1976–2010); the Copenhagen General Population
Study, a cross-sectional/prospective general population study (2003–2010);
and the Copenhagen Ischemic Heart Disease Study, a case-control study
(1991–2010). Individuals were genotyped for variants in GCK (rs4607517),
G6PC2 (rs560887), ADCY5 (rs11708067), DGKB (rs2191349), and ADRA2A
7
(rs10885122), all associated with elevated fasting glucose levels in genomewide association studies.
Results: Risk of IHD increased stepwise with increasing nonfasting glucose
levels. The hazard ratio for IHD in individuals with nonfasting glucose
levels 11 mmol/L (198 mg/dL) versus <5 mmol/L (<90 mg/dL) was 7.3
(95% CI: 4.5–12.1) adjusted for age and gender and 2.6 (1.4−4.8) adjusted
multifactorially. Increasing number of glucose increasing alleles associated with
increasing nonfasting glucose levels, and with increased risk of IHD. The
estimated causal odds ratio for IHD by instrumental variable analysis for a
1 mmol/L (18 mg/dL) increase in nonfasting glucose levels due to genotypes
combined was 1.24 (1.01–1.524), while the corresponding observed hazard
ratio for IHD by Cox regression was 1.18 (1.15–1.22).
Conclusion: Like common nonfasting glucose elevation, plasma glucose
increasing polymorphisms associate with increased risk of IHD. These data
are compatible with a causal association.
32 TRIGLYCERIDE-RICH LIPOPROTEINS AND HIGH-DENSITY
LIPOPROTEIN CHOLESTEROL IN PATIENTS AT HIGH RISK OF
CARDIOVASCULAR DISEASE: EVIDENCE AND GUIDANCE FOR
MANAGEMENT
M.J. Chapman1 , H.N. Ginsberg2 , P. Amarenco3 , F. Andreotti4 , J. Borén5 ,
A. Catapano6 , O.S. Descamps7 , E. Fisher8 , P. Kovanen9 , J.A. Kuivenhoven10 ,
P. Lesnik11 , L. Masana12 , B.G. Nordestgaard13 , K. Ray14 , Z. Reiner15 ,
M.-R. Taskinen16 , L. Tokgözoglu17 , A. Tybjærg-Hansen13 , G.F. Watts18 ,
The European Atherosclerosis Society Consensus Panel. 1 Hôpital de la
Pitié, INSERM UMR-S 939, Paris, France, 2 Columbia University, New York,
NY, USA, 3 Bichat University Hospital, Paris, France, 4 Catholic University
Medical School, Rome, Italy, 5 University of Gothenburg, Gothenburg, Sweden,
6
University of Milan, Milan, Italy, 7 Hopital de Jolimont, Bruxelles, Belgium,
8
New York University, New York, NY, USA, 9 Wihuri Research Institute,
Helsinki, Finland, 10 University of Amsterdam, Amsterdam, The Netherlands,
11
INSERM, Paris, France, 12 Universitat Rovira & Virgili, Reus, Spain,
13
University of Copenhagen, Copenhagen, Denmark, 14 St George’s University
of London, London, UK, 15 University Hospital Center Zagreb, Zagreb, Croatia,
16
Biomedicum, Helsinki, Finland, 17 Hacettepe University Medical Faculty,
Ankara, Turkey, 18 University of Western Australia, Perth, WA, Australia
Aims: First, to critically evaluate the evidence for elevated levels of triglyceriderich lipoproteins (TRL) and low levels of HDL-C as cardiovascular risk factors
in patients at high risk of CVD at LDL-C goal; this lipid profile typically clusters
with central obesity, insulin resistance, dyslipidemia and hypertension. Second,
to propose guidance for clinical management.
Methods and Results: Epidemiological, genetic, pathophysiological and
intervention studies were appraised. The available evidence supports the
contention that elevated TRL and their remnants together with low HDL-C
are causally related to elevated CV risk; indeed, TRL and remnants may
drive atherothrombosis via direct and indirect mechanisms while HDL-mediated
protection may be defective. Thus, the Panel believes that therapeutic targeting
of a high TG/low HDL-C phenotype is in all likelihood beneficial in high-risk
patients with cardiometabolic abnormalities. Lifestyle intervention, including
smoking cessation, exercise, dietary factors and weight loss, is the critical
first step for managing high-risk patients at LDL-C goal with elevated TG
(1.7 mmol/L) and/or low HDL-C (<1.0 mmol/L) according to current European
guidelines. Non-compliance and secondary causes of dyslipidemia must be
addressed. Addition of niacin or a fibrate, or intensification of LDL-C lowering
therapy may then be considered, taking into account relevant safety concerns.
Treatment should be tailored to individual high-risk patients to achieve TG
<1.7 mmol/L and HDL-C 1.0 mmol/l.
Conclusion: The Panel believes this evidence-based guidance for the
management of elevated TG and/or low HDL-C will facilitate reduction of CV
risk that persists in high-risk patients at LDL-C goal.
8
33 PLANT STEROLS AND CARDIOVASCULAR DISEASE:
A SYSTEMATIC REVIEW AND META-ANALYSIS
W. März1,2,3 , G. Silbernagel4 , G. De Backer5 , E. Bruckert6 , R. Carmena7 ,
J. Chapman8 , J. Deanfield9 , O.S. Descamps10 , E.R. Rietzschel11 , K.C. Dias12 ,
B. Genser12 . 1 Synlab Center of Laboratory Diagnostics Heidelberg,
Heidelberg, 2 Institute of Public Health, Social and Preventive Medicine,
Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany,
3
Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical
University of Graz, Graz, Austria, 4 Division of Endocrinology, Diabetology,
Nephrology, Vascular Disease, and Clinical Chemistry, Department of
Internal Medicine, Eberhard-Karls-University, Tübingen, Tübingen, Germany,
5
Department of Public Health, Ghent University, Ghent, Belgium, 6 Division of
Endocrinology and Metabolism, Hôpital de la Pitié-Salpétrière, Paris, France,
7
Division of Endocrinology and Nutrition, Department of Medicine, University
Hospital, Valencia, Spain, 8 Dyslipidemia, Inflammation and Atherosclerosis
Research Unit, National Institute for Health and Medical Research (INSERM),
Hôpital de la Pitié-Salpetriere, Paris, France, 9 Great Ormond Street Hospital,
University College London, London, UK, 10 Epidemiology Unit, School of
Public Health, Université Catholique de Louvain, Brussels, 11 Department
of Cardiovascular Diseases, Ghent University, Ghent, Belgium, 12 BGStats
Consulting, Graz, Austria
Background: It has been suggested that moderately elevated serum
concentrations of plant sterols (similar to those resulting from supplementation)
increased CVD.
Data sources: We conducted a systematic literature review using the
databases MEDLINE, EMBASE and COCHRANE which aimed to investigate
the association between the concentrations of plant sterols (campesterol,
sitosterol) and CVD. We retrieved studies published by April 2010 reporting
serum concentrations of the exposures of interest and (a) risk ratios with respect
to CVD endpoints or (b) summary statistics of concentrations in CVD cases and
controls separately.
Review methods: We extracted the following from each study: design, study
population, summary statistics of concentrations, risk ratios (RR) of CVD
related endpoints, statistical methods and list of confounding variables used in
multivariate modeling. RRs were transformed to contrast the upper vs. the lower
third of the distribution of exposure serum concentrations with respect to the
endpoint “all CVD events pooled”. For studies that reported serum levels of CVD
cases and controls separately we calculated standardized mean differences
(SMDs) and standard errors.
Results: We included 17 studies of different designs. Of those, 8 reported
relative risks and 15 reported summary statistics of serum levels in CVD cases
and controls. For campesterol and sitosterol and their ratios to cholesterol both
types of meta analyses, RRs and SMDs, showed absence of a relationship with
CVD risk.
Conclusion: This systematic review did not show any evidence for an
association between serum levels of plant sterols and risk of CVD.
34 APOLIPOPROTEIN AV INTERNALIZED IN HUMAN ADIPOCYTE
MODULATES CELLULAR TRIGLYCERIDE ACCUMULATION
X.-Y. Zheng, S.-P. Zhao, B.-L. Yu, C.-L. Wu, L. Liu. Department of Cardiology,
the Second Xiang Ya Hospital, Central South University, Changsha, China
Objective: Apolipoprotein AV (apoAV), an important determinant of plasma
triglyceride levels, has been recently reported to modulate triglyceride
metabolism in the hepatocytes. In this study, we investigated whether apoAV
can be internalized by adipocytes and regulate cellular triglyceride storage.
Methods and Results: Human preadipocytes, derived from subcutaneous
adipose tissue of patients undergoing abdominal surgery, were differentiated
into mature adipocytes. Pulse-chase experiments revealed that apoAV was
internalized into human adipocytes, and ~70% of the apoAV internalized during
the pulse remained intracellular within a 24 h chase, while 30% was degraded.
Preincubation with heparin and the receptor associated protein, both of which
prevent the apoAV interaction with the low density lipoprotein receptor-related
protein (LRP), markedly reduced the uptake of apoAV by 61% and 52%,
respectively, which were confirmed by western blot analysis. Using confocal
microscopy we demonstrated that labeled apoAV co-localized around lipid
droplets with a lipid droplet-associated protein. Furthermore it was observed
that treatment of adipocyte with apoAV decreased triglyceride storage by
~30%.
Conclusions: our results demonstrated for the first time that apoAV can be
internalized by human adipocytes, localized to lipid droplets, partially mediated
by LRP. The endocytosis of apoAV may modulate triglyceride accumulation of
adipocytes.
Oral presentations
35 ALIPOGENE TIPARVOVEC GENE THERAPY SIGNIFICANTLY
REDUCES THE RISK OF PANCREATITIS IN LIPOPROTEIN LIPASE
DEFICIENT PATIENTS
S. Greentree1 , J. Méthot2,3 , S. Déry3 , K. Tremblay2,3 , J. de Wal1 , N. van den
Bulk1 , G. Ruiterkamp1 , A. Freidig1 , D. Brisson2,3 , D. Gaudet2,3 . 1 Amsterdam
Molecular Therapeutics (AMT) B.V., Amsterdam, The Netherlands, 2 Dept of
Medicine, Université de Montréal, Montreal, 3 Ecogene-21 and Lipid Clinic,
Chicoutimi, QC, Canada
Introduction: Alipogene tiparvovec (Glybera® ) is developed for therapy in
lipoprotein lipase deficiency (LPLD). It consists of variant LPLS447X in an
adeno-associated virus derived vector, serotype 1. It is administered by onetime intramuscular injection. The most debilitating manifestation of LPLD is
(recurrent) acute pancreatitis, that may cause complications such as diabetes
mellitus and (early) atherosclerosis, and is life threatening. We performed a
historic case control study in LPLD patients who had previously participated in
a clinical study, to determine whether alipogene tiparvovec lowered the risk of
acute pancreatitis and/or severe pancreas-related abdominal pain.
Methods: 22 LPLD patients consented to enrol; of these 17 had previously been
administered alipogene tiparvovec. Historical data for all hospital presentations
due to abdominal pain were collected. Data collection and pancreatitis definition
were according to modified Atlanta Diagnostic Criteria for acute pancreatitis, to
allow for incomplete historical data. Blinded event assessment was done by
an expert adjudication committee. Statistical analysis to model the hazard of
pancreatitis pre/post therapy was executed using a Cox regression gap time
model.
Results: A statistically significant (p = 0.0434) reduction in the risk of acute
pancreatitis was seen when the period from the first pancreatitis event to
administration was compared with the post-therapy period (median = 2.9 years).
The hazard ratio indicated a 63% reduction in risk of acute pancreatitis after
alipogene tiparvovec (95% CI 0.142–0.971).
Conclusion: One-time alipogene tiparvovec was efficacious and resulted in
statistically and clinically significant reduction in risk of severe acute abdominal
hospital presentations.
36 SOCIAL DISRUPTION STRESS ACCELERATES ATHEROSCLEROSIS
IN APOE−/− MICE
M.E. Johansson1 , E. Bernberg2 , M.A. Ulleryd1 , G.M. Bergström2 . 1 Institute
of Neuroscience and Physiology, Unversity of Gothenburg, 2 Wallenberg
Laboratory, University of Gothenburg, Gothenburg, Sweden
Introduction: We have previously shown that different forms of stress have
distinctive effects on atherogenesis in mice. We showed that social stress
increase atherosclerosis in ApoE−/− mice, while more physical forms of stress
do not. Here we evaluated the effect of social disruption (SDR) stress on
atherogenesis and evaluated cytokine release after SDR-stress and five more
physical stressors.
Methods: Male ApoE−/− mice were exposed to SDR-stress during 12 weeks,
and atherosclerotic plaque area was assessed in aorta, aortic root and
innominate artery. Further, male C57BL/6 mice were exposed to SDR-stress or
five physical stressors, and cytokine and corticosterone levels were analyzed
in plasma/serum samples immediately after stress.
Results: We found a correlation between the level of SDR-stress and
atherosclerotic plaque area in aorta and a numerical increased plaque area
in aortic root. Further, SDR-stress increased levels of corticosterone, IL-6 and
CXCL1. Plasma corticosterone increased for all five physical stressors, but IL-6
and CXCL1 only increased in the group exposed to restraint combined with rat
odor.
Conclusions: These findings suggest that SDR-stress is atherogenic, in
contrast to our previous results using the physical stressors. A possible
explanation to this difference is that SDR-stress, but not physical stressors,
leads to release of the pro-inflammatory cytokines IL-6 and CXCL1.
37 DEPRESSION SCORES PREDICT ADHERENCE IN A DIETARY
WEIGHT LOSS INTERVENTION TRIAL
S. Somerset1,2 , L. Graham3 , K. Markwell1,2 . 1 School of Public Health, Griffith
Unversity, 2 Griffith Health Institute, 3 Griffith Health Institute, Griffith Unversity,
Meadowbrook, QLD, Australia
Background: Depression is an independent CHD risk factor and affects BMI,
diet, physical activity, and smoking. Increasing depression prevalence presents
challenges to long-term behaviour change to reduce CHD risk. This study
investigated associations between depression scores and participation in a
community-based weight management intervention trial.
Methods: A group of 64 overweight (BMI > 27), otherwise healthy, adults were
randomised to follow either their usual diet, or an isocaloric diet (saturated
replaced with monounsaturated fat, target 50% total fat, using macadamia
nuts). Depressive symptoms were measured at baseline and ten weeks (Beck
Depression Inventory, BDI-II). All subjects received the same protocol for
physical activity, food diary completion and trial consultations. Anthropometric
79th EAS Congress
and clinical measures (cholesterol, inflammatory mediators) were taken at
baseline and 10 weeks.
Results: During recruitment, pre-existing diagnosed major depression was one
reason for initial exclusion from the trial. There was a significant correlation
(R=-0.38, p < 0.05) between baseline BDI-II scores and duration in trial.
Subjects with a baseline BDI 10 (moderate/severe depression symptoms)
were more likely to dropout before week 10 (p < 0.001). Univariate analysis
of variance confirmed these observations (adjusted R2 = 0.257, p = 0.01).
Body weight remained static over the 10-week period in the intervention group,
corresponding to a relative increase in the control group (adjusted R2 = 0.097,
p = 0.064).
Conclusions: Depression symptoms affect enrolment in and adherence to dietbased risk reduction interventions, and may influence generalisability of results.
Depression scores should be used to characterise, screen and allocate patients
to appropriate treatment pathways.
38 CAN SKINFOLD THICKNESS PREDICT CARDIO-VASCULAR RISK
FACTORS IN YOUTHS?
P. Schwandt, E. Liepold, G.M. Haas. Arteriosklerose-Praeventions-Institut,
Muenchen, Germany
Background: In youths the association of adiposity with skinfold thickness
(SFT) might be closer than with body mass index (BMI). Since data on the
relation to cardiovascular disease (CVD) risk factors is heterogeneous we
examined associations of SFT and BMI with CVD risk factor clustering in 6,888
German children and adolescents.
Subjects and Methods: Data were obtained between 2000 and 2008 from
3,850 children (1,981 boys) aged 6−11 years and 3,038 adolescents (1,639
males) aged 12−18 years participating in the community-based PEP Family
Heart Study Nuremberg. We constructed age- and gender-specific percentile
curves for SFT and calculated ROC-cut-off-points using 3 risk factors out of
7 traditional non-anthropometric risk factors.
Results: Only 4% of the subjects were overweight (85th percentile) and 10%
had SFT 90th percentile. SFT was substantially higher in girls than in boys.
The cut-off percentiles for SFT sum were 87.5 for males, 88.0 mm for females
and for BMI > 90.5 in both genders. SFT was more strongly related to risk factor
clustering than was BMI and even more in children than in adolescents. Thus,
SFT can better predict than BMI CVD risk factors like hypertension, elevated
LDL-C, Non HDL-C, triglycerides, TG/HDL-C ratio as a surrogate of insulin
resistance, fasting glucose, and low HDL-C in youths.
Conclusions: This cross-sectional study demonstrates that skinfold thickness
is more strongly related to cardiovascular risk factors than body mass index in
children and adolescents.
39 IMPACT OF OPTICAL COHERENCE TOMOGRAPHY (OCT),
INTEGRATED-BACKSCATTER-IVUS (IB-IVUS), AND 64-SLICE
MULTIDETECTOR CT (64-SLICE MDCT) ON ACS AND STABLE
LESION CHARACTERISTICS
Y. Ozaki, M. Okumura, K. Hattori, H. Naruse, S.C. Kan, M. Ohota, H. Harigaya,
H. Kawai, M. Ishikawa, T. Hashimoto, J. Ishii. Cardiology, Fujita Health
University School of Medicine, Nagoya, Japan
Background: Several pathological studies suggest that disruption of thincap (<65 mm) fibroatheroma (TCFA) leads to acute coronary syndrome (ACS;
i.e. vulnerable plaque). While optical coherence tomography (OCT; Lightlab,
Westford, MA) offers a high-resolution image (10 mm), Integrated-Backscatter
intravascular ultrasound (IB-IVUS) provides precise tissue characterization and
64-slice MDCT (Aquilion 64 system, Toshiba) could render plaque density profile
(Hounsfield units; HU).
Methods and Results: We prospectively performed 64-slice MDCT, OCT and
IB-IVUS prior to coronary intervention (PCI) in 50 patients (29; ACS and
21; stable angina). While cap thickness by OCT was significantly thinner in
ACS than that in stable angina (40±15 mm vs. 272±145 mm, p < 0.01), the
percentage of lipid area by IB-IVUS was significantly greater in ACS than that
in stable angina (35±10% vs. 25±9%, p < 0.01). The rate of plaque density
with less than30 by 64-slice MDCT was also significantly higher in ACS than
that in stable angina (85% vs. 16%, p < 0.01).
Conclusions: While OCT, IB-IVUS and 64-slice MDCT clearly differentiated
ACS lesions from stable lesions, thin fibrous cap atheroma with lipid rick plaque
would clinically play a role in the mechanism of ACS.
40 NON-INVASIVE MOLECULAR IMAGING USING CONTRAST
ENHANCED ULTRASOUND FOR DETECTION OF ARTERIAL
THROMBOSIS VIA SINGLE-CHAIN ANTIBODY-TARGETED
MICROBUBBLES
X. Wang1,2 , C.E. Hagemeyer1 , F. Jia1 , E. Leitner1 , I. Ahrens1 , K. Peter1 .
1
BakerIDI Heart and Diabetes Institute, 2 Monash University, Melbourne, VIC,
Australia
Background: Molecular imaging is a rapidly evolving field, which allows noninvasive detection of vascular pathologies. Activated platelets are key players
9
in thrombosis, atherosclerosis and inflammation. We hypothesised that singlechain antibodies (scFv) directed against activated platelets and conjugated
to ultrasound contrast bubbles would provide an unique approach for high
sensitivity imaging of thrombus development and facilitate early diagnosis and
treatment of thrombosis.
Methods and Results: Microbubbles were conjugated to either a scFv specific
for activated GPIIb/IIIa (LIBS-MB), or a non-specific scFv (control-MB). Flow
experiments over pre-activated platelets demonstrated strong adhesion of LIBSMB at 50s-1 when compared with control-MB (p < 0.001). Detachment assays at
increase shear rates of 1000s-1 and 6000s-1 dislodged most control-MB while
LIBS-MB remained strongly attached (p < 0.001). Platelet-rich thrombi were
induced in carotid arteries of mice in vivo by ferric chloride injury (6%, 3 min) and
assessed by ultrasound before and 20 min after microbubble injection. There
was no change in greyscale values for animals injected with control-MB, whilst
greyscale values strongly increased after LIBS-MB injection (p < 0.001). After
thrombolysis, ultrasound imaging observed that thrombus areas were reduced
(p < 0.001).
Conclusions: We could demonstrate that anti-GPIIb/IIIa antibody targeted
microbubbles specifically bind to activated platelets in vitro and in vivo, as
well as facilitate monitoring of pharmacological thrombolysis on ultrasound
imaging. This non-invasive and cost effective imaging modality provides a
unique opportunity to detect arterial microthrombi at an early stage allowing
for early diagnosis and therapy.
41 GENETIC VARIATION IN TRANSTHYRETIN, LIPID AND LIPOPROTEIN
LEVELS, AND RISK OF ISCHEMIC VASCULAR DISEASE
L. Hornstrup1 , C.L. Haase1 , R. Frikke-Schmidt1 , A. Tybjærg-Hansen1,2 .
1
Dept. Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital,
2
The Copenhagen City Heart Study, Bispebjerg Hospital, Copenhagen
University Hospital and Faculty of Health Sciences, University of Copenhagen,
Copenhagen, Denmark
Background and Aims: Previous studies have shown that transthyretin (TTR)
can modulate lipid metabolism and possibly contribute to reduced cholesterol
efflux. To further explore this, we hypothesized that genetic variation in TTR
might associate with plasma levels of lipids and lipoproteins, and with risk of
ischemic vascular events in the general population.
Methods: We genotyped 10,287 individuals from the Copenhagen City Heart
Study (CCHS), a prospective study of the general population, for TTR
G6S (rs1800458), the only common, nonsynonymous variant identified by
resequencing TTR in the entire CCHS. We determined the association of G6S
genotype with lipid and lipoprotein levels, and the ability of genotype to predict
risk of ischemic heart disease, myocardial infarction, ischemic cerebrovascular
disease, and ischemic stroke in the general population, with 31-years follow.
Results: Minor allele frequency for the 6S allele was 7.7% in the CCHS.
Homozygosity for the 6S allele (SS genotype) was associated with reductions
in total cholesterol and LDL cholesterol of, respectively, 0.29 mM (5%,
P = 0.03) and 0.33 mM (9%, P = 0.009) compared with non-carriers. Observed
hazard ratios for ischemic heart disease, myocardial infarction, ischemic
cerebrovascular disease, and ischemic stroke were not significantly different
from 1.00 for heterozygotes or homozygotes versus non-carriers.
Conclusion: Homozygosity for the TTR 6S allele was associated with
reductions in plasma levels of total- and LDL cholesterol in the general
population, but not with risk of ischemic vascular events. Larger studies are
warranted to replicate these findings.
42 IDENTIFICATION OF TWO NEW MUTATIONS OF GPIHBP1 IN
SEVERE HYPERCHYLOMICRONEMIA
S. Charriere1,2 , N. Peretti3 , S. Bernard1 , M. Charcosset4 , A. Sassolas4 ,
A. Lachaux3 , P. Moulin1,2 , C. Marçais2,5 . 1 Féderation d’Endocrinologie, Hopital
Louis Pradel, Hospices Civils de Lyon, Bron, 2 Unité 1060 CarMen, INSERM,
Lyon, 3 Service d’Hépatologie, Gastroentérologie et Nutrition, Hopital Femme
Mère Enfant, Hospices Civils de Lyon, 4 Fédération de Biochimie, Groupement
Hospitalier Est, Hospices Civils de Lyon, Bron, 5 Laboratoire de Biologie
Moléculaire, Centre de Biologie Sud, Centre Hospitalier Lyon Sud, Hospices
Civils de Lyon, Pierre-Bénite, France
Introduction: GPIHBP1 (Glycosyl-phosphatidyl-inositol HDL binding protein 1)
is a new endothelial binding site for lipoprotein lipase (LPL), the key enzyme
of triglycerides rich lipoproteins (TGRL) intravascular lipolysis. By sequencing
GPIHBP1 in patients with severe hyperchylomicronemia (TG > 15 mmol/l),
without genetic abnormality previously identified (LPL, apoE, apoC2, apoA5),
we discovered two new homozygous mutations: a C89F mutation in the Ly-6
domain associated with a C14F signal peptide polymorphism, and a G175R
mutation of the C-terminal domain.
Aim and Methods: The aim of the study was to characterize in vitro these
two mutations. CHO745 cells were transiently transfected with wild type (WT)
or mutant GPIHBP1. The expression of GPIHBP1 at the cell surface and LPL
binding were studied by western blotting.
10
Results: The expression of mutant GPIHBP1 at the cell surface was reduced:
−53±6% for C14F, −35±13% for C89F, −45±12% for C14F/C89F haplotype,
and −48±7% for G175R, versus WT. The LPL binding to GPIHBP1 was
preserved for C14F but was altered for C89F (−78±13%), C14F/C89F
haplotype (−91±5%) and for G175R (−43±13%), versus WT.
Conclusions: These two new GPIHBP1 mutations are responsible for a
decrease of both protein expression at the cell surface and LPL binding. There
is a synergic association between C89F mutation and C14F polymorphism
which leads to a major impairment of LPL binding. The G175R mutation is the
first mutation described in the C-terminal domain involved in the GPI anchor
binding of the protein. These results confirm the major role of GPIHBP1 in
TGRL intravascular lipolysis in humans.
43 NEW GAIN OF FUNCTIONS MUTATIONS IN PCSK9 AND THEIR
IMPACT IN FAMILIAL HYPERCHOLESTEROLEMIA
M. AbiFadel1,2 , J.-P. Rabès1,3 , M. Guérin4 , V. Carreau4 , A. Carrié4 ,
M. Varret1 , M.-E. Samson-Bouma1 , L. Tosolini1 , D. Erlich1 , P. Couvert4 ,
D. Bonnefont-Rousselot5 , E. Bruckert4 , A. Prat6 , N. Seidah6 , C. Boileau1,3 .
1
INSERM U698, Bichat-Claude Bernard’s hospital, Paris, France, 2 School
of Pharmacy, Université Saint-Joseph, Beirut, Lebanon, 3 Laboratoire de
Biochimie et de Génétique moléculaire, hôpital Ambroise-Paré, APHP,
Boulogne, 4 INSERM UMR S939; Pitié Salpêtrière hospital, 5 UF de biochimie
des maladies métaboliques; Pitié Salpêtrière hospital, Paris, France,
6
Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of
Montreal, Montreal, QC, Canada
Introduction: Our discovery in 2003 of the first natural mutants of PCSK9
( Proprotein convertase subtilisin kexin 9) in French families with autosomal
dominant hypercholesterolemia (ADH) has revealed the implication of an asyet-unknown actor in cholesterol homeostasis and a new major player in the
regulation of plasma LDL-C levels. PCSK9 has been shown to degrade the
LDL receptor independently of its catalytic activity. Gain of function mutations
are responsible of ADH, while loss of function variants are associated with a
reduction of LDL-C and the risk of coronary heart disease. PCSK9 is now one of
the most promising drug targets to lower LDL-C levels. Thus, the identification
and characterization of new mutations of PCSK9 are very important.
Methods and Results: In order to provide more insight in the genetic causes of
ADH, we are studying further patients with hypercholesterolemia unrelated to
the LDLR or the APOB genes. We have recently identified 2 new mutations
of PCSK9 in French families with severe hypercholesterolemia. These new
mutations are highly conserved between species. We studied the impact of
these mutations on the maturation of PCSK9. Furthermore, FACS analysis
showed that one of these mutants lead to a strong reduction of LDL receptors
on cell surface. Finally we proceeded to a complete lipoprotein analysis and
evaluated, for the first time, the capacity of the plasma of these PCSK9
mutations’ carriers to mediate cellular free cholesterol efflux
Conclusions: These data further contribute to the characterization of PCSK9
mutations and their impact in cholesterol diseases.
44 -203A/C POLYMORPHISM OF CHOLESTEROL 7a-HYDROXYLASE
(CYP7A1) GENE AFFECTS CYP7A1 ACTIVITY AFTER SHORT-TERM
TREATMENT WITH CHOLESTYRAMINE
J. Kovar1,2 , M. Zimolova1,2 , M. Lenicek3 , L. Vitek3 , M. Jirsa1 , R. Poledne1,2 .
1
Institute for Clinical and Experimental Medicine, 2 Centre for Cardiovascular
Research, 3 Dept of Clinical Biochemistry and Laboratory Diagnostics, 1st
Faculty of Medicine, Charles University, Prague, Czech Republic
The cholesterol 7a-hydroxylase (CYP7A1) activity displays a pronounced
diurnal variation. The −203A/C polymorphism of gene encoding CYP7A1 affects
the responsiveness of serum cholesterol to dietary fat and cholesterol. However,
it is not clear yet whether this polymorphism can also affect diurnal variation of
enzyme activity.
The diurnal variation of 7a-hydroxycholest-4-en-3-one [C4], a marker of
CYP7A1 activity, was therefore measured in 16 healthy male volunteers
homozygous either for −203A or for −203C variant. The study of diurnal variation
was carried out on three occasions: after one day treatment with cholestyramine, after one day treatment with chenodeoxycholic acid (CDCA) and without
any treatment. The measurements were carried out from 7 AM to 10 PM.
The area under curve of C4 rose significantly more in subjects homozygous
for −203C variant than in those homozygous for −203A after short term
administration of cholestyramine. Accordingly, at the same time cholesterol
concentration decreased 9% in −203C carriers and was not affected in −203A
carriers. No differences between −203A and −203C carriers were found in
diurnal variation of CYP7A1 activity in control experiment and after CDCA
treatment. These findings seem to be in an agreement with findings of in vitro
study that activity of −203C promoter variant is severalfold higher than that of
−203A variant in dual luciferase assay.
Our results suggest that −203A/C polymorphism of CYP7A1 gene can be
involved in regulation of cholesterolemia response to bile acid sequestrants.
Supported by grant No 1M0510 from MEYS CR.
Oral presentations
45 ACYL CHAIN-DEPENDENT EFFECT OF
LYSOPHOSPHATIDYLCHOLINES ON COX-2 EXPRESSION IN
VASCULAR ENDOTHELIAL CELLS
L. Brkic, M. Riederer, R. Malli, W. Graier, S. Frank. Institute for Molecular
Biology and Biochemistry, Medical University of Graz, Graz, Austria
Background and Aims: Endothelial lipase (EL) is a serum phospholipase produced mainly by vascular endothelial cells. EL generates substantial amounts of
saturated 16:0-lysophosphatidylcholine (LPC) and unsaturated 18:1-, 18:2- and
20:4-LPCs by cleaving its major substrate HDL-phosphatidylcholine. Because
EL is active on the surface of vascular endothelial cells and its expression is
upregulated by inflammation, EL-derived LPCs generated in close proximity of
vascular endothelium might be important modulators of endothelial function.
While 16:0-LPC is an established activator of various signaling cascades,
almost nothing is known about signaling properties of unsaturated LPCs.
Therefore, the aim of the present study was to examine the signaling
properties of unsaturated LPCs in comparison to 16:0-LPC with an emphasis
on modulation of COX-2 expression and activation of MAP kinases.
Results: 16:0-, 18:1-, 20:4-, but not 18:2-LPC raised COX-2 mRNA expression
in endothelial cell line EA.hy926 in a time dependent manner. COX-2 mRNA
induction was reduced with pharmacological inhibitors of certain pathways:
BAPTA/AM (Ca2+ chelator), SB203580 (p38 MAPK inhibitor) and U73122
(specific PLC inhibitor), suggesting the role of Ca2+ and p38 MAPK in LPCelicited COX-2 expression. The activation of p38 MAPK was further confirmed
by Western blot.
COX-2 protein levels were also raised in time dependent manner after
incubating EA.hy926 cells with 16:0-, 18:2-, 20:4-, but not 18:1-LPC.
Conclusions: These results indicate that LPCs raise COX-2 mRNA and protein
levels in time dependent manner. That raise is dependent of Ca2+ and p38
MAP kinase. The exact pathways preceding COX-2 upregulation still need to
be clarified.
46 LIPID ALDEHYDE-MODIFIED AMINOPHOSPHOLIPIDS INDUCE ER
STRESS AND ACTIVATE THE INFLAMMATORY RESPONSE OF
ENDOTHELIAL CELLS
S.S. Davies1,2 , L. Guo2 , Z. Chen2 , B.E. Cox2 , V. Amarnath3 , R.F. Epand4 ,
R.M. Epand4 . 1 Pharmacology, 2 Clinical Pharmacology, 3 Pathology, Vanderbilt
University, Nashville, TN, USA, 4 Biochemistry and Biomedical Services,
McMaster University, Hamilton, ON, Canada
Introduction: Peroxidation of plasma lipoproteins has been implicated in the
endothelial cell activation and monocyte adhesion that initiates atherosclerosis,
but the exact mechanisms underlying this activation remain unclear. Lipid
peroxidation generates lipid aldehydes, including the g-ketoaldehydes (gKA)
also termed isoketals or isolevuglandins, that readily modify the amine
headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde
modification of PE could mediate some of the proinflammatory effects of lipid
peroxidation.
Objective: Determine if aldehyde-modified PE induced endothelial cell
activation and the mechanism for this activation.
Results: We found that PE modified by gKA (gKA-PE) induced THP-1 monocyte
adhesion to human umbilical cord endothelial cells (HUVEC). gKA-PE also
induced surface expression of adhesion molecules and increased MCP-1 and
IL-8 mRNA in HUVEC. To determine the structural requirements for gKA-PE
activity, we tested several related compounds. PE modified by 4-oxo-pentanal
(OPA-PE) induced THP-1 adhesion, but N-glutaroyl-PE and C18:0 N-acyl-PE did
not, suggesting that an N-pyrrole moiety was essential for cellular activity. We
tested the effect of the pyrrole-PEs on membrane parameters and found that
gKA-PE and OPA-PE significantly reduced the temperature for the hexagonal
phase transition (TH ) in artificial bilayers, suggesting that these pyrrole-PE
markedly altered membrane curvature. Additionally, fluorescently labeled gKAPE rapidly internalized to the endoplasmic reticulum (ER) and gKA-PE induced
CHOP and BiP expression, as well as p38MAPK activity, consistent with gKAPE inducing ER stress responses previously linked to inflammatory chemokine
expression.
Conclusion: gKA-PE and related aldehyde-modified PE are potential mediators
of the inflammation induced by lipid peroxidation.
47 ALOX15B IS THE MAJOR 15-LIPOXYGENASE IN HUMAN
MACROPHAGES
S. Wüst1,2 , M. Crucet2,3 , M. Herová1,2 , M. Hersberger1,2 . 1 Division of Clinical
Chemistry and Biochemistry, University Children’s Hospital Zurich, 2 Zurich
Center for Integrative Human Physiology (ZIHP), 3 Institute of Physiology,
University of Zurich, Zurich, Switzerland
The lipoxygenase pathway plays a role in the recruitment and adhesion of
leukocytes by the generation of two classes of arachidonic acid lipid mediators,
the leukotrienes and lipoxins. The leukotrienes occur in the initial phase of
inflammation and have pro-inflammatory effects. The lipoxins are synthesized
later in inflammation and are anti-inflammatory. 3 lipoxygenases are essential
for the production of lipoxins, ALOX12, ALOX15 and ALOX15B. Different animal
79th EAS Congress
models suggested a role for this 12/15-lipoxygenase enzymatic activity in
atherogenesis. To investigate the regulation of the 3 lipoxygenases in human
macrophages we measured the expression of ALOX12, ALOX15 and ALOX15B
mRNA during differentiation of monocytes to macrophages and following
stimulation in macrophages. The results showed a small increase of ALOX15B
during the differentiation of monocytes to macrophages. Expression of ALOX12
and ALOX15 remained on the same low level. Stimulation of macrophages with
a set of cytokines and with hypoxia revealed that IL-4, IL-13, and hypoxia further
increased the ALOX15B mRNA. IL-4 and IL-13 also enhanced the expression
of ALOX15, while none of the stimuli had an impact on ALOX12 expression.
The quantification of the mRNAs showed that ALOX15B is the major mRNA
present in stimulated macrophages. The levels of ALOX15B are higher than
the levels of ALOX15 in macrophages following IL-13 and hypoxia stimulation.
Only stimulation with IL-4 resulted in a similar level of ALOX15 and ALOX15B
mRNAs. These results suggest that ALOX15B plays a major role in human
macrophages and may influence human atherosclerosis eventually together
with ALOX15.
48 INHIBITION OF ALOX15B REDUCES INFLAMMATION IN
ATHEROSCLEROSIS-PRONE MICE
L. Mattsson Hultén1 , A. Lundqvist2 , M.N. Karlsson2 , K. Skålén1 , O. Wiklund2 ,
L. Magnusson1 . 1 Sahlgrenska Center for Cardiovascular and Metabolic
Research, 2 Wallenberg Laboratory, University of Gothenburg, Gothenburg,
Sweden
Introduction: Inflammation in the vascular wall is important for the development
of atherosclerosis. We have shown that the protein ALOX15B is expressed in
human atherosclerotic lesions but not in healthy tissue. The enzyme oxidizes
fatty acids to substances that promote local inflammation and is mostly
expressed in macrophages present in the atherosclerotic tissue.
Methods: To elucidate the role of ALOX15B in the development of
atherosclerosis we have used lentiviral shRNA silencing and bone marrow
transplantation to knockdown ALOX15B gene in vivo in LDL-receptor-deficient
(Ldlr −/− ) mice. The extent of atherosclerosis was analyzed by lipid staining with
Oil Red O and by immunohistochemical techniques.
Results: We have characterized the most specific of four tested shRNAs to
knock down ALOX15B, and RT-PCR data show an 80% inhibition ofALOX15B
in mouse macrophages. We have successfully performed bone marrow
transplantation and shRNA delivery to Ldlr −/− mice and they have been fed a
Western diet to induce atherosclerosis. Knockdown of ALOX15B in vivo resulted
in decreased inflammatory activation and decreased plaque lipid content as well
as increased collagen content suggesting a more stable plaque phenotype.
Conclusion: In vivo experiments show that the levels of ALOX15B influences
parameters shown to affect progression of atherosclerosis indicating that
ALOX15B has an active role in the atherosclerotic process.
49 FARNESOID X RECEPTOR ACTIVATION INDUCES CHOLESTERYL
ESTER TRANSFER PROTEIN EXPRESSION IN HUMANS AND
TRANSGENIC MICE
T. Gautier1 , W. de Haan2 , D. Ye3 , M.J. Bahr4 , T. Claudel1 , T.J.C. Van
Berkel3 , J. Grober5 , L. Lagrost5 , F. Kuipers1 , M. Van Eck3 , P.C.N. Rensen2 ,
U.J.F. Tietge1 . 1 University Medical Center Groningen, Groningen, 2 Leiden
University Medical Center, 3 Leiden/Amsterdam Center for Drug Research,
Leiden, The Netherlands, 4 Hannover Medical School, Hannover, Germany,
5
INSERM UMR866 Lipides, Nutrition, Cancer, Dijon, France
Cholesteryl ester transfer protein (CETP) activity results in a pro-atherogenic
plasma lipoprotein profile. In cholestasis, bile acid (BA)-mediated farnesoid X
receptor (FXR) signalling is activated and plasma HDL cholesterol levels are
low. This study explored the hypothesis that FXR-mediated induction of CETP
contributes to this phenotype.
Patients with cholestasis and high plasma BA had lower HDL cholesterol
levels and significantly higher plasma CETP activity and mass compared to
matched controls with low plasma BA (each: p < 0.01). In addition, BA feeding
in APOE3*Leiden transgenic mice expressing the human CETP transgene
controlled by its endogenous promoter increased cholesterol within apoBcontaining lipoproteins and decreased HDL cholesterol (each p < 0.01), while
hepatic CETP mRNA expression, plasma CETP activity and mass increased
significantly (each: p < 0.01). In vitro studies confirmed that BA and the synthetic
FXR agonist GW4064 augmented CETP mRNA expression in hepatocytes
and macrophages (each: p < 0.001). Furthermore, using liver biopsies we
demonstrate that humans and transgenic mice differ in the relative contribution
of hepatocytes and Kupffer cells to hepatic CETP production, closely related to
the species-specific composition of the BA pool.
Conclusion: This study identifies CETP as a novel FXR target gene using
a combined approach in vivo in patients and transgenic mice as well as in
vitro. Via increased CETP expression FXR activation leads to a pro-atherogenic
lipoprotein profile in CETP expressing species. In perspective, these results
have clinical relevance, especially when the use of FXR agonists as an
emerging treatment strategy for metabolic disease is considered.
11
50 PPARA ACTIVATION DIFFERENTLY AFFECTS MICROPARTICLE
CONTENT IN ATHEROSCLEROTIC LESIONS AND LIVER OF
A MOUSE MODEL OF ATHEROSCLEROSIS AND NASH
M. Baron1 , A.S. Leroyer2 , Z. Majd3 , F. Lalloyer1 , E. Vallez1 , K. Bantubungi1 ,
G. Chinetti-Gbaguidi1 , P. Delerive3 , C.M. Boulanger2 , B. Staels1 , A. Tailleux1 .
1
Inserm U1011 Institut Pasteur de Lille, Lille, 2 Inserm U970, Paris, 3 Genfit SA,
Loos, France
Background: Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are
complex pathologies characterized by lipid accumulation, chronic inflammation
and extensive tissue remodelling. Microparticles (MPs), small membrane
vesicles produced by activated and apoptotic cells, might not only be
biomarkers, but also functional actors in these pathologies. The apoE2-KI
mouse is a model of atherosclerosis and NAFLD. Activation of the nuclear
receptor PPARa decreases atherosclerosis and components of non-alcoholic
steatohepatitis (NASH) in the apoE2-KI mouse.
Objectives:
1. To determine whether MPs are present in atherosclerotic lesions, liver and
plasma during atherosclerosis and NASH progression in apoE2-KI mice,
and
2. to study whether PPARa activation modulates MP concentrations.
Methods: ApoE2-KI mice were fed a Western diet to induce atherosclerosis
and NASH. MPs were isolated from atherosclerotic lesions, liver and blood and
quantified by flow cytometry.
Results: An increase of MPs was observed in the atherosclerotic lesions and
in the liver of apoE2-KI mice upon Western diet feeding. PPARa activation
with fenofibrate decreased MP levels in the atherosclerotic lesions in a PPARadependent manner, but did not influence MP concentrations in the liver.
Conclusion: Here we report that MPs are present in atherosclerotic
lesions and in the liver of apoE2-KI mice. Their concentration increased
during atherosclerosis and NASH development. PPARa activation differentially
modulates MP levels in a tissue-specific manner.
51 C/EBPb CONTROLS EXERCISE-INDUCED CARDIAC GROWTH AND
PROTECTS AGAINST PATHOLOGICAL CARDIAC REMODELING
P. Bostrom1 , N. Mann2 , J. Wu1 , P.A. Quintero2 , E.R. Plovie3 , D.R. Panáková3 ,
R.K. Gupta1 , C.K. Xiao2 , C.A. MacRae3 , A. Rosenzweig2 , B.M. Spiegelman1 .
1
Harvard Medical School, Dana-Farber Cancer Institute, 2 Cardiovascular
Division of the Beth Israel Deaconess Medical Center and Harvard Medical
School, BIDMC, 3 Cardiovascular Division, Brigham & Women’s Hospital,
Boston, MA, USA
The heart has the ability to grow in size in response to exercise, but
little is known about the transcriptional mechanisms underlying physiological
hypertrophy. Adult cardiomyocytes have also recently been proven to hold
the potential for proliferation, a process which could be of great importance
for regenerative medicine. Using a unique RT-PCR based screen against
all transcriptional components, we showed that C/EBPb was down-regulated
with exercise, while the expression of CITED4 was increased. Reduction of
C/EBPb in vitro and in vivo resulted in a phenocopy of endurance exercise with
cardiomyocyte hypertrophy and proliferation. This proliferation was mediated, at
least in part by the increased CITED4. Importantly, mice with reduced cardiac
C/EBPb levels displayed substantial resistance to cardiac failure upon pressure
overload. These data indicate that C/EBPb represses cardiomyocyte growth
and proliferation in the adult mammalian heart and that reduction in C/EBPb is
a central signal in physiologic hypertrophy and proliferation.
Atherosclerosis Supplements 12 (2011) 13–184
Contents lists available at ScienceDirect
Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis
79th EAS Congress, 26−29 June, 2011, Gothenburg, Sweden
52 DUAL METABOLIC DEFECTS ARE REQUIRED TO PRODUCE
HYPERTRIGLYCERIDEMIA IN OBESE SUBJECTS
M. Adiels1 , M.-R. Taskinen2 , J. Westerbacka2 , S. Söderlund2 , J. Kahri2 ,
N. Lundbom2 , J. Lundbom3 , A. Hakkarainen3 , S.-O. Olofsson1 , M. OhroMelander4 , J. Borén1 . 1 Department of Molecular and Clinical Medicine,
Göteborg University, Göteborg, Sweden, 2 Medicine, 3 Medical Imaging Center,
University of Helsinki, Helsinki, Finland, 4 Department of Clinical Sciences,
Lund UniversityUniversity, Lund, Sweden
Objective: Obesity increases the risk of cardiovascular disease and premature
death. However, not all obese subjects develop the metabolic abnormalities
associated with obesity. The aim of this study was to clarify the mechanisms
that induce dyslipidemia in obese subjects.
Methods and Results: Stable isotope tracers were used to elucidate
the pathophysiology of the dyslipidemia in hypertriglyceridemic (n = 14) and
normotriglyceridemic (n = 14) obese men (with comparable body mass index
and visceral fat volume) and in normotriglyceridemic non-obese men (n = 10).
Liver fat was determined using proton magnetic resonance spectroscopy
and subcutaneous abdominal and visceral fat were measured by magnetic
resonance imaging. The results show that serum triglycerides in obese
subjects are increased by the combination of increased secretion and severely
impaired clearance of triglyceride-rich VLDL1 particles. Furthermore, increased
liver and subcutaneous abdominal fat are linked to increased secretion of
VLDL1 particles, whereas increased plasma levels of apolipoprotein C-III are
associated with impaired clearance in obese hypertriglyceridemic subjects.
Conclusion: Dual metabolic defects are required to produce hypertriglyceridemia in obese subjects with similar levels of visceral adiposity. The results
emphasize the clinical importance of measuring both abdominal obesity and
traditional risk factors to properly evaluate the metabolic abnormalities in obese
patients.
53 DO INTAKES OF FATTY ACIDS MODIFY THE ASSOCIATION
BETWEEN GENETIC VARIATION IN FADS-LOCUS AND LIPID
CONCENTRATION?
S. Hellstrand, E. Sonestedt, U. Ericson, B. Gullberg, E. Wirfält, M. Svensson,
M. Orho-Melander. Department of Clinical Sciences, Lund University, Malmö,
Sweden
Background and Aim: Variants in the fatty acid desaturase (FADS) gene
cluster have been associated with LDL, HDL and triglyceride concentrations.
FADS convert the a-linolenic acid (n-3) and linolenic acid (n-6) into longchain polyunsaturated fatty acids (PUFA). The PUFA concentration has
been associated with metabolic syndrome and cardiovascular disease. We
investigated the interaction between PUFA intakes and the FADS polymorphism
rs174547 (T/C) on LDL, HDL and triglyceride concentrations.
Methods: We included 4,635 (45−68 years, 66% females) participants from
the Malmö Diet and Cancer cardiovascular sub-cohort. Diet was assessed by
combining a 168-item dietary questionnaire, 7-day menu book and a 1-h history
interview. PUFA intakes of total energy were divided in tertiles; low, medium and
high. The General Linear Model investigated interactions, adjusted for several
confounders.
Results: The minor C-allele (allele frequency 34%) of rs174547 was
significantly associated with lower LDL (p = 0.03). We observed a significant
interaction between the FADS genotype and long-chain n-3 PUFAs on LDL
(p = 0.01). The C-allele was significantly associated with lower LDL among
individuals with low intakes of long-chain n-3 PUFA (0.14%E, p < 0.001),
but not among those with medium (0.14–0.28%E) or high (>0.28%E) intakes
(p = 0.98 and p = 0.86 respectively). Among those with medium intakes of n-6
PUFA (4.4−5.5%E) the C-allele was significantly associated with lower LDL
(p = 0.05).
Conclusions: Our findings suggest that the intakes of different PUFAs are
important modifiers of the effect size of genetic variation in FADS on LDL
concentrations and could contribute to the inconsistent findings in different
populations.
1567-5688/ $ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
54 EFFECT OF ONE YEAR TREATMENT WITH INSULIN AND ORAL
GLUCOSE-LOWERING AGENTS ON LIPID LEVELS IN NON-OBESE
PATIENTS WITH TYPE 2-DIABETES
S. Søgaard Lund1 , L. Tarnow1 , B. Nielsen1 , B. Hansen1 , M. Frandsen1 ,
O. Pedersen2 , H.-H. Parving3 , A. Vaag1 . 1 Steno Diabetes Center, 2 Hagedorn
Research Institute, Gentofte, 3 Rigshospitalet, Copenhagen, Denmark
Background: In non-obese type-2 diabetes patients (T2DM), metformin has
been suggested to improve cholesterolaemia versus an insulin secretagogue
at similar glycaemic control. The cholesterol-lowering effect of metformin has
been suggested to be enhanced by statin therapy. It is unknown if these effects
persist when combined with insulin therapy. We studied the effect of repaglinide
(an insulin secretagogue) versus metformin both in combination with insulin
therapy on lipid levels in non-obese T2DM patients. The effect of statin use
was also of interest.
Methods: 101 non-obese T2DM patients (BMI 27 kg/m2 ) with HbA1c 6.5%
on oral agents were randomised to 1-year treatment with insulin (biphasic insulin
aspart 70/30) in combination with repaglinide 6 mg or metformin 2 g (doubleblind). Insulin doses were adjusted aiming for HbA1c < 6.5%. Patients who did
not start or stop statin therapy during follow-up were analysed (n = 88).
Results: In statin users, insulin plus metformin (n = 43) significantly lowered
fasting total and Non-HDL cholesterol (Non-HDLC) compared with insulin
plus repaglinide (n = 40) (mean [95% CI] baseline-adjusted difference between
treatments for Non-HDLC: −0.26 mmol/l [−0.49; −0.02], p = 0.039). In patients
not using statins (n = 5), no significant differences in plasma lipid levels were
observed between treatments. Conclusions were similar after adjusting for
changes in statin doses. Insulin doses and HbA1c were similar between
treatments (previously reported).
Conclusion: In non-obese T2DM patients using statins, 1-year treatment
with insulin plus metformin rather than insulin plus an insulin secretagogue
might improve proatherogenic cholesterolaemia. This supports potential
cardioprotective effects of metformin when combined with insulin even in statin
users.
55 REGULATION OF BILE ACID AND CHOLESTEROL METABOLISM
BY ENDOGENOUS THYROID HORMONE IN HUMANS
Y. Bonde1,2 , B. Angelin1,2 , M. Rudling1,2 . 1 Department of Medicin, 2 Department
of Bioscience and Nutrition, Karolinska Institute, Stockholm, Sweden
Objective: Thyroid hormone is a major regulator of lipid metabolism. Animal
data show that thyroid hormone stimulates bile acid synthesis, cholesterol
synthesis, and attenuates intestinal cholesterol absorption. This study aimed
to determine if these findings are relevant to humans.
Methods: Blood samples from 20 hyperthyroid patients taken before start
of treatment and during treatment were analyzed with respect to plasma
lipids, apoAI and B, serum markers for cholesterol absorption (plant sterols),
cholesterol synthesis (lathosterol), and bile acid synthesis (7a-hydroxy-4cholesterol-3-one/C4). Serum levels of FGF19, intestinal derived inhibitor of
hepatic bile acid synthesis, and PCSK9, regulator of hepatic LDL-receptor
numbers, were also measured.
Results: Total plasma cholesterol, LDL-cholesterol, and HDL-cholesterol levels
were 28% (P < 0.001), 38% (P < 0.001), and 17% (P = 0.001) lower, respectively,
in the hyperthyroid state compared to euthyroid state levels. PCSK9 serum
levels were 22% lower (P < 0.01). ApoAI and B serum levels were 14%
(P < 0.001) and 27% (P < 0.001) lower, and plant sterols campesterol and
sitosterol were 25% (P < 0.001) and 18% (P < 0.01) lower, respectively. Serum
lathosterol levels were unchanged. In hyperthyroid state, serum C4 levels were
increased by 41% (P < 0.01) compared to euthyroid state levels. In line with
this, serum FGF19 levels were 29% (P < 0.01) lower in hyperthyroidism.
Conclusions: Data show that bile acid synthesis is increased in hyperthyroidism, possibly due to decreased FGF19 signaling, and that cholesterol
synthesis is unaffected despite of decreased intestinal cholesterol absorption.
Data also indicate increased hepatic LDL-receptor mediated cholesterol uptake,
at least partially due to reduced PCSK9 levels.
14
56 NONFASTING BLOOD CONCENTRATION OF LIPOPROTEIN
SUBFRACTIONS AND RISK OF CORONARY HEART DISEASE IN
A SWEDISH NESTED CASE-CONTROL STUDY
E. Sonestedt1 , S. Chiu2 , O. Melander1 , M. Orho-Melander1 , R.M. Krauss2 .
1
Lund University, Malmö, Sweden, 2 Children’s Hospital Oakland Research
Institute, Oakland, CA, USA
Background: The ion mobility method is unique in its capability for directly
determining concentrations of the full spectrum of lipoprotein particles, from
small HDL to large VLDL, as a function of their particle size. Several studies
have shown significant associations between nonfasting lipid levels and risk of
cardiovascular disease, while the evidence of association between nonfasting
lipoprotein subfractions and cardiovascular risk is limited.
Methods: Plasma lipoprotein subfractions were measured with the ion mobility
method in 1575 subjects (45−74 years) from the Malmö Diet and Cancer cohort
that during 12 years of follow-up were diagnosed with incident coronary heart
disease and 1570 matched controls.
Results: After adjusting for potential confounders, the highest versus lowest
quintile of very small LDL (OR, 1.29; 95% CI, 1.03–1.62; P-trend, 0.007), small
LDL (OR, 1.66; 95% CI, 1.31–2.10; P-trend, <0.001) medium LDL (OR, 1.54;
95% CI, 1.23–1.94; P-trend, <0.001), large IDL (OR, 1.26; 95% CI, 1.00–1.58;
P-trend, 0.008) and VLDL (OR, 1.26; 95% CI, 1.00–1.59; P-trend, 0.005) was
associated with higher risk of coronary heart disease, while no significant
association was observed for large LDL (P-trend, 0.60) and small IDL (P-trend,
0.85). In addition, large HDL (OR, 0.56; 95% CI, 0.43–0.73; P-trend, <0.001),
but not small HDL (P-trend, 0.62), was associated with decreased risk.
Conclusion: In this prospective study measuring lipoprotein subfraction particle
concentration in nonfasting blood, high concentrations of small and medium
LDL particles and low concentration of large HDL particles showed the most
predictive associations with coronary heart disease.
57 MARINE N-3 FATTY ACIDS IN SERUM PHOSPHOLIPIDS AS
BIOMARKERS OF INTAKE INFLUENCE LDL HETEROGENEITY IN
SUBJECTS WITH FAMILIAL LIPOPROTEIN DISORDERS
A. Sala-Vila1 , M. Cofán1 , R. Mateo-Gallego2 , A. Cenarro2 , F. Civeira2 , E. Ros1 .
1
Endocrinology and Nutrition, Hospital Clı́nic de Barcelona, Barcelona, 2 Lipid
Unit, Hospital Universitario Miguel Servet, Zaragoza, Spain
Introduction: Supplementation with marine n-3 fatty acids (FA) or fish oil
shifts plasma lipoprotein composition to a less atherogenic pattern. However,
the issue whether high intake of marine n-3 FA achieved through a nonsupplemented diet is associated with a low concentration of small, dense LDL
has not been investigated thus far.
Objective: To examine the association between the content of marine n-3 FA
in serum phosphatidylcholine (a biomarker of fish intake) and LDL subclass
distribution in 298 Spanish subjects with primary dyslipidemia.
Design: Cross-sectional study.
Patients and Methods: We analyzed fasting serum from patients with
familial combined hyperlipidemia (n = 82) and familial hypercholesterolemia,
with (n = 102) or without (n = 114) defects in the LDL receptor gene. Lipoprotein
particle number and size distribution were assessed with nuclear magnetic
resonance spectroscopy. The FA composition of serum phosphatidylcholine was
determined by gas-chromatography.
Results: In a stepwise linear regression model adjusted for standard cardiovascular risk factors including fasting triglycerides, the phosphatidylcholine
content of marine n-3 FA was inversely and independently associated with
the concentration of total (regression coefficient, [B]= −71.18), medium-small
(B = −0.11) and very-small (B = −62.84) LDL particles (P < 0.01; all), resulting in
a net direct association with LDL size.
Conclusions: In subjects with primary dyslipidaemia, fish intake is associated
with an antiatherogenic LDL pattern independently of triglyceride levels,
suggesting a beneficial effect beyond the known triglyceride-lowering properties
of n-3 FA. This might explain in part low rates of carotid and coronary
atherosclerosis described in populations with high fish intake, such as the
Japanese and Spaniards.
58 CHANGES IN HUMAN LIPOPROTEIN COMPOSITION IN PATIENTS
WITH ACUTE CORONARY SYNDROME
S. Ljunggren1 , H. Karlsson2 , H. Mörtstedt3 , L. Hellström4 , J. Perk4 , C. Besler5 ,
U. Landmesser5 , A. von Eckardstein6 , M. Lindahl1 . 1 Occupational and
Environmental Medicine, Department of Clinical and Experimental Medicine,
Linköping University, 2 Occupational and Environmental Medicine, Linköping
University Hospital, Linköping, 3 Occupational and Environmental Medicine,
Lund University Hospital, Lund, 4 Internal Medicine and Environmental
Medicine and Public Health, Oskarshamn Hospital, Oskarshamn, Sweden,
5
Cardiovascular Center, Zurich University Hospital, 6 Institute for Clinical
Chemistry, Zurich University Hospital, Zurich, Switzerland
The protein composition of lipoproteins may serve as biomarkers or reveal
underlying mechanisms during different states of disease. Here we have
analysed the protein composition of LDL and HDL from patients with acute
coronary syndrome (ACS) and patients receiving statins after previous ACS.
Plasma samples were obtained from males, namely 12 healthy donors, 9
patients with ACS and 7 stable patients receiving statin treatment after previous
ACS. LDL and HDL were isolated by two-step density ultracentrifugation.
LDL proteins were analysed with two-dimensional gel electrophoresis and
mass spectrometry. Paraoxonase 1 (PON-1) in HDL was analyzed with SDSPAGE/Western Blot.
Concentrations of apo A-IV, a1-antitrypsin and transthyretin were significantly
increased (P < 0.05) in LDL from patients with ACS compared to healthy
controls. In patients receiving statins, a1-antitrypsin remained increased while
serum amyloid A4 was decreased. By western blot analysis, a non-significant
increase in PON-1 was found in HDL from patients with ACS. Interestingly, a
truncated form of PON-1 was detected in all patients with ACS but not in any
of the controls.
In conclusion, we confirm previous findings that LDL-associated transthyretin
is a possible biomarker of myocardial infarction. Moreover, the increased
concentration of the inflammatory marker a1-antitrypsin in LDL from both ACS
patients and stable patients after ACS indicate that the enrichment does not
only reflect an acute phase response. The presence of a truncated form of
the antioxidant protein PON-1 in HDL may explain previous findings showing
increased amounts but lower activity of PON-1 in patients with ACS.
59 RELATIONSHIPS BETWEEN HSCRP REDUCTION AND LDL-C, NONHDL-C, APOB AND HDL-C IN HYPERLIPIDEMIC PATIENTS TREATED
WITH EZETIMIBE/SIMVASTATIN + EXTENDED-RELEASE NIACIN
W.V. Brown1 , S. Fazio2 , J.R. Guyton3 , Q. Dong4 , J.E. Tomassini5 , A. Shah4 ,
A.M. Tershakovec5 . 1 Emory University School of Medicine, Atlanta, GA,
2
Vanderbilt University Medical Center, Nashville, TN, 3 Duke University Medical
Center, Durham, NC, 4 Merck, Rahway, NJ, 5 Merck, North Wales, PA, USA
Objective: To determine the relationship between hsCRP and lipoproteins other
than LDL-C. This is important in light of the anti-inflammatory functions of
HDL-C and elevated hsCRP levels in patients with high cardiovascular risk.
Method: An analysis of a randomized, double-blind study in type IIA/IIB
hyperlipidemic (HL) patients treated with ezetimibe/simvastatin (E/S) 10/20 mg
+extended-release niacin (N) to 2 g versus E/S or N for 24 weeks, and versus
E/S for 64 weeks. Correlations between hsCRP and lipids were assessed at
baseline, 24 and 64 weeks.
Results: Baseline hsCRP and HDL-C levels were inversely correlated
(p < 0.001); all other baseline correlations were weak and nonsignificant (table).
At 24 weeks, hsCRP levels significantly correlated with LDL-C, non-HDL-C,
and apoB levels in the E/S+N group, but only with those of non-HDL-C and
apoB in the E/S group. At 64 weeks, correlations between hsCRP and LDL-C,
non-HDL-C and apoB in the E/S+N group were still significant, whereas no
correlation reached significance in the E/S group. In the E/S+N group, hsCRP
and HDL-C levels were inversely and significantly correlated at 24 and 64
weeks. Changes in hsCRP significantly correlated only with apoB at 24 weeks
and HDL-C at 64 weeks with E/S+N. Correlations were unchanged with N
alone.
Conclusion: We observed an inverse association between HDL-C and
baseline, study-end and changes in hsCRP levels with E/S+N therapy in HL
patients. This suggests that combined LDL-lowering and HDL-raising therapies
may have enhanced HDL-C anti-inflammatory effects versus E/S or N alone.
Spearman correlation coefficients
Baseline
Study-end
Relationship
hsCRP and
Pooled
treatments
E/S+N
E/S
N
E/S+N
Changes
E/S
N
24 wks
n = 668
n = 337
n = 186
n = 145
n = 337
n = 186
n = 145
LDL-C
−0.037
0.226§
0.045
0.049
0.050
0.106
0.063
HDL-C
−0.153§
−0.080
0.020
−0.001
0.075
−0.122
0.146†
−0.097
non-HDL-C
−0.141‡
0.254§
0.075
0.095
0.072
0.042
apoB
0.072
0.250§
0.182†
0.103
0.114†
0.108
0.079
64 wks
n = 430
n = 156
−
n = 272
n = 156
−
LDL-C
−0.085
n = 272
0.184‡
−0.007
−
0.038
0.116
−
HDL-C
−0.152‡
−0.050
−
−0.132†
−0.025
−
non-HDL-C
0.004
−0.141†
0.241§
0.033
−
0.098
0.104
−
apoB
0.051
0.221§
0.116
−
0.105
0.133
−
E/S = ezetimibe/simvastatin 10/20 mg; N = up to 2 g of Niaspan; pooled treatments at 24 wks = E/S+N, E/S
and N and at 64 wks = E/S+N and E/S (Included patients on N at 24 wks who switched to E/S+N or E/S for
40 wks more).
† p < 0.05, ‡ p < 0.01, § p < 0.001 for baseline, study-end levels and changes at 24 and 64 wk levels. For apoB,
sample sizes are: n = 661 for pooled treatments, n = 333 for EIS+N, n = 184 for E/S, n = 144 for N at 24 wks,
and n = 424 for pooled treatments and n = 269 for E/S+N at 64 wks.
79th EAS Congress
60 COMPLETE SEQUENCING OF APOB GENE OF PATIENTS WITH
CLINICAL DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLAEMIA
A.C. Alves1,2 , M. Bourbon1,2 . 1 Unidade de I&D − Grupo de Investigação
Cardiovascula, DPSDC, Instituto Nacional de Saúde Dr. Ricardo Jorge,
2
Center for Biodiversity, Functional & Integrative Genomics, Lisbon, Portugal
Familial hypercholesterolemia (FH) is an inherited disorder of cholesterol
metabolism caused mainly by mutations in LDLR gene, but mutations in APOB
and PCSK9 genes also cause FH. These 3 genes are currently studied
in “Portuguese FH Study”. Only 48% of patients studied have a detectable
mutation in the 3 genes mention above so, other mutations in these genes or
other gene defects must exist to explain the cause of hypercholesterolemia in
the remaining families.
The main aim of this project was the whole sequencing of APOB gene, in 65
index patients without mutations in LDLR or PCSK9 genes or in fragments of
exon 26 and 29 of APOB gene, by pyrosequencing, in order to identify the
genetic cause of the hypercholesterolemia in these patients.
A pool of the 65 DNAs was sequenced by pyrosequecing method and a total
of 227688 nucleotide reads were obtained, corresponding to a mean coverage
of 35x/fragment/individual.
A total of 87 alterations were detected, being 27 described SNPs. From the 26
alterations detected, only 12 were found and 4 of these had not been previously
described. After family studies only one alteration did not co-segregate in the
family, providing further evidence that 3 of the alterations found can be mutations
causing disease, but functional studies are required to prove pathogenicity.
Pyrosequencing allows the rapid sequencing of a large number of individuals,
but apart from its high cost, it has some limitations as a large number of false
positives and so requires an improvement of technique.
61 VARIABILITY OF DIURNAL TRIGLYCERIDE LEVELS IN MEN AND
WOMEN
B. Klop1 , A.J.H.H.M. van Oostrom2 , J.P.H. van Wijk3 , A. Alipour1 , E. Birnie1 ,
J.W.F. Elte1 , J.S. Cohn4 , M. Castro Cabezas1 . 1 Sint Franciscus Gasthuis,
Rotterdam, 2 Sint Antonius Ziekenhuis, Nieuwegein, 3 Universitair Medisch
Centrum Utrecht, Utrecht, The Netherlands, 4 Heart Research Institute,
Camperdown, NSW, Australia
Objective: Both increased fasting and non-fasting triglycerides (TG) predict
cardiovascular events. However, TG vary largely in the fasting state and possibly
even more postprandially. Only limited data are available on the intra-individual
variability of non-fasting TG.
Methods: Capillary triglycerides (cTG) of 272 subjects were measured in a
free living situation at six standardized time points (fasting, before lunch, after
lunch, before dinner, after dinner and at bedtime) for three days. Coefficients
of variation (CV) for cTG were calculated for each time-point. Subjects were
divided into tertiles based on their three-day average fasting cTG.
Results: The CV for cTG gradually increased during the day with a median
ranging from 19.2% (IQR 10.2–33.4) to 25.0% (IQR 13.2–40.4). CVfasting
was significantly lower compared to CVafterdinner and CVbedtime (P < 0.05),
but CVbeforelunch , CVafterlunch and CVbeforedinner did not differ significantly with
other time-points. Subjects from the first tertile showed a significantly lower
CVfasting and CVbeforelunch compared to subjects from the second and third
tertile (P < 0.001). Within the second and third tertile there were no significant
differences in CV between the different time-points. Average CV was increased
with 20.2% in men compared to women for fasting and non-fasting time-points
(P < 0.05).
Conclusions: Men show a higher intra-individual variability of fasting and nonfasting TG than women. Variability of non-fasting TG compared to fasting TG is
similar and only slightly higher during the evening. Measuring non-fasting TG
instead of fasting TG could be easier for patients and help in cardiovascular
risk assessment.
62 POST-PRANDIAL LIPIDEMIA IS REDUCED BY TREATMENT OF
OBSTRUCTIVE SLEEP APNOEA: A RANDOMISED, PLACEBO
CONTROLLED TRIAL OF CONTINUOUS POSITIVE AIRWAY
PRESSURE
D.R. Sullivan1 , N. Lai1 , C. Phillips2 , R. Grunstein2 , B. Yee2 , P. Liu2 . 1 Clinical
Biochemistry, Royal Prince Alfred Hospital, 2 Woolcock Institute, University of
Sydney, Sydney, NSW, Australia
Although studies have proven that obstructive sleep apnoea (OSA) causes
some of the observed hypertension, there are no robust data to support a causal
relationship with dyslipidemia. We conducted a randomised, placebo-controlled
crossover trial to assess whether post-prandial lipidemia (a strong marker
of cardiovascular risk) would improve because of treatment with continuous
positive airway pressure (CPAP).
Adult patients with OSA who met all inclusion and exclusion criteria were
recruited from three sleep clinics. Lipid profiles including triglycerides (TAG)
were measured at 7 time points over a 24-hour period (inclusive of sleep)
on each of three occasions − at baseline prior to treatment and then after
2 months of either sham (placebo) CPAP or therapeutic CPAP (with 1 month
15
washout). Patients consumed western style meals (55% carbohydrate, 30% fat,
15% protein) at set times during the daytime (wake) period.
Mean compliance of the 29 patients was lower on sham CPAP than therapeutic
CPAP (3.5±2.3 vs 4.4±2.2 hrs/night; p < 0.05). However intra-individual usage
between devices was correlated (r = 0.55, p < 0.01). TAG concentrations peaked
at 2 time points (2pm and 3am). Intention-to-treat analysis revealed that peak
TAG levels (mean±SEM) were significantly worse on sham CPAP (2 pm:
3.45±0.09 mmol/L; 3am: 3.48±0.09 mmol/L) than therapeutic CPAP (2 pm:
2.97±0.09 mmol/L; 3 am: 3.08±0.09 mmol/L; p for difference <0.01 at both
time points. Moreover, TAG, HDL and Total Cholesterol levels across the whole
24 hour period were improved by CPAP (p < 0.01). Free fatty acids were not
different between treatments
63 INFLUENCE OF THE GLUCOSE TOLERANCE TEST ON
PRO-ATHEROGENIC MODIFICATION OF LDL AND ITS RELATION
TO PARAOXONASE ACTIVITY
H. Soran, V. Charlton-Menys, N. Younis, M. France, M. Bannerjee,
K. Cruickshank, P. Durrington. University of Manchester, Manchester, UK
Background: Diabetes is associated with increased oxidation and glycation
of LDL, modifications which increase its atherogenicity. Serum paraoxonase
PON1) activity is decreased in diabetes and this may contribute to the
greater susceptibility of LDL to oxidation and glycation. Whereas it is known
that in vitro LDL undergoes both oxidation and glycation and PON1 activity
decreases slowly on incubation with glucose, nothing is known about how
rapidly excursions of blood glucose affect these parameters in vivo.
Methods: We determined the effect of the glucose tolerance test in 24 women
with gestational diabetes. Blood samples were taken at baseline, 30 min and
2 h after oral glucose (75 g).
Results: Blood glucose was higher after glucose (5.1 [0.2] mmol/L (mean[SD])
at baseline, 8.0 [0.3] mmol/L at 30 min (p < 0.001) and 6.9 [0.4] mmol/L at
2 h (p < 0.001)). Serum PON1 activity decreased from 124 [23] nmol/ml/min at
baseline to 109 [19] nmol/ml/min at 30 min (p < 0.005) and 107 [20] nmol/ml/min
(p < 0.001) at 2 h. Oxidised LDL decreased at 30 min from 77 [5] U/l at baseline
to 66 [4] U/l (p < 0.005) and still lower at 2 h (63 [5] U/l; p < 0.001). Glycated
LDL increased from 3.1 [0.2] mg/dL to 3.6 [0.2] mg/dL at 30 min (p < 0.001) but
returned to baseline at 2 h (3.0 [0.2] mg/dL (NS).
Conclusions:
1. PON1 activity decreased acutely in response to increases in blood glucose
and this effect persisted up to 2 h.
2. Simultaneously there was a transient rise in immunoassayable glycated LDL,
whereas oxidised LDL diminished.
64 SERUM LEVELS OF LIPIDS, OXIDATIVE STRESS AND HEPATIC
HISTOLOGY IN OBESE PATIENTS UNDERGOING BARIATRIC
SURGERY
O. Amin Hussein1,2,3 , I. Shams1,3 , K. Abu Jabal1 , I. Dashkovsky4 , S. Szvalb5 ,
I. Waksman4 . 1 Internal Medicine A, Ziv Medical Center, Safed, 2 Faculty
of Medicine, Technion Institute of Technology, Haifa, 3 Laboratory for
Atherosclerosis Research, 4 Surgery Department, 5 Pathology Department, Ziv
Medical Center, Safed, Israel
Background: Nonalcoholic fatty liver disease (NAFLD) has been consistently
associated with obesity and insulin resistance.
The aim of this study is to evaluate the relation between hepatic histological
parameters in a severely obese individuals undergoing bariatric surgery and
serum lipids, oxidative stress and paraoxonase (PON) activity.
Methods: One hundred thirty seven patients undergoing bariatric surgery were
enrolled. Serum blood tests including fasting glucose, alanine aminotransferase,
total cholesterol, triglycerides, LDL-C and HDL-C, were collected before surgery
and liver biopsy was done during the procedures.
Serum PON arylesterase and lactonase activities were measured. Serum lipid
peroxides (PD) were measured spectrophotometrically. Serum malondialdehyde
(MDA) was examined by using the lipid peroxidation assay for thiobarbituric acid
reactive substances. Both MDA and PD were measured at baseline and after
oxidation with AAPH. Biopsies were graded for steatosis and inflammation and
staged for fibrosis according to Brunt EM.
Results: Advanced grade of steatosis was seen in patients with high glucose,
ALT, triglycerides and low HDL-C (p < 0.01). Advanced grade of inflammation
was seen in patients with high glucose, triglyceride and low HDL-C serum
levels (p < 0.05). Advanced fibrosis stage was found in patients with high serum
glucose and ALT levels (p < 0.05). PON lactonase, PON arylesterase, peroxides
and MDA contents in basal or oxidized serum were not different by inflammation
and steatosis grades.
Conclusion: In obese patients, advanced grades of steatosis and inflammation
and advanced stage of fibrosis is seen in patients with high serum level of
glucose and triglycerides and low levels of HDL-C.
16
65 MILDER PHENOTYPE OF RELATIVES OF INDEX PATIENTS CAN
MISDIAGNOSE FAMILIAL HYPERCHOLESTEROLEMIA
A.M. Medeiros1,2 , A.C. Alves1,2 , M. Bourbon1,2 , on behalf of the Investigators
of the Portuguese FH Study. 1 Instituto Nacional de Saúde Dr. Ricardo Jorge,
2
BioFIG − Center for Biodiversity, Functional & Integrative Genomics, Lisbon,
Portugal
Familial Hypercholesterolemia (FH) is a genetic disorder characterized by high
levels of LDLc in plasma, accelerated atherosclerosis and increased risk of
premature coronary heart disease (pCHD). FH results from mutations in three
genes involved in lipid metabolism: LDLR, APOB, PCSK9. It is known that FH
patients’ phenotype is heterogeneous varying with different conditions, as gene
and type of mutation.
The present study pretends to characterize the biochemical profile of FH
patients genetically identified in Portugal. The Portuguese FH Study identified
420 patients: 182 index (60 children, 122 adults) and 238 relatives (56
children, 182 adults) with a genetic defect. Biochemical parameters (total
cholesterol (TC), LDLc, HDLc, triglycerides, ApoB, ApoAI) were analyzed
with SPSS software using ANOVA tests. TC and LDLc levels are statistically
higher in index patients than in relatives identified in cascade screening: index children, TC=315.96±61.51 mg/dl and LDLc=239.61±60.43 mg/dl
vs TC=277.52±66.40 mg/dl and LDLc=209.05±53.44 mg/dl for relatives
children (p = 0.002, p = 0.014); index adults TC = 369.56±78.94 mg/dl and
LDLc = 287.72±78.93 mg/dl vs TC=332.93±75.54 mg/dl and LDLc=246.26
±71.79 mg/dl for relatives adults (p < 0.001, p = 0.001). Although CT and LDLc
mean values are above FH criteria a considered number of relatives have
both TC and LDLc bellow these values (16%). Only 40% of relatives adults
genetically identified are in treatment and 13% have pCHD vs 79% of index
adults in treatment and 25% have pCHD.Genetic diagnosis of FH in Portugal
allows early identification of FH patients, in particular relatives with mild
phenotype that would not be identified by clinical criteria alone, allowing early
implementation of therapeutic measures that will reduce their cardiovascular
risk.
66 THE MOLECULAR BASIS OF AUTOSOMAL DOMINANT
HYPERCHOLESTEROLEMIA: ASSESSMENT IN A LARGE COHORT
OF HYPERCHOLESTEROLEMIC CHILDREN
A. van der Graaf, H.J. Avis, D.M. Kusters, M.N. Vissers, B.A. Hutten,
J.C. Defesche, R. Huijgen, S.W. Fouchier, F.A. Wijburg, J.J. Kastelein,
A. Wiegman. AMC, Amsterdam, The Netherlands
Background: Autosomal dominant hypercholesterolemia (ADH) is characterized by elevated low-density lipoprotein cholesterol (LDL-C) levels and
premature cardiovascular disease (CVD). Mutations in the genes encoding
for the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and
proprotein convertase subtilisin/kexin 9 (PCSK9) underlie ADH. Nevertheless,
a proportion of individuals that exhibit the ADH phenotype do not carry
mutations in any of these three genes. Estimates about the percentage of such
cases amongst the ADH phenotype vary widely. We therefore investigated a
large pediatric population with an unequivocal ADH phenotype to assess the
molecular basis of hereditary hypercholesterolemia and define the percentage
of individuals with unexplained dyslipidemia.
Methods and Results: We enrolled individuals with LDL-C levels above the
95th percentile for age and gender and an autosomal dominant inheritance
pattern of hypercholesterolemia from a large referred pediatric cohort of 1430
children. We excluded children with thyroid dysfunction, nephrotic syndrome,
autoimmune disease, liver disease, primary biliary cirrhosis, obesity (BMI
>75th percentile for age and gender), as well as children referred via a cascade
screening program and those from families with a known molecular diagnosis.
Of the 269 children that remained after applying the exclusion criteria, 255
(95%) carried a functional mutation (LDLR 95%; APOB 5%).
Conclusion: In the vast majority of children with an ADH phenotype, a
causative mutation can be identified, strongly suggesting that most of the large
effect genes underlying ADH are known to date.
67 LOW LIPOPROTEIN(A) CONCENTRATION IS ASSOCIATED WITH
SHORTER SURVIVAL: A POPULATION-BASED COHORT STUDY
(THE JMS COHORT STUDY)
M. Sawabe1 , N. Tanaka2 , M.N. Mieno3 , S. Ishikawa4 , K. Kayaba5 ,
K. Nakahara6 , S. Matsushita6 , The JMS Cohort Study Group. 1 Bioresource
Center for Geriatric Research, Tokyo Metropolitan Geriatric Hospital, Itabashi,
Japan, 2 Department of Biostatistics, Harvard School of Public Health, Boston,
MA, USA, 3 Department of Medical Informatics, 4 Center for Community
Medicine, Jichi Medical University, Shimotsuke, 5 Saitama Prefectural
University, Koshigaya, 6 Department of Internal Medicine, Tokyo Metropolitan
Geriatric Hospital, Itabashi, Japan
Background: Experimental studies support the anti-neoplastic effect of apo(a),
but several clinical studies have reported contradictory results. To determine
whether a low lipoprotein(a) [Lp(a)] concentration may promote carcinogenesis,
data from the Jichi Medical School cohort study, a multi-center population-based
cohort study conducted in Japan, was analyzed.
Methods: The subjects were 10,692 study participants (4,122 men and 6,570
women). The average age at registration was 55.2 years, and the median
observation period was 4,559 days. A total of 899 participants died during the
observation period.
Results: The estimated hazard ratio was high for both low and very high
Lp(a) levels. We defined three Lp(a) groups: low Lp(a) group [Lp(a) < 80 mg/L],
intermediate Lp(a) group [80 Lp(a) < 550], and very high Lp(a) group
[Lp(a) 550]. A Kaplan-Meier plot showed higher cumulative death rates for the
low Lp(a) group than for the intermediate Lp(a) group for all-cause, cancer and
miscellaneous-cause deaths (p < 0.001, p = 0.03, and p = 0.03, respectively).
A Cox proportional hazards analysis showed that a low Lp(a) level was a
significant risk for all-cause, cancer and miscellaneous-cause deaths (p < 0.001,
p = 0.006, and p = 0.004, respectively). The hazard ratio (1.43) of a low Lp(a)
level for all-cause deaths almost reached that (1.52) of a male sex.
Conclusions: These results support the anti-neoplastic effect of Lp(a). Thus,
Lp(a)-lowering drugs presently under development might promote cancer or allcause deaths. This novel finding also supports the epidemiological significance
of a low Lp(a) concentration.
68 THE FUNCTION OF APOA5 IN THE METABOLISM OF TRIGLYCERIDERICH LIPOPROTEINS (TRL) PARTIALLY DEPENDS ON LRP1
K. Brügelmann1 , A. Bartelt1 , J. Heeren1 , M. Merkel2 . 1 IBMII: Molecular Cell
Biology, University Medical Center Hamburg-Eppendorf, 2 Department of
Internal Medicine, Asklepios Clinic St. George, Hamburg, Germany
Objective: Apolipoprotein A5 is a strong modulator in the metabolism of TRL.
It was shown that apoA5 activates lipoprotein lipase and thereby accelerates
plasma TG hydrolysis. In vitro studies suggest that apoA5 may also mediate
lipoprotein uptake by members of the LDL receptor (LDLR) family. This study
investigates interactions of apoA5 with lipoprotein receptors in vivo and in
primary hepatocytes in vitro.
Methods: The TG-lowering effect of the human apoA5 transgene (A5tr) was
investigated in mice with LDLR deficiency (LDLR−/− ), in mice lacking hepatic
LRP1 (hLRP1−/− ) and respective littermate wildtype controls (hLRP1+/+ ) as
well as in LRP1-LDLR-knockout mice (LDLR−/− -hLRP1−/− ) and hLRP1 controls
(LDLR−/− -hLRP1+/+ ). The secretion of apoA5 protein in plasma and in cell
supernatants of primary hepatocytes was measured by Elisa.
Results: In LDLR−/− mice apoA5 decreased TRL-TG by 46%. In hLRP1+/+ -A5tr
mice TG levels are 30% decreased compared to hLRP1+/+ -Wt. In contrast, in
hLRP1−/− A5tr mice, the decreasing effect of apoA5 is abolished. In LDLR−/− hLRP1+/+ -A5Tr, TG levels are 25% decreased compared to LDLR−/− -hLRP1−/− A5tr mice. In mice lacking hLRP1 as well as in LDLR−/− -hLRP1−/− the A5tr
plasma concentration was 36% increased in contrast to hLRP1+/+ and 45%
in contrast to LDLR−/− -hLRP1−/− -A5tr mice. To further elucidate a possible
interaction of LRP1 and apoA5 experiments with primary hepatocytes are
in progress. Preliminary data showed an increase of apoA5 protein in cell
supernatants in the absence of LRP1.
Conclusion: These data suggest that LRP1 is involved in the secretion of
apoA5 and/or in the trapping of TRL remnants into the liver.
69 HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA IN NORWAY,
SWEDEN, DENMARK AND FINLAND − SUBSTANTIAL VARIATION
IN FREQUENCY OF THE DIAGNOSIS
A. Græsdal1 , M.P. Bogsrud2 , Y. Stålstrøm3 , K. Retterstøl2 , G. Langslet2 ,
L. Ose2 . 1 Vestfold Indremedisinske Senter, Sandefjord, 2 Lipid Clinic, Oslo
University Hospital, Rikshospitalet, Oslo, 3 Gambro, Tønsberg, Norway
Background and Aims: Homozygous familial hypercholesterolemia (HFH) is
caused by homozygosity or compound heterozygosity in genes encoding the
low density lipoprotein (LDL) receptor. The prevalence is roughly estimated to be
1 per million among European Caucasians. Due to extreme cholesterol values
myocardial infarction can occur in early childhood and most of the patients will
die before the age of 30 if not treated with cholesterol lowering medications
and LDL-apheresis. The aim of this study is to compare the frequency of the
diagnosis and relevant treatment in the Nordic countries.
Methods: In Norway all patients with HFH are registered at the Lipid Clinic in
Oslo. In the other Nordic countries no central clinic exists, but we have contacted
the hospitals to obtain an overview of the number of HFH patients.
Results: In Norway 8 of 4.8 million inhabitans have the diagnosis − one has
underwent liver transplantation, the remaining 7 are treated with LDL-apheresis.
In Finland none of 5.3 million inhabitans is known to be diagnosed. In Sweden
8 of 9.3 million inhabitans have been diagnosed and are treated with apheresis.
In Denmark 2 of 5.5 million inhabitans are diagnosed, one is in apheresis
treatment, and one is planned for apheresis treatment.
Conclusion: The great variation in frequency of patients with HFH between
the Nordic countries in this study, is likely to represent an underdiagnosis of
this severe condition. We propose that all patients wtith HFH are registered
nationally and that the effect of the apheresis treatment is recorded.
79th EAS Congress
70 A VARIANT NEAR THE MELANOCORTIN-4 RECEPTOR (MC4R)
GENE REGULATE POSTPRANDIAL LIPID METABOLISM IN A
HEALTHY CAUCASIAN POPULATION
P. Perez-Martinez, J. Delgado-Lista, A. Garcia-Rios, N. Delgado-Casado,
C. Marin, P. Gomez-Luna, F. Perez-Jimenez, J. Lopez-Miranda. Lipid and
Atherosclerosis Unit, IMIBIC/Reina Sofia University Hospital/University of
Cordoba/CIBEROBN, Córdoba, Spain
The melanocortin-4 receptor (MC4R) is an essential regulator of food intake
and energy homeostasis. Previous data suggests an influence of MC4R activity
on triglyceride levels. Our aim was to determine whether the presence of the
rs12970134 polymorphism near the MC4R gene could influence postprandial
lipoprotein metabolism. 88 healthy volunteers were selected, 53 homozygous
for the common genotype (G/G) and 35 carriers for the minor A-allele (G/A and
A/A). They were given a fat-rich meal containing 1 g fat and 7 mg cholesterol
per kg body weight and vitamin A 60000 IU/m2 body surface. Fat accounted
for 60% of calories, and protein and carbohydrates for 15% and 25% of energy
respectively. Blood samples were taken at time 0, every 1 h until 6 h, and
every 2.5 h until 11 h. Total cholesterol and TAGs in plasma, and cholesterol,
TAGs and retinyl palmitate in triacylglycerol-rich lipoproteins (TRLs) (large and
small) were determined by ultracentrifugation. Individuals carrying the G/G
genotype displayed a higher postprandial response of large-TRL TAGs than did
carriers of the minor A-allele, as demonstrated by a significant genotype-by time
interaction (P = 0.023). G/G individuals also presented higher concentrations
of plasma TAGs and total cholesterol during the postprandial period than did
carriers of the A-allele (P = 0.033 and P = 0.019, respectively). Furthermore G/G
subjects showed greater postprandial response of small TRL-apo B48 than did
carriers of the A-allele (P = 0.032). These results suggest that the rs12970134
polymorphism near the MC4R gene may partly explain the interindividual
differences in postprandial lipoprotein response in healthy subjects.
71 MOLECULAR DIAGNOSIS OF FAMILIAL HYPERCHOLESTEROLEMIA:
THE UTILITY OF A COUNTRY SPECIFIC PROTOCOL
M. D’Agostino1,2 , M. Romano1,2 , M.D. Di Taranto3 , G. Marotta4 , M. Gentile4 ,
A. Iannuzzi5 , P. Rubba4 , G. Fortunato1,2 . 1 Dip. Biochimica e Biotecnologie
Mediche, Università degli Studi di Napoli Feredico II, 2 CEINGE S.C.a
r.l. Biotecnologie Avanzate, 3 Fondazione IRCCS SDN, 4 Dipartimento di
Medicina Clinica e Sperimentale, Università degli Studi di Napoli Federico II,
5
Dipartimento di Medicina Interna, AORN A. Cardarelli, Naples, Italy
Introduction: Familial Hypercholesterolemia (FH) is a disease characterized
by high levels of total and LDL cholesterol. Mutations in the LDL receptor
(LDLR) gene are the main cause of FH. More than 1100 mutations in the LDLR
gene including single-nucleotide mutations, duplications, deletions and splicing
alterations have been reported so far.
Materials and Methods: We enrolled 102 unrelated patients from Southern
Italy with clinically diagnosed FH. The promoter and 18 exons of the LDLR gene
were amplified by PCR and directly sequenced. To confirm splicing mutations,
we performed RT-PCR analysis on mRNA.
For detection of large rearrangements, copy number quantification of the 18
exons of the LDLR gene was performed by the SALSA MLPA kit. Long-PCR
have been carried out to confirm large rearrangements.
Results and Conclusions: Direct sequencing of the promoter and encoding
regions of the LDLR gene revealed mutations in 71/102 patients (mutation
rate 69.9%). MLPA analysis revealed gross deletions in 5/31 subjects (total
mutation rate 74.5%). Among the 34 different mutations found, 6 mutations
(2 splicing alterations and 4 missense mutations) account for more than 50%
of cases. Although the LDLR mutation number is very high, genomic clusters
of mutations have been reported in different country indicating that specific
diagnostic protocols can be useful in the genetic screening allowing time and
cost reduction. Therefore, we propose a specific diagnostic protocol that firstly
searches for the six most frequent mutations and then analyzes the other
genomic regions.
Acknowledgements: CEINGE Convenzione Regione Campania, DGRC
1901/2009 and Fondazione IRCCS SDN.
72 INSULIN RESISTANCE IS ASSOCIATED WITH THE METABOLIC
SYNDROME BUT NOT WITH PERIPHERAL ARTERIAL DISEASE
A. Vonbank1,2,3 , C.H. Saely1,2,3 , P. Rein1,2,3 , C. Boehnel1,3 , S. Greber1 ,
H. Drexel1,2,3,4 . 1 VIVIT Institute, 2 Internal Medicine, Academic Teaching
Hospital Feldkirch, Feldkirch, Austria, 3 Private University of the Principality of
Liechtenstein, Triesen, Liechtenstein, 4 Drexel University College of Medicine,
Philadelphia, PA, USA
Background: Insulin resistance (IR) is the key feature of the metabolic
syndrome (MetS); its association with peripheral arterial disease (PAD) is
unclear. We hypothesised that insulin resistance is associated with both the
MetS and sonographically proven PAD.
Methods: IR was determined using the HOMA index in 197 patients with
sonographically proven PAD as well as in 214 controls who did not have a history
17
of PAD and in whom coronary artery disease was ruled out angiographically;
the MetS was defined according to NCEP-ATPIII criteria.
Results: HOMA-IR scores were higher in MetS patients than in subjects without
the MetS (5.9±6.2 vs. 2.9±3.9; p < 0.001). However, HOMA IR did not differ
significantly between patients with PAD and controls (4.2±5.4 vs. 3.3±4.3;
p = 0.124). When both, the presence of the MetS and of PAD was considered,
HOMA-IR was significantly higher in patients with the MetS both among those
with PAD (6.1±5.7 vs. 3.6±5.2; p < 0.001) and among controls (5.8±6.8 vs.
2.3±1.8; p < 0.001), whereas it did not differ significantly between patients
with PAD and controls among patients with the MetS (5.8±6.8 vs. 6.1±5.7;
p = 0.587) nor among those without the MetS (2.3±1.8 vs. 3.6±5.2; p = 0.165).
Similar results were obtained with the IDF or harmonized consensus definitions
of the MetS.
Conclusion: We conclude that IR is significantly associated with the MetS but
not with sonographically proven PAD.
73 APOLIPOPROTEIN PROFILES IN ATHEROSCLEROTIC DISEASE
M. Formato1 , A.J. Lepedda1 , E. Zinellu1 , A. Cigliano1 , F. Piredda2 ,
P.P. Bacciu2 , A. Guarino3 , R. Spirito3 . 1 Dipartimento di Scienze Fisiologiche,
Biochimiche e Cellulari, 2 Service of Vascular Surgery, University of Sassari,
Sassari, 3 Centro Cardiologico ‘F. Monzino’ IRCCS, University of Milano,
Milano, Italy
Aims: Recently, the apolipoprotein composition of the major lipoprotein classes
as VLDL, LDL, and HDL in physiological conditions has been studied by
means of proteomic approaches. The aim of this work was to characterize the
apolipoprotein pattern in atherosclerotic patients by means of two-dimensional
electrophoresis and mass spectrometry.
Methods: For this purpose we purified plasma VLDL, LDL, and HDL from
patients undergoing carotid endarterectomy and from healthy normolipidemic
subjects by single isopycnic density gradient ultracentrifugation. Samples were
loaded into 4−7 IPG strips and isoelectric focused. Proteins were then reduced
and alkylated before SDS-PAGE. After staining and image analysis, spots of
interest were excised and protein identified by peptide mass fingerprinting after
tryptic digestion.
Results: We identified 21 spots corresponding to about 96% of 52 protein
spots detected in VLDL, 22 spots corresponding to about 92% of 43 spots in
LDL and 20 corresponding to about 96% of 60 spots in HDL. By image analysis
we found many differences in the relative abundance of some identified VLDL
and LDL apolipoproteins between patients and healthy subjects. Conversely,
HDL showed quite similar apolipoprotein profiles.
Conclusions: In summary, we have set up a method to compare 2DE
apolipoproteins profiles of plasma VLDL, LDL and HDL fractions. This
method seems to be suitable to detect potential changes associated to the
atherosclerotic process, and it could provide insight into the development of
novel diagnostic tools and/or future therapeutic agents.
This work was supported by “Fondazione Banco di Sardegna” (Sassari, Italy)
grant.
74 SERUM INTESTINAL APOB-48 IS INCREASED IN PATIENTS WITH
SEVERE HYPERTRIGLYCERIDEMIA
A. Abed1 , P. Giral2 , R. Bittar3 , D. Bonnefont-Rousselot3 , J.-M. Lacorte4 .
1
INSERM U872, 2 Endocrinologie − Métabolisme, 3 Biochimie Métabolique,
APHP Pitié Salpêtrière, 4 Centre de Recherche des Cordeliers, INSERM U872,
Paris, France
Background: Since several years, relationships were established between high
serum levels of postprandial Triglyceride-Rich-Lipoprotein (TRL) and carotid
intima-media thickness, cardiovascular disease and death. TRL contain apoB100 when they are secreted by the liver and apoB-48 when they are produced
by intestine. Our aim was to quantify serum apoB-48, a marker of intestinal
lipoproteins, in patients with severe hypertriglyceridemia.
Methods: Serum apoB-48 was quantified with specific human apoB-48 ELISA
kit in 41 hypertriglyceridemic patients (2.46 g/L to 20.51 g/L) well phenotyped.
Six patients with severe hypertriglyceridemia (fasting triglycerides >10 g/L)
participated to a very short diet intervention consisting of one or two days
of a high-fat (65%), low-carbohydrate, isocaloric diet. Fasting triglycerides and
apoB-48 were quantified every day.
Results: We established a positive correlation between serum fasting
triglycerides and apoB-48 amount in patients with hypertriglyceridemia. After
a very short high-fat diet intervention, two patients, among six with severe
hypertiglyceridemia, were considered as “fat-diet responders” with a two-fold
increase in fasting triglycerides level after one day of high-fat diet. At the same
time, concentration of serum apoB-48 reached a two-fold increase without any
change in apoB-100 level. Four patients were “non-fat-diet responders” with
no change of fasting triglycerides and apoB-48. Post-heparin lipoprotein lipase
activity was normal in all patients
Conclusion: In severe hypertriglyceridemic patients, considerated as “fat-diet
responders”, we observed a relation between increase of fasting triglycerides
18
and serum apoB-48 level suggesting that, in these patients, intestinal TRL are
involved in fasting hypertriglyceridemia.
75 EFFICACY OF EZETIMIBE/STATINS AND STATIN MONOTHERAPY
AND FACTORS ASSOCIATED WITH TREATMENT RESPONSE:
POOLED ANALYSIS OF >21,000 SUBJECTS FROM 27 TRIALS
P.P. Toth1 , D. Morrone2 , W. Weintraub2 , M.E. Hanson3 , R.S. Lowe3 , J. Lin3 ,
A. Shah3 , A.M. Tershakovec3 . 1 Preventive Cardiology, Sterling Rock Falls
Clinic, Sterling, IL, 2 Christiana Care Center for Heart & Vascular Health,
Newark, DE, 3 Merck, Whitehouse Station, NJ, USA
Objective: Evaluate lipid-altering efficacy of ezetimibe co-administered
with statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, pravastatin,
rosuvastatin, simvastatin) and statin monotherapy. Identify factors related to
treatment response.
Methods: Patient level data were combined from 27 double-blind, placebocontrolled or active comparator studies that randomized adult hypercholesterolemic patients to statin (N = 10,517) or ezetimibe+statin (N = 11,714) for
6−24 weeks. Analyses evaluated percent changes from baseline LDL-C
(primary efficacy parameter) and other lipids; attainment of specified lipid
targets; associations between response to treatment (percent change from
baseline lipids, CRP) and factors potentially influencing treatment response
(treatment, age, sex, race, BMI, diabetes, baseline lipids, CHD status, statin
potency, first/second-line therapy [statin-naı̈ve or statin-washout/ongoing statin
at study entry], trial within first/second-line therapy).
Table 1
Parameter
Statin
baseline
mean#
Treatment difference§
Ezetimibe + Statin
% change†
% change†
baseline
mean#
%
p†
Total C (mg/dL)
234.2
−17.0"
231.5
−27.1"
−10.1
<0.0001
LDL-C (mg/dL)
151.2
−23.6*
148.7
−38.7*
−15.1
<0.0001
Non-HDL-C (mg/dL)
184.7
−21.4"
182.0
−35.0"
−13.5
<0.0001
ApoB (mg/dL)
144.2
−18.1"
142.7
−28.9"
−10.8
<0.0001
Triglycerides (mg/dL)
153.0
−15.4*
152.0
−19.9"
−4.7
<0.0001
HDL-C (mg/dL)
49.6
2.1*
49.5
3.7*
1.6
<0.0001
Total-C/HDL-C
5.0
4.9
−28.6"
−10.9
<0.0001
CRP (mg/L)
2.2
−17.8"
−8.2+
2.1
−16.7"
−8.6
<0.0001
LDL-C, statin potency, age, diabetes, and others (table). Factors related to other
efficacy parameters were identified.
Conclusions: This study demonstrated that adding ezetimibe to statins results
in additional improvement in key lipid parameters and attainment of LDL-C, nonHDL-C and ApoB treatment targets; factors associated with treatment efficacy
were identified.
76 METABOLIC BENEFITS OF ECCENTRIC ENDURANCE EXERCISE
IN OVERWEIGHT AND OBESE INDIVIDUALS
P. Rein1,2,3 , C.H. Saely1,2,3 , A. Vonbank1,2,3 , S. Aczel1,2 , C. Boehnel1,3 ,
V. Kiene1 , V. Drexel1,3 , T. Bochdansky1 , H. Drexel1,2,3,4 . 1 VIVIT Institute,
2
Internal Medicine, Academic Teaching Hospital Feldkirch, Feldkirch, Austria,
3
Private University of the Principality of Liechtenstein, Triesen, Liechtenstein,
4
Drexel University College of Medicine, Philadelphia, PA, USA
Background: The interplay of muscle contraction with an external force can
result in one of three types of muscle activity: shortening or “concentric” when
muscle contraction is stronger than the external force; lengthening or “eccentric”
when the external force is stronger; and isometric when both forces are equal.
Eccentric endurance exercise (e.g. hiking downwards) is less strenuous than
concentric exercise (e.g. hiking upwards) but its metabolic effects are largely
unknown; no data exist in overweight and obese individuals.
Methods: We allocated 43 overweight and obese sedentary individuals to an
exercise intervention program, consisting of hiking downwards a pre-defined
route in the Austrian Alps over two months. For the opposite way, a cable
car was used where compliance was recorded electronically. The difference in
altitude was 540 metres; the distance was covered three to five times a week.
Fasting and postprandial metabolic profiles were obtained at baseline and after
the two months period.
Results: Compared with baseline, eccentric exercise significantly lowered
fasting glucose (98±11 vs. 96±14 mg/dl; p = 0.042) and improved glucose tolerance (242±48 vs. 219±60 mg*dl-1 *h; p = 0.001). Furthermore,
eccentric exercise significantly improved triglyceride tolerance (1841±893
vs. 1468±656 mg*dl-1 *h; p = 0.001) and the postprandial leukocyte count
(69.4±11.7 vs. 67.0±13.0 G*L-1 *h; p = 0.044).
Conclusion: We conclude that eccentric exercise is a promising new exercise
modality with favourable metabolic effects. This little strenuous exercise could
become especially important, as a large proportion of patients suffer from
comorbidities that confer a low tolerance for higher-intensity training protocols.
Apo = apolipoprotein; CRP = C-reactive protein; HDL-C = high density lipoprotein cholesterol; LDL-C = low
density lipoprotein cholesterol; Total C = total cholesterol.
† Percent change from baseline (least squares mean [median for triglycerides and (back-transformed value
of the log postbaseline/baseline) − 1) × 100% for CRP]) based on ANCOVA model with terms for treatment,
first/second line, race, gender, CHD, statin potency, age, BMI, baseline LDL-C, baseline HDL-C, baseline TRIG,
diabetes, study within first/second line, and baseline of parameter being evaluated (if not already noted).
§ differences in least squares mean values (Hodges-Lehmann estimate of medians for triglycerides and
geometric mean percent changes for CRP) between ezetimibe–statin and statin groups.
‡ adjusted odds ratio – based on logistic model with terms for first/second line, treatment, and baseline value.
# median for triglycerides and geometric mean for CRP.
*p 0.0001, **p 0.01.
Table 2
odds ratio‡
95% CI‡
33.3
3.96
3.67, 4.28
75.3
4.22
3.94, 4.52
20.7
40.6
3.79
3.53, 4.07
<130 mg/dL
52.9
75.3
4.12
3.85, 4.42
<80 mg/dL
15.8
28.7
3.09
2.85, 3.35
<90 mg/dL
29.3
45.4
2.87
2.68, 3.08
Target level
LDL
Non-HDL-C
ApoB
% attainment
Statin
Ezetimibe + Statin
<70 mg/dL
15.1
<100 mg/dL
51.9
<100 mg/dL
For notes, see Table 1.
Table 3
Factors associated with LDL-C reduction (F-value)
Treatment*
1st /2nd
line*
baseline
LDL-C*
statin
potency*
age*
diabetes*
study w/in
1st /2nd line*
baseline
TG*
race*
gender**
(4415.8)
(4296.3)
(429.9)
(380.4)
(231.9)
(79.2)
(36.4)
(19.0)
(15.1)
(8.4)
For notes, see Table 1.
Results: Ezetimibe+statins produced significantly greater percent reductions
in total-C, LDL-C, non-HDL-C, ApoB, triglycerides, CRP, and lipid ratios, and
percent increases in HDL-C and ApoA1 than statins at statin potencies
compared (table). Significantly greater attainment of LDL-C (<70 mg/dL,
<100 mg/dL), non-HDL-C (<100 mg/dL, <130 mg/dL), and ApoB (<80 mg/dL,
<90 mg/dL) was observed for combined treatment versus statin monotherapy.
CRP < 1 mg/L and <3 mg/L was also assessed. Factors significantly associated
with LDL-C reductions included treatment, first/second-line therapy, baseline
77 EFFECTS OF PA ON THE RISK OF CORONARY ARTERY DISEASE
IN HYPERCHOLESTEROLEMIC PATIENTS IN RELATED WITH
NONATTAINMENT THE LIPID MANAGEMENT GOALS FOR LDL-C
OR NON HDL-C: SUB-ANALYSIS OF THE JELIS
J. Sasaki1 , M. Yokoyama2 , M. Matsuzaki3 , Y. Saito4 , H. Origasa5 , Y. Ishikawa6 ,
S. Oikawa7 , H. Itakura8 , H. Hishida9 , T. Kita10 , A. Kitabatake11 , N. Nakaya12 ,
T. Sakata13 , K. Shimada14 , K. Shirato15 , Y. Matsuzawa16 . 1 International
University of Health and Welfare Graduate School of Public Health Medicine,
Fukuoka, 2 Hyogo Prefectural Awaji Hospital, Sumoto, 3 Department of Medicine
and Clinical Science, Division of Cardiovascular Medicine, Yamaguchi
University Graduate School of Medicine, Yamaguchi, 4 Chiba University,
Chiba, 5 Division of Biostatistics and Clinical Epidemiology, University of
Toyama, Toyama, 6 Kakogawa Municipal Hospital, Kakogawa, 7 Division of
Endocrinology and Metabolism, Department of Medicine, Nippon Medical
School, 8 Shinagawa Eeast One Medical Clinic, Tokyo, 9 Division of Cardiology,
Department of Internal Medicine, Fujita Health University School of Medicine,
Aichi, 10 Kobe City Medical Center General Hospital, Hyogo, 11 Hiraoka
Hospital, Osaka, 12 Nakaya Clinic, Tokyo, 13 Hisatune Hospital, Fukuoka,
14
Division of Cardiovascular Medicine, Department of Medicine, Jichi Medical
University, Tochigi, 15 Saito Hospital, Miyagi, 16 Sumitomo Hospital, Osaka,
Japan
Aim: To investigate the effects of EPA on coronary artery disease (CAD)
in patients with nonattainment the lipid management goals for low-density
lipoprotein cholesterol (LDL-C) or non high-density lipoprotein cholesterol (non
HDL-C) under treatment with statins.
Methods: The patients were distributed into 4 subgroups using the
management goal for LDL-C recommended by the Japan Atherosclerosis
Society guideline (2007) and the goal for non HDL-C defined 30 mg/dL higher
than LDL-C: A) attained both goals; B) attained the LDL-C but not the non
HDL-C goal; C) attained the non HDL-C but not the LDL-C goal; and D) didn’t
attain any of the two goals. The incidences of CAD in the 4 subgroups were
compared, and the effects of EPA on the risk of CAD in these subgroups were
examined.
Results: In the non EPA group, the incidence of CAD in patients who didn’t
achieve the goals for LDL-C or non HDL-C was significantly higher compared
with that in patients achieved those goals. Patients in subgroups B, C, and D
were at a significantly higher risk for CAD than those in subgroup A (B: HR 1.98,
P = 0.0329, C: HR 2.10, P = 0.0181, D: HR 3.36, P = 0.0000). EPA reduced the
risk of CAD by 24% in subgroups B, C, and D (P = 0.0071).
79th EAS Congress
Conclusion: We reconfirmed non HDL-C as a residual risk marker of CAD
after LDL-C lowering therapy.
EPA was useful to reduce the occurrence of CAD in the patients who didn’t
attain the goals for LDL-C and/or for non HDL-C.
78 INFLUENCE OF APOE GENOTYPE IN THE PHENOTYPE OF
CLINICAL DIAGNOSED PORTUGUESE FH PATIENTS
A.M. Medeiros1,2 , T. Santos1,2 , A.C. Alves1,2 , M. Bourbon1,2 , on behalf of the
Investigators of the Portuguese FH Study. 1 Instituto Nacional de Saúde Dr.
Ricardo Jorge, 2 BioFIG − Center for Biodiversity, Functional & Integrative
Genomics, Lisbon, Portugal
APOE gene is polymorphic, comprises three frequent alleles (e2, e3, e4) which
create six different genotypes and six protein isoforms with different affinity to
LDLR. Isoform E2 has less affinity to receptor while isoform E4 have much
efficient bond and compete with apoB.
The present study pretends to evaluate the distribution of APOE alleles/genotype of index patients with clinical diagnosis of Familial Hypercholesterolemia (FH) and investigate if a specific allele/genotype is cause
of hypercholesterolemia. APOE genotyping was performed using SnaPShot
Multiplex System after PCR amplification. Biochemical parameters were
determined by routine methods and results were analyzed with SPSS software
using t-test.
For this analysis, children and adults were divided according to their
genetic diagnosis. Total of 353 patients were analyzed: 140 patients with
mutation in LDLR, APOB or PCSK9 (44 children (G1), 96 adults (G2))
and 213 without a detectable mutation (70 children (G3), 143 adults
(G4). Distribution of 6 genotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4,
E4/E4) was as following: G1, 0%, 2.3%, 4.5%, 68.2%, 20.5%, 4.5%; G2,
1.0%, 1.0%, 4.2%, 62.5%, 28.1%, 3.1%; G3, 0%, 1.4%, 5.7%, 55.7%,
35.7%, 1.4%; G4, 0%, 1.4%, 5.6%, 63.5%, 25.9%, 3.5%. Statistical
analysis revealed that presence of at least one e4 is associated with high
levels of LDLc in G3/G4 patients (LDLcG3=182.96±49.46 mg/dl, (e4/eX,
X = 2,3,4) vs LDLcG3=152.65±60.60 mg/dl (eY/eZ, Y,Z = 2,3), p = 0.025;
LDLcG4=227.69±43.17 mg/dl, (e4/eX, X = 2,3,4) vs LDLcG4=179.70±64.78
mg/dl (eY/eZ, Y,Z = 2,3), p < 0.001)) but not in G1/G2 patients (p = 0.100,
p = 0.832). Presence of e4 can be the cause of hypercholesterolemia presented
by patients without genetic diagnosis of FH.
79 COMPARISON OF THE EFFECTS OF SIMVASTATIN VERSUS
ROSUVASTATIN VERSUS SIMVASTATIN/EZETIMIBE ON
PARAMETERS OF CARBOHYDRATE METABOLISM
E. Moutzouri, E. Liberopoulos, M. Kostapanos, H. Milionis, M. Elisaf. University
of Ioannina School of Medicine, Ioannina, Greece
Background: Statin treatment may be associated with adverse effects on
glucose metabolism. Whether this is a class effect is not known. In contrast,
ezetimibe monotherapy may beneficially affect insulin sensitivity. The aim of
this study was to compare the effects of 3 different regimens of equivalent lowdensity lipoprotein (LDL) cholesterol lowering capacity on glucose metabolism.
Methods: A total of 153 hypercholesterolemic patients were randomly
allocated to receive open-label simvastatin 40 mg, rosuvastatin 10 mg or
simvastatin/ezetimibe 10/10 mg for 12 weeks. The primary end point was
changes in homeostasis model assessment of insulin resistance (HOMA-IR).
Results: A marginally significant (p = 0.04) increase in HOMA-IR was observed
in all 3 groups. No significant difference in HOMA-IR change was observed
between groups. Similarly, fasting insulin levels were significantly increased in
all 3 groups (p = 0.04). No significant increase in fasting plasma glucose and
glycated hemoglobin levels from baseline was seen in any group. Changes in
serum lipids were similar across groups.
Conclusion: All 3 hypolipidemic regimens of equivalent LDL cholesterol
lowering capacity resulted in similar increases in HOMA-IR and fasting insulin.
80 CHANGES IN LDL-C LEVELS AND GOAL ATTAINMENT ASSOCIATED
WITH ADDING EZETIMIBE ONTO SIMVASTATIN, ATORVASTATIN
AND ROSUVASTATIN COMPARED WITH TITRATING STATINS
J.M. Foody1 , P.P. Toth2 , S. Sajjan3 , D.R. Ramey4 , D.R. Neff4 ,
A.M. Tershakovec4 , H. Hu3 , J.E. Tomassini4 , K. Tunceli5 . 1 Division of
Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, MA,
2
Sterling Rock Falls Clinic, Sterling, IL, 3 Merck, W Point, 4 Merck, North Wales,
PA, 5 Merck, Whitehouse Station, NJ, USA
Objective: To assess the effect of adding ezetimibe to simvastatin, atorvastatin
and rosuvastatin therapies vs titrating these statins on LDL-C changes and goal
attainment in CHD/CHD risk-equivalent patients in a large, national managed
care database.
Methods: CHD/CHD risk-equivalent patients initially on statin monotherapy
were identified from 11/01/02−11/30/09. % change from baseline in LDL-C and
the proportion of patients attaining LDL-C <1.8 and <2.6 mmol/l were evaluated
in 17,830 eligible patients [18 yrs with baseline and follow-up LDL-C values, no
concomitant use of other lipid-lowering therapy (LLT) and on LLT for 42 days].
19
Results: LDL-C lowering and goal attainment improved substantially in patients
treated with ezetimibe added onto simvastatin, atorvastatin or rosuvastatin
therapies (n = 2,312) vs those (n = 13,053) who titrated these statins. In
multivariable models, % change from baseline in LDL-C was −13.1 to −14.8%
greater for those who added ezetimibe onto simvastatin, atorvastatin or
rosuvastatin vs those who titrated these statins. The likelihood of attaining
LDL-C <1.8 and <2.6 mmol/l increased by 2.6 to 3.2-fold and 2.5 to 3.1-fold
respectively in patients who added ezetimibe onto simvastatin, atorvastatin and
rosuvastatin vs titrating these statins.
Conclusion: In a large managed care database, patients who added ezetimibe
onto simvastatin, atorvastatin or rosuvastatin had greater LDL-C reductions
and goal attainment than those who titrated these statin therapies. Our study
suggests that high-risk CHD patients in need of more intensive LDL-C lowering
therapy may benefit by adding ezetimibe onto statin therapy vs titrating to higher
statin doses.
Simvastatin
Atorvastatin
Rosuvastatin
Add-on†
Titrate‡
Add-on†
Titrate‡
Add-on†
Titrate‡
(N = 540)
(N = 4170)
(N = 1504)
(N = 7653)
(N = 268)
(N = 1230)
BL Mean mmol/l
(95% CI)
3.1
(3.0, 3.1)
2.8
(2.8, 2.8)
2.9
(2.9, 3.0)
2.7
(2.7, 2.7)
2.9
(2.8, 3.0)
2.6
(2.6, 2.7)
% change FU
(95% CI)
−26.0
−9.8
−27.0
−9.7
−27.0
−8.7
(−28.3, −23.8) (−10.5, −9.0) (−28.1, −25.9) (−10.3, −9.2) (−29.7, −24.3) (−10.3, −7.2)
% attainment
(95% CI)
<2.6 mmol/l EL 21.7
(18.2, 25.1)
38.3
(36.8, 39.8)
32.7
(30.3, 35.0)
45.0
(43.9, 46.1)
34.0
(28.3, 39.6)
50.8
(48.0, 53.6)
<2.6 mmol/l FU 77.2
(73.7, 80.8)
62.3
(60.9, 63.8)
83.7
(81.8, 85.6)
69.8
(68.8, 70.9)
81.7
(77.1, 86.3)
70.7
(68.1, 73.2)
<1.8 mmol/l EL 4.6
(2.9, 6.4)
6.8
(6.0, 7.5)
4.0
(3.0, 5.0)
9.7
(9.0, 10.4)
3.4
(1.2, 5.5)
14.8
(12.8, 16.8)
<1.8 mmol/l FU 29.8
(26.0, 33.7)
13.5
(12.3, 20.3)
35.4
(33.0, 37.8)
18.1
(17.2, 18.9)
36.2
(30.4, 42.0)
25.7
(23.3, 28.1)
Difference %
change (95% CI)
−13.1 (−15.4, −10.8)
−14.8 (−16.3, −13.4)
−14.4 (−18.0, −10.7)
Odds (95% CI)
attainment
<2.6 mmol/l FU
2.8 (2.1, 3.6)
3.2 (2.7, 3.9)
2.6 (1.8, 3.8)
Odds (95% CI)
attainment
<1.8 mmol/l FU
2.9 (2.2, 3.8)
3.1 (2.7, 3.7)
2.5 (1.8, 3.4)
† Add-on = patients who had a Rx for simvastatin, atorvastatin or rosuvastatin and added-on ezetimibe 10 mg
to statin therapy; ‡ Titrate = patients who had a Rx for simvastatin, atorvastatin or rosuvastatin and titrated to
a higher dose;
BL = Baseline, defined as period of up to 6 mos before add-on or titration date, but not extending back before
statin monotherapy Rx, BL LDL-C is the last LDL-C measurement before above date after at least 42 days on
therapy, FU = Follow-up, defined as up to 12 mos after the add-on or titration date; FU LDL-C is the last LDL-C
measurement during the 12 mos period after at least 42 days on therapy after the relevant date; % change from
baseline was assessed by ANCOVA and % patient attainment by logistic regression. Analyses were adjusted
for age, gender, BL LDL-C and statin potency; logistic regression was also adjusted for BL goal attainment.
81 TEMPORAL RESPONSES OF CHOLESTYRAMINE TREATMENT
ON FGF19 LEVELS, BILE ACID AND CHOLESTEROL SYNTHESIS,
SERUM TRIGLYCERIDES AND BILE ACID LEVELS
B. Sjöberg1,2 , S. Straniero1,2 , B. Angelin1,2 , M. Rudling1,2 . 1 Department of
Medicine, 2 Department of Biosciences and Nutrition, Karolinska Institutet,
Stockholm, Sweden
Objective: Bile acid (BA) synthesis has a diurnal rhythm partly regulated
from the intestine via fibroblast growth factor (FGF)19. We here unraveled
the temporal changes in serum triglycerides, cholesterol and BAs following
acute and chronic interruption of enterohepatic circulation of BAs with
cholestyramine.
Methods: Healthy volunteers received a single day or chronic treatment with
cholestyramine in two consecutive experiments. Lipids, BAs and markers for
BA and cholesterol synthesis were monitored in serum. All reported responses
were significant.
Results: BA synthesis was induced within 6 hrs after the first dose in parallel
with diminished levels of FGF19. The diurnal rhythm of BA synthesis remained
by day 2, although at a higher level, while the diurnal rhythm of cholesterol
synthesis was broken and remained so two days after cessation of treatment. A
single day of treatment doubled serum triglycerides and increased BA synthesis
4-fold by the morning of day 2. Concomitantly, total serum BAs were reduced.
In contrast, chronic treatment increased BA synthesis 10-fold and diminished
FGF19 while triglycerides and BAs were unchanged.
Conclusion: Our results indicate that hypertriglyceridemia induced by
cholestyramine treatment is linked to reduced systemic BA levels present
at initiation of treatment. Chronic cholestyramine treatment strongly induces
BA synthesis that compensates the initially reduced levels of BAs and
completely eliminates the initial hypertriglyceridemia. The powerful induction
20
of BA synthesis during chronic treatment may involve the cholestyramineinduced elimination of secondary BAs, strong inhibitors of BA synthesis, such
as deoxycholic acid.
82 COMBINATION EZETIMIBE/SIMVASTATIN + EXTENDED-RELEASE
NIACIN THERAPY IMPROVES ATTAINMENT OF RECOMMENDED
LDL-C, NON-HDL-C AND APOB LEVELS IN HYPERLIPIDEMIC
PATIENTS
S. Fazio1 , J.R. Guyton2 , J. Lin3 , J.E. Tomassini4 , A. Shah3 , A.M. Tershakovec4 .
1
Vanderbilt University Medical Center, Nashville, TN, 2 Department of Medicine,
Duke University Medical Center, Durham, NC, 3 Merck, Rahway, NJ, 4 Merck,
North Wales, PA, USA
Objective: Concomitant targeting of LDL-C, non-HDL-C and apoB levels is
suggested for subjects with high cardiovascular risk.‡§
Methods: Analysis of a randomized, double-blind study. The effect of
ezetimibe/simvastatin (E/S) + extended-release niacin (N) vs E/S and/or N on
the attainment of specified LDL-C, non-HDL-C and apoB levels in type IIA/IIB
hyperlipidemic (HL) subjects, and subgroups with high CHD risk (+/−AVD),
diabetes (DM), metabolic syndrome (MetS/nonDM) and nonDM/nonMetS was
assessed. Patients received E/S 10/20 mg+N to 2 g or E/S 10/20 mg for
64 weeks, or N to 2 g for 24 weeks then E/S 10/20 mg+N 2 g or E/S 10/20 mg
for 40 weeks more.
Results: A significantly greater number of patients on E/S+N attained
concomitant levels of LDL-C, non-HDL-C and apoB (cluster 1: <100, <130
and <90 mg/dl for high CHD risk; cluster 2: <70, <100 and <80 mg/dl for very
high CHD risk; see table) compared with N and E/S at 24 weeks and E/S
at 64 weeks. Attainment rates at 24 and 64 weeks in all subgroups were
significantly greater with E/S+N vs N, and numerically to statistically greater
with E/S+N vs E/S. Greater attainment of all three specified lipid levels was
most consistent with single level attainment of non-HDL-C.
Conclusion: Our results indicate that combination E/S+N is an effective
therapeutic option for improving attainment of lipid goals in HL patients.
Attainment of non-HDL-C levels best supported concomitant attainment of all
three lipid endpoints. The outcome benefits of E/S+N await results of ongoing
clinical trials.
% of patients attaining treatment levels
24 weeks
E/S+N †
64 weeks
E/S †
N†
E/S+N †
E/S †
Cluster 1: LDL-C <100 mg/dl; non HDL-C <130 mg/dl; ApoB <90 mg/dl‡
Full cohort
77.3
54.6*
6.7*
77.3
57.1*
High risk w/o AVD
76.8
47.1*
2.0*
78.3
69.0
High risk w/ AVD
64.4
71.9
5.6*
66.7
59.3
DM
74.1
60.0
2.1*
79.6
75.0
MetS/nonDM
72.6
47.3*
7.1*
73.0
50.0*
NonDM/nonMetS
82.5
61.4*
8.4*
80.4
59.2*
Cluster 2: LDL-C <70 mg/dl; nonHDL-C <100 mg/dl; ApoB <80 mg/dl§
Full cohort
62.1
31.2*
1.8*
58.3
28.6*
High risk w/o AVD
62.5
38.2*
2.0*
60.9
41.4
High risk w/ AVD
46.7
40.6
2.7*
54.5
37.0
DM
63.8
40.0*
2.1*
61.2
41.7
MetS/nonDM
53.4
27.5*
3.6*
54.9
30.5*
NonDM/nonMetS
68.9
32.5*
1.2*
60.1
22.4*
†
Number of patients assessed for E/S+S, E/S and N at 24 wks and E/S+N and
E/S at 64 wks respectively in: Full cohort (383, 205, 163, 321, 182); High risk
w/o AVD (56, 34, 25, 46, 29); High risk w/ AVD (45, 32, 18, 33, 27); DM (58, 30,
23, 49, 24); MetS/nonDM (146, 91, 56, 122, 82); NonDM/nonMetS (177, 83,
83,148, 76); ‡ suggested for high-risk patients and § highest-risk patients with
cardiometabolic risk (Brunzell JD et al, JACC 2008, 51:1512−24); *95% CI for
OR ratio for treatment comparison (E/S+N vs E/S or E/S+N vs N) excludes 1.
Some variability in the results may be due to the small sample sizes in the
subgroups. % attaining single treatment levels for LDL-C <70 mg/dl, non-HDL-C
<100 mg/dl and ApoB <80 mg/dl in the full cohort at 24 wks were E/S+N (72.9,
78.8, 64.0%); E/S (49.5, 55.7, 32.2%); N (1.8, 5.4, 2.5%) and at 64 wks were
E/S+N (65.3, 75.3, 64.8%); E/S (38.2, 51.7, 35.7%). % attainment for LDL-C
<100 mg/dl, non-HDL-C <130 mg/dl and ApoB <90 mg/dl in the full cohort at
24 wks were E/S+N (90.5, 90.0, 77.3%); E/S (92.5, 90.1, 54.6%); N (18.7,
28.9, 7.4%) and at 64 wks were E/S+N (86.2, 86.2, 77.6%); E/S (86.0, 85.0,
57.7%).
83 SEASONAL VARIATIONS IN TRANS-SIALIDASE ACTIVITY IN HUMAN
BLOOD
A.N. Orekhov1,2 , A.S. Feoktistov1,3 , E.A. Orekhova1 , V.A. Smutova1,4 ,
I.A. Sobenin1,2,5 , A.V. Pshezhetsky4 . 1 Institute for Atherosclerosis Research,
Russian Academy of Natural Sciences, 2 Institue of General Pathology and
Pathophysiology, Russian Academy of Medical Sciences, 3 School of Biology,
M.V. Lomonosov Moscow State University, Moscow, Russia, 4 Departments of
Pediatrics and Biochemistry, University of Montreal, Montreal, QC, Canada,
5
Institute of Clinical Cardiology, Russian Cardilogy Research Centre, Moscow,
Russia
We have discovered multiple-modified low density lipoprotein (mLDL) in the
blood of atherosclerotic patients. mLDL is able to induce accumulation of
lipids in human monocytes and in the smooth muscle cells cultured from
unaffected intima of human aorta. Along with small size, high density and
increased electronegative charge, mLDL is characterized by decreased level
of sialic acid. Negative correlation between the level of sialic acid in LDL and
its ability to induce accumulation of intracellular cholesterol has been found.
Thus, desialylation is an atherogenic modification of LDL. In the blood of
atherosclerotic patients we have discovered a soluble enzyme belonging by
its donor-acceptor properties to trans-sialidases. Lipoprotein-deficient serum
obtained by ultracentrifugation was used for isolation of trans-sialidase by the
affinity chromatography with a-2,8-Neu5Ac-sepharose. After washing, sorbentbound protein fraction was eluted with 50 mM sialic acid. We have revealed
seasonal differences in trans-sialidases activity in blood serum of 61% observed
patients. It is known that coronary heart disease risk increases in winter. More
than 43% of patients had higher level of trans-sialidase activity in winter as
compared with summer period. The opposite situation occurred in 17% of
patients. Thereby we could see that the most part of observed people have
higher blood serum atherogenicity in winter period. This correlates well with
seasonal dynamics of heart disease risk. Thus, trans-sialidases activity and
concentration of mLDL in human blood serum can serve as the additional factors
for estimation of cardiovascular disease risk.
Supported by Russian Ministry of Education and Science.
84 CHOLESTEROL METABOLISM IN PATIENTS WITH DIABETES
MELLITUS TYPE 1 AND IMPACT OF WEIGHT REDUCTION
J. Lesná, V. Bláha, A. Tichá, R. Hyšpler, F. Musil, L. Sobotka, A. Šmahelová.
Charles University in Prague, University Hospital in Hradec Králové, Hradec
Králové, Czech Republic
Background and Aims: A redundant fat mass represents a risk factor for
malfunction of cholesterol metabolism. The aim of this study was to elucidate
the relationship between the reduction of fat tissue and attenuation of insulin
resistance with consequent changes in cholesterol metabolism.
Materials and Methods: Plasma levels of cholesterol, non-cholesterol sterols
and squalene were estimated in obese 1. type diabetics (n = 14, BMI > 30kg/m2 ,
age 29−62y); measurements were repeated after one week of fasting, a month
of a diet containing 150 g of saccharides per day and a year of a diet with 225 g
saccharides per day. Simultaneously, a group of non-obese 1. type diabetics
(n = 14, BMI < 24, 21−57 y) was investigated. Total cholesterol, HDL, LDL and
TAG were estimated enzymatically. Gas chromatography with flame ionisation
detector was used to estimate squalene and non-cholesterol sterols.
Results: Durig the one-month of weight-reduction program obese diabetics
lost on average 7 kg of body weight (including 3 kg of fat tissue).
Statistically significant differences (p < 0.05) were found for total cholesterol
(before:4.96±0.92 mmol/l;month after:4.47±1.05 mmol/l) and campesterol
(before:12.69±5.14 mmol/l; month after:10.08±4.2 umol/l). In obese diabetics
the positive correlation between therapeutic insulin dose and total cholesterol
level was found (p = 0.007).
After 12 months of standard diabetic diet the significant rise of markers
of cholesterol absorption and fall of markers of endogenous synthesis was
revealed.
Conclusion: During short term fasting endogenous cholesterol synthesis
was decreased, followed by increase back up to the original values during
the subsequent month. Results suggest decreased endogenous synthesis
and increased absorption of cholesterol in diabetics, who underwent weight
reduction.
The study was funded by investigation programme of Ministry of Education,
Youth and Sport 0021620820.
85 EZITIMIBE ALONE AND IN COMBINATION LOWERS
CONCENTRATIONS OF SMALL, DENSE LOW DENSITY
LIPOPROTEINS IN TYPE 2 DIABETES MELLITUS
K. Winkler1 , S. Jacob2 , T. Schewe1 , G. Putz1 , M.M. Hoffmann1 , T. Konrad3 .
1
Clinical Chemistry, University Freiburg Medical Center, Freiburg im Breisgau,
2
Cardio-Metabolic Institute, Villingen-Schwenningen, 3 Institute for Metabolic
Research of the Goethe-University Frankfurt, Frankfurt/Main, Germany
Aims: The effectiveness of the cholesterol absorption inhibitor ezetimibe on
LDL subfraction and finally on atherosclerotic risk profile is controversially
79th EAS Congress
discussed mostly due to inappropriate study population, laboratory methods
and data interpretation. Therefore, concentrations of atherogenic small, dense
LDL (sdLDL) were determined by ultracentrifugation in patients with type 2
diabetes mellitus (T2DM) with an elevated cardiovascular risk profile
Methods and Results: Multicenter, randomized, double-blind study investigating the 6-week effect of ezitimibe 10 mg (E), simvastatin 20 mg (S) and
combination of ezitimibe/simvastatin 10/20 mg (S/E) on concentrations of sdLDL
separated from fresh plasma by endpoint gradient ultracentrifugation in patients
with T2DM (Eudra-CT 2006–005906−30). Fifty-six patients were screened for
sdLDL, 41 were randomized, and 40 patients (12 E; 14 S and 14 SE) completed
the study. Total and LDL cholesterol were reduced by 14% (p = 0.004) and
15% (p = 0.006) with E, 22% (p < 0.001) and 32% (p < 0.001) with S, and
32% (p < 0.001) and 44% (p < 0.001) with S/E, respectively. However, E almost
exclusively reduced concentrations of sdLDL by 20% (p = 0.043) whereas S and
S/E reduced sdLDL by 24% (p = 0.020) and 33% (p = 0.003), and non-sdLDL
by 28% (p = 0.004) and 42% (p < 0.001), respectively.
Conclusions: Ezetimibe alone and in combination reduced concentrations of
atherogenic sdLDL. Thus, a potentially proatherogenic effect of ezetimibe by
increasing sdLDL could not be confirmed but was rather disproved.
86 LIPOPROTEIN(A) AND FAMILY HISTORY OF CARDIOVASCULAR
DISEASE IN CHILDREN
O. Guardamagna1 , F. Abello1 , P. Assandro1 , G. Anfossi2 , M. Pirro3 .
1
Department of Pediatrics, 2 Department of Clinical and Biological Sciences,
Unit of Internal Medicine and Metabolic Disease, University of Turin, Turin,
3
Department of Clinical and Experimental Medicine, Unit of Internal Medicine,
Angiology and Arteriosclerosis Diseases, University of Perugia, Perugia,
Italy
Objective: Increased lipoprotein(a) [Lp(a)] levels and family history of
cardiovascular disease (CVD) represent emergent CVD risk factors.
Aim of the study was to investigate the impact of lipid profile and Lp(a) on CVD
family history.
Methods: Lipid profile, Lp(a) levels and a two generation genealogical tree were
evaluated among 231 children affected by familial dyslipidemias and 32 children
showing only elevated Lp(a) levels. Lp(a) levels were stratified according to
presence, age of occurrence, number and type of CVD in their kindreds.
Results: Among 231 children affected by familial dyslipidemias lipoprotein and
Lp(a) levels did not differ between children with or without family history of CVD.
However, the percentage of children with elevated Lp(a) (85th percentile) was
higher among those with a positive family history for early CVD (p = 0.01).
Lp(a) was a significant independent predictor of the number of premature
cardiovascular events (b = 0.17, p = 0.01) and of cerebrovascular events in the
kindreds (OR 2.5, 95% CI: 1.05–6.03, p = 0.039), independent of plasma lipid
fractions and other risk factors. Furthermore in the group of children showing
only elevated Lp(a) levels a positive family history of CVD was detected in 85%
families with an higher incidence of ischemic stroke alone or plus coronary heart
disease (64.7%).
Conclusion: The association between Lp(a) and family history of early CVD
is due to a threshold effect among those with the highest Lp(a) levels.
Furthermore multiple cardiovascular events and cerebrovascular events are
significantly predicted by any increase in plasma Lp(a) levels, independent of
other cardiovascular risk factors.
87 EZETIMIBE/SIMVASTATIN 10/40MG VERSUS ATORVASTATIN
40MG IN HIGH CARDIOVASCULAR RISK PATIENTS WITH PRIMARY
HYPERCHOLESTEROLEMIA: A RANDOMIZED, DOUBLE-BLIND,
ACTIVE-CONTROLLED, MULTICENTER STUDY
F. Civeira1 , A.G. Dan2 , P.L.K. Hing3 , M.E. Hanson4 , R. Massaad5 , C. Milardo4 ,
J. Triscari4 . 1 Hospital Universitario Miguel Servet, Zaragoza, Spain, 2 University
Hospital Colentina of Bucharest, Bucharest, Romania, 3 School of Medicine
and Health Sciences, Monash University, Johor Bahru, Malaysia, 4 Merck
Sharp & Dohme Corp., a div. of Merck & Co., Inc., Whitehouse Station, NJ,
USA, 5 Merck Sharp & Dohme Corp., Brussels, Belgium
Objective: To assess the LDL-C lowering efficacy of switching to
ezetimibe/simvastatin 10/40 mg vs doubling atorvastatin to 40 mg in patients
treated with atorvastatin 20 mg but with LDL-C 2.59 & 4.14 mmol/L.
Methods: Adults at high cardiovascular risk with hypercholesterolemia
(N = 250), pretreated with atorvastatin 20 mg, were randomized to ezetimibe/simvastatin 10/40 mg or atorvastatin 40 mg for 6 weeks. Percent change in
LDL-C and other lipids was assessed using a longitudinal data analysis method
with terms for treatment, time, time-by-treatment interaction, stratum, and timeby-stratum interaction. Percentage of patients achieving LDL-C < 1.81 mmol/L,
<2.00 mmol/L or <2.59 mmol/L was assessed using a logistic regression model
with terms for treatment and stratum. Tolerability was assessed.
Results: Switching to ezetimibe/simvastatin resulted in significantly greater
changes in LDL-C (−26.81% vs. −11.81%), total cholesterol (−15.97% vs.
−7.73%), non-HDL-C (−22.50% vs. −10.88%), Apo B (−17.23% vs. −9.53%),
and Apo A-I (2.56% vs. −2.69%) vs doubling the atorvastatin dose (all p
21
0.002), but not HDL-C, triglycerides or hs-CRP. Significantly more patients
achieved LDL-C < 1.81 mmol/L (29% vs. 5%), <2.00 mmol/L (39% vs. 9%)
or <2.59 mmol/L (69% vs. 41%) after switching to ezetimibe/simvastatin vs.
doubling the atorvastatin dose (all p < 0.001). The overall safety profile appeared
generally comparable between treatment groups.
Conclusions: In high cardiovascular risk patients with hypercholesterolemia
already treated with atorvastatin 20 mg but not at LDL-C < 2.59 mmol/L,
switching to combination ezetimibe/simvastatin 10/40 mg provided significantly
greater lipid-altering and greater achievement of LDL-C targets compared with
doubling the atorvastatin dose to 40 mg. Both treatments were generally welltolerated.
88 EZETIMIBE MONO-THERAPY AMELIORATES VASCULAR FUNCTION
IN PATIENTS WITH HYPERCHOLESTEROLEMIA THROUGH
DECREASING OXIDATIVE STRESS
H. Kurobe1 , K.-I. Aihara2 , Y. Hirata3 , T. Motoki1 , M. Sugano1 , T. Kanbara1 ,
T. Nakayama1 , T. Kitaichi1 , M. Akaike3 , M. Sata3 , T. Matsumoto2 , T. Kitagawa1 .
1
Department of Cardiovascular Surgery, 2 Department of Medicine and
Bioregulatory Sciences, 3 Department of Cardiovascular Medicine, IHBS, the
University of Tokushima Graduate School, Tokushima-shi, Japan
Background and Objective: Ezetimibe, an inhibitor of cholesterol intestinal
absorption, is a lipid lowering agent. However, anti-atherogenic effects of
ezetimibe have not been fully elucidated. Therefore, the objective in this
study is to clarify the vascular protective effects of ezetimibe in patients with
hypercholesterolemia.
Methods: Ezetimibe was administrated to 20 patients with hypercholesterolemia (Group E). And age- and sex-matched 20 patients with hypercholesterolemia also followed as controls (Group C). The changes of metabolic profiles
and cardiovascular surrogate markers before and 12 weeks after ezetimibe
treatment were statistically evaluated.
Results: Ezetimibe treatment significantly reduced serum levels of low-density
lipoprotein cholesterol (LDL-C) and malondialdehyde-modified low density
lipoprotein (MDA-LDL). In addition, the values of body mass index, body weight,
waist circumference, plasma HbA1c, urinary 8-OHdG and urinary albumin
were significantly decreased in group E, but not in Group C. On the other
hand, high-density lipoprotein cholesterol (HDL-C) and adiponectin levels were
significantly increased in group E, but not in group C. The values of brachialankle pulse wave velocity (ba-PWV), mean arterial blood pressure (m-ABP),
and % of flow-mediated dilation (FMD) were significantly improved in only group
E. Furthermore, ultrasonic studies demonstrated amelioration of the vascular
stiffness of common carotid arteries in group E, but not in group C. These
vascular protective effects of ezetimibe were statistically correlated with the
decreased values of MDA-LDL but not LDL-C.
Conclusion: Ezetimibe exerts lipid lowering independent-vascular protection in
patients with hypercholesterolemia through decreasing oxidative stress.
89 IMPROVED PLASMA LIPID PROFILE AND REDUCTION IN HEPATIC
STEATOSIS WITH RIMONABANT THERAPY IN MORBIDLY OBESE
PATIENTS
A.S. Wierzbicki1 , S. Pendleton2 , Z. MacMahon3 , A. Dar4 , M.A. Crook1 ,
A.J. Botha5 . 1 Metabolic Medicine/Chemical Pathology, 2 Dietetics & Nutrition,
3
Metabolic Medicine, 4 Gastroenterology, 5 Surgery, Guy’s & St. Thomas’
Hospitals, London, UK
Introduction: Bariatric protocols desire weight loss prior to surgery. Little data
exists of the efficacy of orexigenic drugs in the morbidly obese.
Aim: To investigate the efficacy of rimonabant in patients prior to bariatric
surgery.
Methods: A retrospective cohort analysis was performed of patients referred
for bariatric surgery from 2006−9. Patients had anthropometric, biochemical
and physiological investigations and attended a >6 month course of life style
therapy.
Results: 376 patients were identified and 222 complied with the protocol.
Patients receiving rimonabant therapy were aged 51±11 years, 40% male
and had a BMI of 49.2±8.8 kg/m2 . Diabetes was present in 36%, nocturnal
hypoventilation in 24%, hypertension in 66%, lymphoedema in 10% and
depression in 11%. 35 patients discontinued rimonabant therapy in <1 month
mostly due to depression or nausea. Rimonabant therapy for a mean 12.3
months resulted in a 4.6±11 kg (median 4.5 (p = 0.11); 1.7±4 BMI points
(p = 0.07)) weight loss with a reduction in waist circumference of 7±10 cm
(P = 0.008); blood pressure by 6.6/3.8 mmHg (P < 0.001), insulin by 39%
(p = 0.01); homeostasis index (HOMA-IR) by 55% (P = 0.005); and HbA1c by
0.5% (p = 0.01). Alanine transaminase levels were reduced by 17% (p = 0.002);
total cholesterol by 15% (P < 0.001), triglycerides by 37% (P = 0.02) and
LDL-C by 17% (p = 0.002) and HDL-C increased by 7% (P 0.10). 8 patients
discontinued rimonabant therapy due to side-effects over this period.
Conclusions: Rimonabant therapy, if initially tolerated, resulted in a nonsignificant weight loss in morbidly obese patients allied but significant
improvements in hyperlipidaemia, hepatic steatosis, and insulin resistance.
22
90 UPDATE OF THE BIOCHEMICAL AND MOLECULAR RESULTS
OF PORTUGUESE PATIENTS WITH FAMILIAL COMBINED
HYPERLIPIDAEMIA
T. Santos1,2 , Q. Rato3 , I.M. Gaspar4 , M.T. Rico5 , J.M. Silva6 , M. Bourbon1,2 .
1
Grupo de Investigação Cardiovascular, Instituto Nacional de Saúde Dr.
Ricardo Jorge, 2 BioFIG, Center for Biodiversity, Functional and Integrative
Genomics, Lisbon, 3 Serviço de Cardiologia, Hospital S. Bernardo, Setúbal,
4
Serviço de Genética Médica, Hospital Egas Moniz, 5 Serviço de Medicina
Interna, Hospital Sta. Marta, Lisbon, 6 Serviço de Medicina II, Hospital da
Universidade de Coimbra, Coimbra, Portugal
FCHL is a complex disorder with a highly atherogenic profile. The aim of this
study is the biochemical/molecular characterization of FCHL patients.
Molecular study of LPL, APOAIV , APOAV , APOCIII and USF1 (s1,s2) was
performed in 35 index patients by PCR amplification and sequencing. Total
cholesterol (TC), HDL-c, sdLDL, triglycerides (TG), apoB and apoCIII were
measured in automated analysers. sdLDL was also analysed by electrophoresis
with Lipoprint® . ApoAIV and ApoAV were measured by ELISA. For all patients,
biochemical characterization of apolipoproteins and sdLDL before treatment
was not possible to determine.
Prior to medication the levels of TC (305±62 mg/dL) and TG (380±269 mg/dL)
were high but HDL-c was normal (44±11 mg/dL). Even under medication these
patients presented high levels of CT (205±58 mg/dL), TG (233±109 mg/dL)
and ApoCIII (15±4 mg/dL) and normal levels of HDL-c (44±9 mg/dL). ApoAIV
(17±10 mg/dL), ApoAV (156±140ng/mL) and ApoB (89±41 mg/dL) levels were
within normal range in majority of cases. Lipoprint® analysis of 8 patients under
medication revealed an atherogenic pattern.
Two new alterations were found. One patient with TC=271 mg/dl and
TG=275 mg/dL carried APOAIV Q359_E362 (values without medication).
Another patient with TC=419 mg/dL and TG=1095 mg/dL presented APOAV
D332fsX336 and, even under medication, had high sdLDL (39 mg/dL, cut-off
value 35 mg/dl) and an atherogenic pattern with Lipoprint® analysis (pattern B).
Some patients had a novel alteration APOCIII 3269C>A that was proven later
that was a polymorphism.
Patients with FCHL have increased cardiovascular risk that can be
prevented with an early genetic identification and an extensive biochemical
characterization that can be important to evaluate the efficacy of treatment.
91 THE EFFECTS OF COMBINING ROSUVASTATIN WITH SARTANS
OF DIFFERENT PPARg ACTIVATING CAPACITY ON LOW-DENSITY
LIPOPROTEIN SUBFRACTIONS AND LIPOPROTEIN-ASSOCIATED
PHOSPHOLIPASE A2
C.V. Rizos1 , E.N. Liberopoulos1 , C.C. Tellis2 , M. Florentin1 , M.S. Elisaf1 ,
A.D. Tselepis2 . 1 Internal Medicine, 2 Laboratory of Biochemistry, University of
Ioannina, Ioannina, Greece
Aims: Sartans differ in their capacity to partially active the peroxisome
proliferator-activated receptor g (PPARg). The aim of this study was to evaluate
whether this characteristic is associated with changes in low-density lipoprotein
(LDL) subfraction phenotype or plasma lipoprotein-associated phospholipase
A2 (LpPLA2 ) when co-administrating a sartan with a statin.
Methods: Patients (n = 151) with mild hypertension, impaired fasting plasma
glucose (FPG), elevated triglycerides (150 mg/dL) and LDL cholesterol
(160 mg/dL) were given dietary intervention and rosuvastatin (10 mg/day).
Patients were randomized to: (1) a sartan which partially activates PPARg
(telmisartan 80 mg/day; RT group), (2) a sartan with low efficacy in activating
PPARg (irbesartan 300 mg/day; RI group) and (3) a sartan with no efficacy
in activating PPARg (olmesartan 20 mg/day; RO group). Patients were reevaluated 6 months after treatment onset.
Results: The lipidemic profile was similarly altered in all groups. Small dense
LDL cholesterol decreased vs baseline in the RT (−67%), RI (−58%) and RO
(−61%) groups (p < 0.001). All regimens increased LDL particle size vs baseline
(RT +1.4%; p = 0.002, RI +1.0%; p < 0.04 and RO +1.4%; p = 0.001). Plasma LpPLA2 activity equally decreased vs baseline in all groups (RT −38%, RI −38%,
RO −43%) (p < 0.001). Plasma Lp-PLA2 mass similarly decreased in all groups
vs baseline (RT −28%; p = 0.001, RI −32%; p = 0.01 and RO −27%; p = 0.001).
No difference for any parameter change was noticed among groups.
Conclusions: Co-administrating rosuvastatin with sartans of different degree
of PPARg activating capacity was not associated with differential changes in
LDL subfractions or plasma LpPLA2 activity and mass.
92 THE PRESENCE OF REMNANT LIPOPROTEINS IN THE FASTING
PLASMA WITH TG LEVELS LESS THAN 150 MG/DL
K. Nakajima1 , T. Nakano1 , Y. Tokita1 , T. Nagamine1 , S. Yatsuzuka2 ,
Y. Shimomura2 , A. Tanaka3 . 1 Gunma University, Maebashi, 2 Hidaka Hospital,
Takasaki, 3 Kagawa Nutrition University, Tokyo, Japan
Introduction: Remnant-like lipoprotein particles (RLP) have been measured by
cholesterol as RLP-C for CHD risk assessment. However, RLP-TG is a better
marker to detect the presence of remnant lipoproteins in plasma with TG levels
less than 150 mg/dL.
Method: Serum RLP-TG levels in health-check populations, cardiovascular
disease, diabetes and oral fat load cases were determined. Serum TC, TG,
HDL-C, LDL-C and RLP-C concentrations were also determined in the same
cases.
Results: Cut-off value (75 percentile) of RLP-TG determined in the fasting
normal control cases in Japanese population was 13 mg/dl in men and 10 mg/dL
in women. Ninety fifth percentile of RLP-TG less than TG levels 150 mg/dL
was 20 mg/dL. In patients with diabetes, metabolic syndrome, cardiovascular
disease, RLP-TG levels were significantly higher than those in normal control
subjects. RLP-TG levels increased significantly in postprandial plasma with TG
levels less than 150 mg/dL. When fasting TG levels were less than 150 mg/dl,
the frequency of the cases in normal control group RLP-TG >20 mg/dL was
4.8% and the frequency in disease cases were significantly higher.
Conclusion: RLP-TG levels were shown to be significantly higher in cases
with diabetes, metabolic syndrome, cardiovascular disease. Moreover, the
frequency of higher RLP-TG levels above 20 mg/dL in the fasting state when
TG < 150 mg/dL was significantly higher in disease cases than in normal
controls. These results revealed that TG therapy should be targeted to reduce
less than 150 mg/dL of TG using RLP-TG as a parameter.
93 HYPERLIPIDEMIA AND OXIDATIVE STRESS ARE THE MAIN
DETERMINANTS OF INTIMA-MEDIA THICKNESS IN CHILDREN
WITH CHRONIC KIDNEY DISEASE
S. Spasic1 , J. Kotur-Stevuljevic1 , A. Peco-Antic2,3 , M. Kostic2,3 , D. Vasic3,4 ,
A. Stefanovic1 , A. Vujovic1 , Z. Jelic-Ivanovic1 , V. Spasojevic-Kalimanovska1 ,
D. Kornic5 . 1 Department of Medical Biochemistry, Faculty of Pharmacy,
University of Belgrade, Belgrade, Serbia, 2 Nephrology Department, University
Children’s Hospital, Belgrade, Serbia, 3 Faculty of Medicine, University of
Belgrade, Belgrade, Serbia, 4 Institute for Cardiovascula Disease, Clinical
Center of Serbia, Belgrade, 5 Biochemistry Laboratory, Health Center
‘Pancevo’, Pancevo, Serbia
Objective: Roles of dyslipidemia and oxidative stress in early phases of
atherosclerosis were tested in renal disease (RD) children. We have used
intima-media thickness of common carotid arteries (IMT CCA) as a measure of
early atherosclerosis.
Methods: Fifty-two pediatric patients were enrolled in the study (10 chronic
renal insufficiency − CRI, 22 renal transplant − RT and 20 haemodialysis − HD
patients) and 36 healthy children (CG). Lipid status was assessed by measuring
total, LDL, HDL, free-cholesterol and tryglicerides (TC, LDL-C, HDL-C, FC, TG).
Oxidative status was determined through malondialdehyde (MDA) concentration
and superoxide-dismutase (SOD) activity. IMT measurement was performed
by using Siemens SONOLINE B-mode ultrasound apparatus. Multiple linear
regression analysis with backward selection has been used to estimate
influence of dyslipidemia, oxidative status and renal function markers on
patients’ IMT value.
Results: Renal disease children had disturbed lipid content, which was the
most pronounced in HD children. They have significantly higher FC, and TG
compared to healthy children (FC: 1.50±0.391 vs. 1.06±0.227, p < 0.001;
TG: 1.934±0.917 vs. 0.703±0.359, p < 0.001). Oxidative stress was markedly
increased (MDA: CRI 1.498±0.26226, RT 1.548±0.3969, HD 1.773±0.3426,
CG 0.975±0.3329, p < 0.001) and antioxidative defense was compromised
(SOD:CG 120±21, CRI 84±25, RT 93±12, HD 119±37 p < 0.001) in RD
children. Multiple linear regression analysis revealed that model which has
included lipid and oxidative status parameters had more than 90% of influence
in variability of IMT values.
Conclusion: Early atherosclerosis in renal disease children is caused, at least
in part, by dyslipidemia and oxidative stress.
94 IN SILICO MODELLING OF HUMAN LIPOPROTEIN METABOLISM
M. Jansen1 , K. Winkler1 , G. Pütz1 , P. Pfaffelhuber2 . 1 Department of Clinical
Chemistry, University Hospital Freiburg, 2 Department of Mathematics and
Physics, University of Freiburg, Freiburg im Breisgau, Germany
Introduction: Lipoproteins play an important role in the genesis of
atherosclerosis and cardiovascular diseases. High density lipoproteins (HDL)
and low density lipoproteins (LDL) are of great interest in lipid research.
However, lipoprotein analysis is elaborate and expensive. A model predicting
the risk of cardiovascular disease depending on the lipid metabolism may be
of great benefit.
Methods: Here, we present a refined version of a previously described model
of the human lipoprotein metabolism. The model describes the behaviour of
particular lipoproteins dependent on a set of reaction rates. Due to the large
number of possible lipoprotein compositions, the computation is mostly done
by the stochastic Gillespie algorithm and only in special cases deterministically.
The now presented model is based on a dataset of lipoprotein measurements
(circa 2000 patients) and contains several changes and refinements to the prior
approach. One important enhancement is the remodelling of the cholesteryl
ester transfer protein (CETP) dynamics.
79th EAS Congress
Results: Our model is able to reproduce measured lipid data of humans with
normal lipoprotein values. Furthermore, certain lipoprotein-associated diseases
like hypercholesterolemia may be simulated by modifying the associated
parameters of the metabolism. As particular lipoproteins are simulated, we are
able to get information about the distribution and metabolic rates of lipoproteins
in all density classes. Hence it is possible to simulate kinetic measurements.
Discussion: The model is capable to investigate associated diseases of lipid
metabolism and to provide a powerful tool for possible therapeutic approaches.
However, there is still need for further improvements.
95 ATORVASTATIN INCREASES CHOLESTEROL LEVELS IN
APOE/LDLR-DEFICIENT MICE
M. Slanarova1 , E. Brcakova1 , L. Vecerova1 , Z. Strasky1 , J. Rathouska1 ,
R. Hyspler2 , A. Ticha2 , P. Nachtigal1 . 1 Charles University, Faculty of Pharmacy,
2
Charles University, Medical Faculty and Faculty Hospital, Hradec Kralove,
Czech Republic
It has been demonstrated that statins are able to increase cholesterol levels
in hypercholesterolemic mice. Therefore we wanted to determine possible
mechanism of atorvastatin effects on cholesterol levels in blood and metabolism
in liver in ApoE/LDLR-deficient mice.
Two-month-old female ApoE/LDLR-deficient mice were randomly subdivided
into 2 groups. Control group was fed with the chow diet and in other group
was added chow diet atorvastatin at the dosage of 50 mg/kg/day for 2 months.
Serum lipoproteins were prepared using ultracentrifugation. Total concentration
of cholesterol was measured spectrophotometrically. The cholesterol synthetic
flux was assessed by means of GC-MS system. The expression of HMG-CoAreductase, ABCA1, SR-B1, ABCG5 and ABCG8 was detected by means of
Western blot method. The administration of atorvastatin resulted in a significant
increase in a total cholesterol and VLDL cholesterol in blood. Atorvastatin
treatment did not affect the expression of HMG-CoA-reductase but decreased
synthetic flux of cholesterol into the liver, however increased its concentration
in liver tissue. The expression of SR-B1 and expression of ABCG5 receptor
was not significantly changed after atorvastatin treatment. On the other hand
ABCA1 and ABCG8 expression was reduced after atorvastatin treatment.
In conclusion, atorvastatin is able to increase total and VLDL cholesterol levels
in ApoE/LDLR-deficient mice. We propose, that this effect is related to increased
cholesterol content in liver, decreased cholesterol efflux into bile and increased
VLDL release into blood circulation.
This work was supported by grant from The Grant Agency of Charles University
in Prague number 137310/C, grant SVV-2010–261−00 and Research project
MZO 00179906
96 DETERMINATION OF SDLDL PARTICLES IN PATIENTS WITH
FAMILIAL HYERCHOLESTEROLAEMIA AND FAMILIAL COMBINED
HYPERLIPIDAEMIA
A. Gomes1,2 , T. Santos1,2 , M. Bourbon1,2 . 1 Unidade de I&D, Grupo de
Investigação Cardiovascular, DPSDC, Instituto Nacional de Saúde Dr. Ricardo
Jorge, 2 Center for Biodiversity, Functional & Integrative Genomics, Lisbon,
Portugal
Several studies demonstrated that sdLDL are an emerging cardiovascular (CV)
risk factor. The objective of this study was sdLDL measurement in patients with
genetic diagnosis of Familial Hypercholesterolaemia (FH) and clinical diagnosis
of Familial Combined Hyperlipidaemia (FCHL) to establish a relation between
sdLDL, CV risk and the efficacy of therapeutics.
Lipid profile was determined using a polyacrylamide gel electrophoresis system
that separates the particles in serum that contain cholesterol. The lipidogram
obtained classifies the patients as being profile A (low CV risk) or B (high CV
risk) depending on the sdLDL concentration.
The lipid profile was obtained from 43 FH adults and 46 FCHL adults, index
and relatives. FH and FCHL patients without medication and with high sdLDL
(>6 mg/dl) have significant higher levels of total cholesterol, LDL and ApoB
and FCHL patients also have significant higher triglycerides, compared to
FH and FCHL patients with sdLDL levels under recommended values. Under
medication FH patients have significant higher ApoB levels and lower HDL,
and FCHL patients have significant higher total cholesterol, LDL and ApoB
levels. Interestingly, 71.4% of FCHL patients under medication presented high
CV risk profile, showing that statins seem not to decrease sdLDL levels and
neither CV risk. Also FCHL patients are not well medicated or do not respond
to usual medication to decrease cholesterol. These preliminary results indicate
that sdLDL could be a good biomarker for treatment control but further studies
are needed to evaluate the effect of medication in sdLDL levels in FH and FCHL
patients.
23
97 ACUTE STIMULATION OF ENDOGENOUS ESTROGEN IN WOMEN
ALTERS CHOLESTEROL METABOLISM AND REDUCES PCSK9
AND LDL CHOLESTEROL LEVELS
L. Persson1 , P. Henriksson2 , E. Westerlund2 , O. Hovatta3 , B. Angelin1 ,
M. Rudling1 . 1 Department of Endocrinology, Metabolism and Diabetes,
Department of Medicine, and Molecular Nutrition Unit, Center of Biosciences,
Department of Biosciences and Nutrition, Karolinska Institutet at Karolinska
University Hospital − Huddinge, 2 Division of Cardiovascular Medicine,
Department of Clinical Sciences, Karolinska Institutet at Danderyd Hospital,
3
Department of Clinical Science, Intervention and Technology, Karolinska
Institutet at Karolinska University Hospital − Huddinge, Stockholm, Sweden
Objective: Treatment with estrogen influences cholesterol metabolism.
Whether changes in endogenous estrogen regulate cholesterol metabolism in
humans is less clear. To explore this we studied 31 women during in vitro
fertilization (IVF) pre-treatment.
Method: Blood was sampled at maximal suppression of estrogen using
GnRH (900 mg/day for two weeks), and during stimulation of estrogen using
recombinant FSH (75–300 IU/day for six days).
Results: Stimulation of endogenous estrogen resulted in decreased levels of
total cholesterol (−13%), mainly in VLDL (−22%) and LDL (−20%) fractions.
ApoB level was also reduced (−13%), suggesting a higher catabolism
and clearance of apoB containing particles, presumably through hepatic
LDL-receptors (LDLRs). Serum proprotein convertase subtilisin/kexin type-9
(PCSK9), a posttranscriptional suppressor of LDLRs, was reduced by 15%.
The level of growth hormone was increased 3-fold during estrogen stimulation
and may participate in the regulation of PCSK9. HDL cholesterol and CETP
activity did not change during estrogen stimulation, although the level of apoAI
was increased (8%). Serum triglycerides increased by 40%, mainly within LDL
(37%) and HDL (58%) fractions. Estrogen stimulation did not change plasma
markers of bile acid, cholesterol synthesis or cholesterol absorption.
Conclusions: We conclude that endogenous estrogen in women strongly
alters cholesterol metabolism. Our findings support the concept that estrogen
increases the clearance of plasma apoB containing particles, presumably due
to increased LDLR numbers. Our data further indicate that increased LDLRs
numbers are probably caused by reduced PCSK9 serum levels, presumably
due to increased estrogen-stimulated secretion of pituitary growth hormone.
98 TARGET LIPID LEVELS AND RESERVES IN HYPOLIPIDEMIC
THERAPY IN THE PATIENTS AT VERY HIGH RISK OF
CARDIOVASCULAR EVENTS
J. Petrus, F. Málek, J. Dvořák, V. Skalnı́ková, P. Neužil. Nemocnice na
Homolce, Praha, Czech Republic
Background: The expert societies defined target lipid levels according to the
level of cardiovascular risk. The aim to reduce LDL cholesterol for the subjects
at high risk of cardiovascular events is <2.5 mmol/l, or <2.0 mmol/l if feasible.
Aim of the study: to identify lipid levels and proportion of subjects at high
cardiovascular risk reaching target lipid values.
Patients population and methods: Hypolipidemic therapy and lipid levels were
evaluated in 529 patients (351 men and 178 women) who were reffered for the
elective cardiac catheterization between Jan and May 2010. The type and dose
of hypolipidemic therapy was obtained from patients medical records. Statin
dose was recalculated to equivalent dose of atorvastatin according to study of
Law and co-workers published in BMJ 2003.
Results: Hypolipidemic therapy with statins was used in 77% patients, 61%
were given atorvastatin with mean calculated daily dose of 24.30±16.1 mg. LDL
cholesterol target <2.5 mmol/l reached 46% subjects, LDL cholesterol target
<2.0 mmol/l 26%, HDL cholesterol level >1.0 mmol/l reached 56% of patients.
Conclusion: The study showed inadequate lipid control in the patients at high
risk of cardiovascular event: the target for LDL cholesterol <2.5 mmol/l was
reached in less than half, and LDL cholesterol target <2.0 mmol/l was reached
in about quarter of the patients. Hypolipidemic therapy with statins was used
in only 77% of the subjects at high CV risk with low calculated daily dose of
atorvastatin.
24
99 OXIDIZED-LDL AND APO B48 IN DIABETIC KIDNEY DISEASE
N. Fanton1 , T. Moreno2 , M.J. Alférez3 , M. Jiménez3 , I. Coca4 , D. Vianello1 ,
A. Zambon1 , P. Valdivielso2 . 1 Medical and Surgical Sciences, University of
Padova, Padova, Italy, 2 Medicina Interna, 3 Nefrologia, Hospital Virgen de
la Victoria, Malaga, 4 Medicina Interna, Hospital Sta Barbara, Puertollano,
Spain
Background and Aims: Post-prandial hyperlipidemia is a risk factor for
atherosclerosis. Fasting plasma apo B48 is a promising marker of post-prandial
lipid metabolism. We investigated the association of apo B48 with oxidized LDL
(ox-LDL), and the presence of peripheral arterial disease (PAD) in diabetics
with and without kidney disease (DKD).
Patients and Methods: 40 age and sex-matched subjects were enrolled, 20
with DKD and 20 without, defined by albumin excretion rate and estimated
glomerular filtration rate (eGFR by Cockroft-Gault formula). Lipoproteins,
subclinical PAD by ankle-brachial index (ABI), post-prandial lipaemia by fasting
apo B48 (ELISA), levels of ox-LDLs (ELISA), albuminuria and eGFR were
evaluated.
Results: Demographic and anthropometric data, glycaemic control and lipid
profile were similar between patients with and without nephropathy. Apo B48
was significantly associated with TG content in VLDL, LDL and HDL (all
p < 0.01), with plasma ox-LDL (p = 0.016), and glycated haemoglobin A1c
(p < 0.01). Apo B48 (p < 0.01) and ox-LDL (p < 0.05) were higher in patients
with eGFR < 60 ml/min vs. those with eGFR 60. Progressively lower eGFR
was associated with increasing apo B48 (p = 0.014, non-parametric ANOVA).
Estimated-GFR <60 ml/min was associated with significantly lower ABI vs eGFR
>60 ml/min. Levels of ox-LDL were significantly lower in patients on statin vs off
statin (P < 0.05) regardless of eGFR.
Conclusions: This study provides evidence of a significant association between
apo B48, ox-LDL and a proatherogenic lipid phenotype in patients with DKD,
suggesting a significant effect of statins on LDL oxidation in these patients
100 SERUM LEVELS OF VASPIN AND CARDIOVASCULAR RISK
FACTORS IN MORBID OBESE PATIENTS
A. Golpaie, Z. Karbaschian, N. Tajik, M.J. Hosseinzadeh-Attar. Nutrition and
Biochemistry, Tehran University of Medical Sciences, Tehran, Iran
Introduction: Obesity, the most common nutritional disorder in the world,
is associated with an increased mortality and morbidity of cardiovascular
disease (CVD). Adipokines are protein products of adipose tissue with paracrine
and endocrine actions, which have been implicated in the pathogenesis
of cardiovascular disease. Visceral adipose tissue-derived serpin (vaspin)
was identified as a novel adipokine related to obesity and its metabolic
consequences. The association of vaspin with atherosclerosis is still obscure.
The present study aimed to investigate the relationship of this adipokine with
the cardiovascular risk factors in morbid obese patients.
Methods: 37 morbid obese patients aged 37±9.1 yr were studied. BMI, waist
circumference, vaspin, HDL, LDL, TG and total cholesterol were measured.
Pearson’s correlation was used to evaluate the relationship between variables.
P values <0.05 were considered as significant.
Results: Mean BMI, waist circumference and waist to hip ratio were 44.81±4.7,
123.1±14.2 cm, and 0.9±0.1 respectively. The levels of serum vaspin, LDL,
HDL, TG, total cholesterol were 0.34±0.2 ng/ml, 111.9±28.8, 38.7±8.9,
179±118.3, and 188.3±39.7 mg/ml respectively. Similar to some other studies
in this study vaspin was not associated with waist circumference and lipid
profiles.
Conclusion: Although there are conflicting results in terms of the association of
vaspin with CVD, we could not show any relationship between CVD risk factors
and vaspin concentrations.
101 PRO-ATHEROGENIC POSTPRANDIAL PROFILE: MEAL
INDUCED CHANGES OF LIPOPROTEIN SUB-FRACTIONS AND
INFLAMMATION MARKERS IN OBESE BOYS
A. Zambon1 , L. Pinelli2 , M.G. Surano2 , M. Carlon1 , S. Cordioli2 , S. Gasperotti2 ,
D. Vianello1 , C. Maffeis2 . 1 Medical and Surgical Sciences, University of
Padova, Padova, 2 Department of Mother and Child, Biology-Genetics, Section
of Pediatrics, University of Verona, Verona, Italy
Background and Aims: Obesity is a pro-atherogenic condition. Postprandial
lipoprotein profile and circulating cytokines changes may contribute to promote
the atherothrombotic process. We investigated postprandial metabolic response
in obese boys.
Patients and Methods: Ten prepubertal obese boys consumed two meals
with the same energy and protein content but different carbohydrate/fat
ratio: 1) moderate fat (MF): 61% carbohydrate, 27% fat; 2) high fat (HF):
37% carbohydrate, 52% fat. IL-6, IL-10, TNFa, hs-CRP, leptin, adiponectin,
lipoprotein distribution by ultracentrifugation, and LDL oxidation (ox-LDL) by
ELISA were evaluated at baseline and postprandially at several time points.
Results: Glucose and insulin AUC were significantly (p < 0.05) lower after HF
meal. HF meal was followed by a significant decrease in the cholesterol carried
in the HDL fractions, while cholesterol in the small, dense LDL and in the VLDL
particles increased, as compared to baseline (p < 0.05 for all). No differences
were found in the cholesterol distribution after MF meal. HDL-C concentration
was lower (p < 0.05) at 300 min. after HF vs. MF meal. Ox-LDL concentration
increased after HF meal but not after MF meal [9.3(2.2) vs 1.8(2.2)% from
baseline, p < 0.02)]. A positive association (r = 0.33, p < 0.05) was observed
between the densest LDL particles and the ox-LDL plasma levels. A reduction of
IL-6 was found at 120min after the MF [−23.3(5.5) vs −8.4(3.8)% from baseline,
p < 0.05)] compared with the HF meal.
Conclusion: A simple change of ~25% of energy load from fat to carbohydrate
in a meal significantly improves postprandial pro-atherogenic factors in obese
boys.
102 RISK FACTORS FOR NON-ALCOHOLIC FATTY LIVER DISEASE IN
RUSSIAN FEDERATION IN NATIONAL-WIDE DIREG-L-01903 STUDY
O.M. Drapkina, V.T. Ivashkin. Cardiology, Moscow Medical University, Moscow,
Russia
Aim: To assess the risk factors for non-alcoholic fatty liver disease (NAFLD) in
Russian Federation in the national population-based DIGER study.
Methods: In total of 30 787 primary care patients (56% females, mean age
47.8±16 yrs) were enrolled into open multicenter national-wide prospective
study. Careful clinical examination, serum biochemistry (including ALT, AST,
g-GT, lipid spectrum, glucose and hepatitis screening) and abdominal ultrasound
diagnostics with precise liver assessment were performed in 30 754 patients.
Results: NAFLD was found in 8215 (27) % of included patients. In patients aged
from 18 to 29 years abdominal obesity was identified as risk factor, because in
was found in 45% NAFLD patients in comparison with 14% of patients without
NAFLD, p < 0.001. The significance of abdominal obesity as NAFLD risk factor
is decreased with advanced age due to relatively higher prevalence of obesity
in patients without NAFLD aged from 40 to 80 years. NAFLD was diagnosed
in 64.3% of patients with type I diabetes, 69.8% patients with type II diabetes,
45.2% of patients with arterial hypertension, 61.5% of patients with obesity and
in 66.9% in those with metabolic syndrome.
Conclusion: Taking into account high prevalence (27%) of NAFLD in Russian
Federation the attention should be given for NAFLD risk factors such as arterial
hypertension, dyslipidemia and hypercholesterolemia in all age groups as well
as abdominal obesity in patients younger than 39 years. Metabolic factors
clustering might explore an important link between metabolic syndrome and
NAFLD.
103 THE LIVER SIEVE IN CHOLESTEROL METABOLISM INFLUENCES
BOTH LIFESTYLE AND FAMILIAL ATHEROSCLEROSIS
R. Fraser1,2 , B.R. Dobbs3 , V.C. Cogger4 , D.G. Le Couteur4 . 1 Pathology,
University of Otago, Christchurch, 2 Medical Director, Canterbury Medical
Research Foundation, 3 Surgery, Canterbury District Health Boatd,
Christchurch, New Zealand, 4 Geriatrics, University of Sydney, Sydney, NSW,
Australia
We first demonstrated ultra-filtration by the fenestrated liver sinusoidal
endothelial cells (LSEC) of chylomicrons and their smaller remnants (natural
nano-vectors of dietary cholesterol) before their entry into the space of Disse
(1975−8). This filtration preceded remnant contact of specific apoE hepatocytereceptors internalising dietary cholesterol to inhibit HMGCoA reductase and
hepatic cholesterol synthesis. Thus sieve porosity balances food cholesterol
with cholesterol synthesised by the liver.
LSEC porosity and remnant diameter, composition and structure relate to
factors affecting lipid metabolism and atherosclerosis. These include ratios
and types of dietary lipids, drugs (eg nicotine, alcohol, cocaine, pantethine),
hormones, diabetes, ageing, surfactants and susceptibility of different species to
dietary lipids and experimental atherosclerosis. Decrease in fenestral diameters
and frequency or an increase in remnant size leads to both post-prandial
triglyceride-rich lipoproteins and liver-derived lipoprotein-cholesterol.
A Boston developmental pharmaceutical group (Alnylam) is perfecting artificial
lipidoid vectors transporting siRNA to silence rogue proteins, so potentially
treating genetic diseases such as familial-hypercholesterolaemia by silencing
rogue enzymes destroying the LDL receptor. To pass normal fenestrae (about
100nm diameter) they developed the ideal vector size of about 50 nm. (Akinc,
2009). This reinforces our 2003 suggestion that the failure of an adenovirusvector gene replacement therapy in one of four cured hemophiliac dogs was due
to LSEC defenestration from this dog’s cirrhosis (Mount et al. 2002). Smaller
miRNA-HDL complexes have since been described (2011).
Thus liver sieve porosity plays a role not only in the pathogenesis of
atherosclerotic diseases from poor lifestyles but has importance for potential
therapy for familial-atherosclerosis.
106 EXTENDED RELEASE NIACIN/LAROPIPRANT LOWERS
ATHEROGENIC LIPIDS ACROSS PATIENT SUBGROUPS
C.M. Ballantyne1 , H.E. Bays2 , A.K. Shah3 , C. Sisk3 , Q. Dong3 , D. Maccubbin3 .
1
Baylor College of Medicine, Houston, TX, 2 L-MARC, Louisville, 3 Merck
Sharp & Dohme Corp., Whitehouse Station, NJ, USA
Background: Extended-release niacin/laropiprant (ERN/LRPT) produces
favorable effects on LDL-C, HDL-C and TG and other lipid/lipoprotein
parameters associated with cardiovascular risk in patients with primary
hypercholesterolemia or mixed dyslipidemia.
Objective: We examined the consistency of ERN/LRPT effects on non-HDL-C,
Apolipoprotein (Apo) B, and Lp(a) across several studies and among key
subgroups of dyslipidemic patients.
Methods: This analysis of 4 phase III, randomized, double-blind, studies
of ERN/LRPT (Table) examined the percent change from baseline in nonHDL-C, ApoB, and Lp(a), and the consistency of treatment effects based upon
differences in gender, race (white, non-white) and baseline age (<65, 65 years)
[95% confidence intervals].
Results: ERN/LRPT produced significantly greater improvements in nonHDL-C, ApoB, and Lp(a), compared with placebo/active comparator in each
study cohort (Table). These effects were generally consistent across key
subgroups within each study.
P069
P067
P022
Dyslipidemia;
67% on statins
P020
ERN/LRPT, extended release niacin/laropiprant; ERN, extended release niacin; Pbo, placebo; Sim, simastatin; Lp(a), lipoprotein a; Apo B, apolipoprotein
B non-HDL-C, non-high-density lipoprotein cholesterol; CI, confidence interval; T2 DM, type 2 diabetes mellitus.
*p < 0.0001.
−19.8 (−22.8,
−16.8)*
−17.1 (−19.7,
−14.5)*
ERN/LRPT vs. Pbo −25.0 (−28.6,
−20.2)*
Add on/36 weeks
−6.4 (−9.3, −3.5)*
−8.2 (−10.8,
−5.6)*
ERN/LRPTvs. Statin −20.5 (−24.5,
dose doubled
−16.9)*
−12.4 (−14.5,
−10.2)*
−12.2, (−14.3,
−10.1)*
−21.4 (25.0,
−17.5)*
ERN/LRPT + Sim
vs. Simva
Washout,
coadministration/
12 weeks
Add on/12 weeks
<19.8 (<22.4,
<17.3)*
<18.8 (<21.2,
<16.5)*
ERN/LRPT vs. Pbo −20.8 (−24.7,
−16.7)*
add on/24 weeks
ApoB (mg/dL)
Lp(a) (mg/dL)
Population
Lipid effects across studies
Objective: To study the efficacy of the combination ezetimide–orlistate, on
lipidemic profile in overweight and obese patients.
Material and Methods: A total of 46 subjects with BMI > 29 and
hypercholesterolemia >220 mg/dl has been studied. Everyone was under
isothermal diet, while 16 were ezetimide 10 mg daily (group I), 16 orlistate
120 mg ×3 (group II), and 14 a combination of these two (group III). All were
determined at baseline and after four months, BMI, waist circumference and
body weight, while the biochemical control was reflected in the lipid profile:
total cholesterol, triglycerides, HDL and LDL cholesterol.
Results: In all groups there was a decrease in both total and LDL cholesterol,
and triglycerides, it is worth noting that there was no serious side effects.
However, in Group III, recorded price reduction of LDL cholesterol by 32.8%, is
statistically much greater than that of Group I (17.5%) and II (15%) (p < 0.05).
Also in Group III, as in II there was a significant reduction in both the waist
circumference, and body weight, in contrast to group I, where there were no
corresponding significant changes.
Conclusions: It turns out therefore that the combination of orlistate–ezetimide
has an additive effect on lipidemic profile of hypercholesterolemic obese patient
has no significant side effects and improves the anthropometric parameters.
Treatments
105 STUDY ON EFFECT OF COMBINING ORLISTATE–EZETIMIDE ON
LIPIDEMIC PROFILE ON OBESE PATIENTS
S. Patiakas, C. Charalampous. Microbiological Laboratory of General Hospital
of Kastoria, Thessaloniki, Greece
25
ERN/LRPT (n = 800)
ERN (n = 543)
Pbo (n = 270)
Dyslipidemia
ERN/LRPT (n = 195)
statin naive or ERN/LRPT + Sim
washed off
(n = 610) Sim (n = 693)
Dyslipidemia;
ERN/LRPT +
100% on statins Statin (N = 606)
Sim (n = 610)
T2 DM; 80% on ERN/LRPT (n = 464)
statins
Pbo (n = 342)
Study design/
duration
Objectives: It is difficult to identify a single parameter of plasma lipid
disturbances correlating with the progression of atherosclerosis in patients
presenting clinical symptoms of the disease.
Purpose: Thus the project attempted to determine, on the basis of routinely
used assessments of lipid parameters, a sensitive indicator which would
associate with the severity of coronary heart disease in patients undergoing
hypolipidemic treatment.
Methods: ApoB100, apoA1, LDL, HDL, triglicerydes (TG) and total cholesterol
(TC) levels were assessed with the use of typical laboratory techniques
in140 consecutive patients referred for coronary angiography. Coronary
atherosclerosis was evaluated on the basis of single stenosis significance as
well as quantitatively by applying the Gensini score.
Results: The study revealed that the level of TC/apoB100 ratio was significantly
lower in patients with advanced coronary arteriosclerosis (defined as a “3
vessel disease”, or as a arteriosclerosis over 95 percentyl of Gensini score)
receiving hypolipidemic treatment (irrespective of the treatment’s intensity)
compared to the patients without any lesions in coronary arteries taking
statins. There were no differences in TC, LDL, HDL, TG, apoB100 and apoA1
between studied groups. It was also found that in group without clinical
markers of coronary and/or peripheral atherosclerosis, patients taking lipidlowering therapy in comparison to subjects without statins treatment had
significantly higher TC/apoB100 ratio. This suggests a tendency for relatively
higher levels of apoB100 compared to total cholesterol levels in patients with
angiographically severe coronary atherosclerosis. The mechanisms responsible
for this phenomenon remains a matter of speculation. A large study group is
required in order to confirm the results of the analysis
Comparison
Between Treatment Group Difference in Percent Change from Baseline
(95% CI)
104 TOTAL CHOLESTEROL TO APOLIPOPROTEIN B100 RATIO IN
ADVANCED STABLE ANGINA PATIENTS TREATED WITH STATINS
P. Burchardt1 , B. Zuchowski1 , A. Palasz1 , J. Zurawski2 , H. Wysocki1 . 1 Division
of Cardiology-Intensive Therapy, 2 Poznan University of Medical Sciences,
Poznan, Poland
Non-HDL-C (mg/dL)
Protocol
number
79th EAS Congress
Conclusion: In addition to favorable beneficial effects on LDL-C, HDL-C, and
TG levels, ERN/LRPT produced significant reductions in non-HDL-C, ApoB, and
Lp(a), which were generally consistent across the patient subgroups examined.
ERN/LRPT represents an effective, comprehensive therapeutic option for the
treatment of dyslipidemia across a range of patient types.
107 LIPID PARAMETERS IN ACUTE CORONARY SYNDROMES VERSUS
STABLE CORONARY ARTERY DISEASE
A. Vonbank1,2,3 , C.H. Saely1,2,3 , P. Rein1,2,3 , C. Boehnel1,3 , V. Jankovic1,3 ,
J. Breuss1,3 , H. Drexel1,2,3,4 . 1 VIVIT Institute, 2 Internal Medicine, Academic
Teaching Hospital Feldkirch, Feldkirch, Austria, 3 Private University of the
Principality of Liechtenstein, Triesen, Liechtenstein, 4 Drexel University College
of Medicine, Philadelphia, PA, USA
Background: Differences in lipid parameters between patients with acute
coronary syndromes (ACS) and patients with stable coronary artery disease
(CAD) are unclear and are addressed in the present study.
Methods: We enrolled 582 patients with angiographically proven stable CAD,
of whom 26.9% had diabetes mellitus type 2 (T2DM) and 182 patients with
ACS, of whom 35.8% had T2DM.
Results: When compared to patients with stable CAD, HDL cholesterol
(45.8±15.5 mg/dl vs. 50.2±16.1 mg/dl; p < 0.001) and apolipoprotein A1
(139.0±30.4 mg/dl vs. 154.6±31.0 mg/dl; p < 0.001) were significantly lower in
26
patients with ACS in the total population as well as among subjects with T2DM.
Analysis of covariance confirmed an independent impact of the ACS state on
these lipid parameters after multivariate adjustment both in the total population
and among subjects with T2DM. In contrast, total cholesterol, LDL cholesterol
and apolipoprotein B neither in the total population (p = 0.583, p = 0.884 and
p = 0.834 respectively) nor among subjects with T2DM (p = 0.133, p = 0.234,
and p = 0.371, respectively) differed significantly between ACS and stable CAD
patients. Triglycerides were significantly higher in patients with ACS than in
patients with stable CAD in total study population (155.8±121.1 mg/dl vs.
140.8±90.6 mg/dl; p = 0.037) but not in patients with T2DM (p = 0.972).
Conclusion: We conclude that HDL cholesterol and apolipoprotein A1 are
lower in the ACS state than with stable CAD; this particularly holds true among
patients with T2DM.
108 OXIDATIVE STRESS AND ANTIOXIDANT PARAMETERS IN
PATIENTS WITH HYPERLIPIDEMIA
O. Enkhtaivan, M. Malchinkhuu, L. Tserenkhuu. Pathophysiology, Health
Sciences University of Mongolia, Ulaanbaatar, Mongolia
Hyperlipidemia is important risk factor for cardiovascular diseases. The lipid
oxidation theory for atherogenesis proposes that oxidation products are
responsible for foam cell formation and development of lesion. The major target
for oxidation is suggested to be intimal low-density lipoprotein (LDL). The aim
of this study was the role of oxidative stress status and arterial stiffens in
hyperlipidemia.
The serum total antioxidant capacity (TAC), malondialdehyde (MDA) levels
and cardio-ankle vascular index were measured in 31 hyperlipidemia patients
(serum total cholesterol level >240 mg/dl, LDL >160 mg/dl) and 56 healthy
subjects.
The concentration of TAC (101.38±33.34, p < 0.01) was significantly decreased,
serum MDA (119.75±36.06, p < 0.01) and CAVI (7.71±1.40, p < 0.01) were
increased in hyperlipidemia patients in comparison with control subjects
(TAC-131.56±38.08, MDA-88.17±26.76 and CAVI-6.75±1.14). The serum
total cholesterol level was positive correlation with CAVI (r = 0.364, p < 0.01)
and MDA (r = 0.452, p < 0.01). However, there was negative correlation with
TAC (r = −0.382, p < 0.01). Between CAVI and MDA was positive correlation
(r = 0.612, p < 0.01).
These data suggest that an increase of CAVI related to endothelium
dysfunction. Also, these changes are associated with an increase of lipid
oxidation in hyperlipidemia patients.
109 ROLE OF BLOOD CHOLESTEROL TRANSPORT SYSTEM
DISTURBANCES IN ATHEROSCLEROSIS IN RHEUMATOID
ARTHRITIS (RA)
T. Popkova, D. Novikova, A. Novikov, E. Alexandrova, E. Nasonov. Research
Institute of Rheumatology of RAMS, Moscow, Russia
Objective: To study role of inflammation and disturbances of blood cholesterol
transport system in atherosclerotic damage development in RA.
Material and Methods: 84 RA pts with mean age 48 years and mean disease
duration 87 months were included. Control group consisted of 20 humans
of comparable age and sex without rheumatic diseases. Cholesterol (Ch),
triglycerides (TG) and high-density lipoprotein cholesterol (HDL C) serum levels
were evaluated with colorimetric and photometric methods, CRP, apoA1 and
Lp(a) levels were assessed by immunonephelometric method.
Results: Increase of Lp(a) and TG concentrations in RA was more frequent
than in control (31.2 (6.6; 44.8) mg/dl vs 1.6 (0.9; 2.3) mg/dl and 1.1 (0.7; 1.5)
mmol/l vs 1.0 (0.3; 1.1) mmol/l, respectively). Ch and HDL C levels did not
differ. Intima-media complex (IMC) thickness in RA and control was the same.
Atherosclerotic plaques (AP) in RA were more frequent than control (38%. vs
3%, p = 0.009). RA pts showed negative correlation between IMC thickness and
HDL C, as well as between HDL C values, apoA1, CRP. Lp(a) level correlated
with DAS28 (r = 0.32). In pts with high Lp(a) level AP were more frequent, activity
of RA, apo B were higher than in pts with normal level of this lipoprotein. Lp(a)
values in pts with AP (65.3 (44.7; 66.8) mg/dl) were higher than in pts without
AP (8.2 (3.1; 11.4) mg/dl, p < 0.05).
Conclusion: Chronic inflammation in RA plays an important role in
disturbances in blood cholesterol transport system. Lp(a) level increase is a
risk factor of atherosclerotic vascular damage.
110 ASSOCIATIONS OF APOE POLYMORPHISMS WITH BASELINE
LIPIDS AND RESPONSE TO ROSUVASTATIN VARY IN
CHINESE PATIENTS WITH AND WITHOUT FAMILIAL
HYPERCHOLESTEROLAEMIA
M. Hu, V.W.L. Mak, E.W.M. Poon, B. Tomlinson. The Chinese University of
Hong Kong, Shatin, Hong Kong S.A.R.
Objectives: To evaluate associations of apolipoprotein E (APOE) genotype
with baseline lipid levels and response to rosuvastatin in Chinese patients with
hypercholesterolaemia.
Methods: A total of 386 patients with hypercholesterolaemia, including 166 with
familial hypercholesterolaemia (FH), with good adherence to rosuvastatin 10 mg
daily, were genotyped for the APOE rs7412 and rs429358 polymorphisms. The
lipid profile was examined before and after at least 4 weeks of therapy.
Results: FH patients were younger and had less metabolic syndrome features
than non-FH patients. The genotype distributions of APOE polymorphism were
similar in patients with and without FH. In non-FH patients, individuals carrying
the e2 allele had lower and those carrying the e4 allele had higher LDLcholesterol levels than subjects with the e3/e3 genotype but this was not
statistically significant (3.59±1.25: 4.17±0.97: 4.28±0.91 mmol/L, P > 0.05).
However, an opposite association between APOE polymorphisms and LDLcholesterol levels was observed in patients with FH (e2-carriers vs. e3e3
vs. e4-carriers = 6.97±1.81: 6.57±1.55: 5.72±1.27 mmol/L, P = 0.005). With
statin therapy, patients with FH had 2.6% smaller reductions in LDL-cholesterol
compared to patients without FH (P < 0.05). In non-FH patients, subjects with
the e2 allele had greater LDL-cholesterol reduction whereas e4 carriers had
the poorest response (57.2±18.6%: 53.5±12.8%: 50.0±12.6%, P < 0.05 for
trend). There was no association between APOE genotype and LDL-cholesterol
response to rosuvastatin in patients with FH.
Conclusions: This study demonstrates different associations of APOE
polymorphisms with baseline LDL-cholesterol levels and response to statins
in Chinese patients with different phenotypes of FH and non-FH.
111 MECHANISM OF HYPERCHOLESTEROLEMIA IN PHHC RAT
M. Zimolová1,2 , M. Schmiedtová1,2 , M. Heczková1 , R. Poledne1,2 , M. Jirsa1 ,
J. Kovář1,2 . 1 Institute for Clinical and Experimental Medicine, 2 Centre of
Cardiovascular Research, Prague, Czech Republic
Prague Hereditary Hypercholesterolemic (PHHC) rat develops polygenic
hypercholesterolemia after dietary cholesterol. However, its pathogenesis has
not been characterized yet.
Objectives: To explain pathogenesis of hypercholesterolemia and identify
genes involved in its development in PHHC rat.
Methods: Firstly, nascent VLDL of PHHC and Wistar rats were characterised.
The rate of disappearance of 125 I-labelled VLDL from Wistar and PHHC rats
was determined in vivo. Secondly, male PHHC and parental Wistar rats were
fed chow or 1% cholesterol (CHOL) diet for three weeks. Hepatic transcriptome
was analysed using GeneChip arrays.
Results: On CHOL diet, cholesterol and TG accumulated in liver of PHHC
and Wistar rats; however cholesterolemia rose only in PHHC rats. Nascent
VLDL of PHHC rats carry twice as much cholesterol as VLDL of Wistar rats
on CHOL diet and are catabolised more slowly. The gene expression of both
strains responded to CHOL diet exactly in the same way – genes of cholesterol
synthetic pathway were downregulated. On the other hand, several genes were
found to be differently expressed between both strains independently of the diet.
Of these genes, the only one apparently involved in lipoprotein metabolism,
Apof, was sequenced and 17bp insertion was found in PHHC rats.
Conclusions: PHHC rats secrete cholesterol-enriched VLDL from liver and
these VLDL are catabolised more slowly. This can explain the accumulation of
cholesterol in serum of PHHC rats. The analysis of transcriptome revealed
several new candidate genes for hypercholesterolemia. Their exact role in
pathogenesis remains to be determined.
Supported by 1M0510 MEYS CR.
112 COMPARATIVE EFFECT OF APHERESIS VS
ATORVASTATIN/APHERESIS ON MARKERS OF INFLAMMATION
IN PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA
M. Puntoni1 , F. Sbrana2 , F. Bigazzi2 , F. Minichilli1 , T. Sampietro1 . 1 Institute
of Clinical Physiology – CNR, 2 Fondazione G. Monasterio CNR-Regione
Toscana, Pisa, Pisa, Italy
Objective: Patients with Familial Hypercholesterolemia (FH) have increased
cardiovascular events. Clinical trials have demonstrated that lowering circulating
lipid levels by LDL-apheresis has beneficial effects on prognosis. However,
whether apheresis vascular effects in FH are related to modulation of
pro- and anti-inflammatory cytokines, and whether the combination of apheresis
with atorvastatin is able to enhance the putative anti-inflammatory effect of
apheresis remains unknown. We examined, in a intra-patient study, the effect
of atorvastatin/apheresis vs. apheresis alone on the releasing of circulating
pro- and anti-inflammatory markers.
Methods: 9 heterozygous patients (56±11 years) with FH (mean cholesterol
385±42 mg/dL) were treated with apheresis alone and afterwards with
apheresis plus atorvastatin 40 mg/d. Lipid profiles, serum C-reactive protein,
CK, GOT, GGT, the anti-inflammatory markers IL-4 and IL-10 and the proinflammatory markers INFg and IL-6 were determined before and at 2, 4, 6
and 8 days after apheresis and atorvastatin/apheresis.
Results: Treatment with atorvastatin/apheresis significantly reduced lipid profile
more than LDL-apheresis alone at each scheduled time. When compared
to apheresis alone, combined treatment statistically decreased cholesterol by
more than 25−35% at all times and relatively increased IL-4 concentration. The
79th EAS Congress
levels of cholesterol in atorvastatin/apheresis patients were inversely correlated
with those of IL-4 and IL-10 and positively correlated with IFNg.
Conclusion: The combination of atorvastatin with LDL-apheresis decreased
serum cholesterol levels more than apheresis alone. Apheresis had an antiinflammatory effect and the effect of the drug reducing cholesterol levels affects
the balance between pro- and anti-inflammatory cytokines in favor of antiinflammation contribute.
113 POLIDISTRECTUAL VASCULAR INVOLVEMENT IN FAMILIAL
HYPERCHILOMICRONEMIA
F. Sbrana1 , M. Puntoni2 , F. Bigazzi1 , M. Margelli3 , C. Petersen1 , R. Bonini3 ,
T. Sampietro2 . 1 Fondazione G. Monasterio CNR-Regione Toscana, 2 Institute
of Clinical Physiology − CNR, Pisa, 3 S.I.M.T. Lucca, Lucca Hospital − Azienda
U.S.L. 2, Lucca, Italy
A 72-year-old man was referred to our clinic with a lipid profile, under
combination therapy with a statin and ezetimibe, characterized by severe
hypertriglyceridemia (7230 mg/dl), hypercholesterolemia (374 mg/dl), low HDLcholesterol (17 mg/dl), and normal circulating Lp(a). The patient had undergone
post-traumatic splenectomy and presented a history of systemic hypertension
treated medically with well-controlled blood pressure. The patient had also
presented in the past abdominal pain with subsequent diagnosis of chronic
pancreatitis complicated by diabetes mellitus, well compensated when he came
to our attention.
Following a pathological exercise test, a coronary angiogram was performed
which showed a significant stenosis of the right coronary artery, which was
treated successfully with percutaneous coronary intervention. Clinical workup revealed ectasia of the abdominal aorta (28 mm), non significant bilateral
carotid artery disease, and peripheral artery disease of the femoral-popliteal
axis symptomatic for intermittent claudication.
A lipidogram was also performed and electrophoretic lipoprotein patterns did not
vary 2 hours after heparin infusion, pointing to the existence of lipoprotein lipase
deficit. Electrophoresis also showed a broadband of chylomicrons at baseline,
at the beginning, and at the end of heparin infusion.
Hyperchilomicronemia is a rare genetic disorder with an incidence of 1 per
1000000. Following diagnosis, our patient began plasma exchange therapy with
subsequent improvement of his lipid profile. At the present time, he is regularly
followed up at our clinic and non invasive imaging has excluded any significant
progression of atherosclerosis after 2 years of therapy.
114 AVERAGE LIPID PARAMETERS IN THREE CENTERS OF CENTRAL
AND EASTERN EUROPE: HAPIEE PROJECT
K. Makarenkova1 , S. Malyutina1 , G. Simonova1 , M. Bobak2 , Y. Nikitin1 ,
HAPIEE Group. 1 Institute of Internal Medicine SB RAMS, Novosibirsk, Russia,
2
Department of Epidemiology & Public Health, University College of London,
London, UK
Purpose: To analyze major lipid parameters in Siberian population in
dependence of age and gender and to compare them with similar parameters
of Czech Republic and Poland.
Material and Methods: Within the HAPIEE project during the period 2002–
2005 there have been studied a representative sample of urban population of
Novosibirsk (Russia), Krakow (Poland) and six centers of Czech Republic. Total
number of subjects was 25099.
Results: Average levels of total and LDL cholesterol within all centers in
Czech Republic, Poland and Russia were relatively high – 5.97 mmol/l and
3.75 mmol/l respectively. Acceptable values of triglycerides and HDL cholesterol
were 1.68 mmol/l and 1.46 mmol/l respectively.
The lowest levels of total and LDL cholesterol in men and women were
registered in Czech Republic – 5.7 mmol/l and 3.45 mmol/l respectively, however
triglycerides level was higher – 1.89 mmol/l, and HDL cholesterol was lower –
1.39 mmol/l. In Russia subjects of both genders have the highest levels
of total (6.29 mmol/l) and LDL (4.06 mmol/l) cholesterol, but triglycerides
level was the lowest (1.53 mmol/l) and the highest level of HDL cholesterol
(1.54 mmol/l). In Poland lipid parameters have intermediate values: average
total cholesterol – 5.84 mmol/l, triglycerides – 1.65 mmol/l, HDL-C – 1.44 mmol/l,
LDL-C – 3.65 mmol/l and respectively atherogenic indexes.
Conclusion: In Russia, compared with the Czech Republic and Poland, there
are the highest levels of total and LDL cholesterol; however the atherogenic
indexes are particularly similar because of high HDL cholesterol levels.
115 SMOKING INTENSITY MAY HINDER THE BENEFICIAL EFFECT
OF STATIN THERAPY ON CIRCULTING OXIDIZED LOW-DENSITY
LIPOPROTEIN LEVELS AND CAROTID AHTEROMATOUS
STENOSIS
S. Kougialis, E. Skopelitis, D. Levisianou, A. Zervou, T. Gialernios, N. Kosmas,
E. Mouka, A. Galanopoulou, H. Mpilinis, A. Polydorou. General Hospital of
Nikea, Nikea, Greece
Background and Aims: Oxidized low-density lipoprotein (oxLDL) is a marker
of oxidative stress in atheromatosis. Statins reduce atheromatosis. The aim
27
of the present study was to investigate possible beneficial effect of statins on
circulating oxLDL and the degree of carotid atheromatosis in relation to smoking
habits.
Materials and Methods: A total of 100 patients (76 males, median age 68
years) with carotid stenosis were enrolled in the study. Those with stenosis
>70% (n = 50) underwent angioplasty. Those with <70% (n = 50) were treated
conservatively. Both, were given atrorvastatin in doses adequate to maintain
LDL-cholesterol <100 mg/dl and were followed at 1, 3, 6 and 12 months by
measuring anthropometrics, complete lipid profile, and oxLDL in every visit.
Stenosis was evaluated by ultrasonography at baseline and 12 months.
Results: Smokers were 54 and non-smokers 46. OxLDL (60.68±24.09
vs 45.48±24.89 mg/dl, p = 0.0036) and carotid stenosis (29.68±25.59 vs
23.06±21.71%, p = 0.002) were significantly reduced at 12 months in
smokers and in non-smokers (69.33±25.11 vs 40.36±5.6 mg/dl, p = 0.001)
(24.67±26.22 vs 20.00±21.45%, p = 0.004). Smoking was divided into
subgroups (mild: 10, moderate: 10−20, severe: 20 cigarettes/day).
Significant reduction of oxLDL (48.24±8.74 vs 41.54±9.00 mg/dl, p = 0.027)
and stenosis (27.63±25.68 vs 23.42±21.74%, p = 0.009) were observed only
in mild group. In moderate and severe groups, oxLDL and stenosis were not
significantly reduced.
Conclusion: Significant reduction of oxLDL and stenosis was shown in smoking
and non-smoking patients with atheromatosis treated with statins. Yet, the
beneficial effect of statins in carotid atheromatosis was evident in mild and
retracted in moderate and severe smoking.
116 OBESITY AND METABOLIC SYNDROME
P. Hlubik1 , H. Stritecka1 , J. Hlubik2 . 1 Military Hygiene, University of Military
Health Sciences, Hradec Kralove, 2 Faculty of Electrical Engineering, CVUT,
Praha, Czech Republic
Objective: Metabolic syndrome risk level is elevated not only in patients with
established CVD, but also with DM II and obesity. The important part of
metabolic syndrome is abdominal obesity. Increase of body fat is possible to
evidence by using bio-impedance method (BIA).
Methods: Our objective was the investigation of the change of selected
anthropometrical and biochemical parameters, especially those, which are
generally used as risk indices for the origin and development of cardiovascular
diseases: BMI, waist circumference, % of body fat, serum concentrations of total
cholesterol, HDL and LDL, TAG during controlled regime of weight reduction
in 20 obese patients with arterial hypertension. Reduction was done only no
pharmacological way − modification dietary habits.
Results: Metabolic syndrome risk decreased after the loss of body weight. After
finishing the targeted weight reduction we proved that there is a statistically
significant change in anthropometrical and biochemical parameters.
Conclusions: The results of the study proved that there is a positive effect of a
targeted reduction diet on obese persons. Goal-directed reduction has positive
effect of risk of genesis and development of atherosclerosis and CVD.
Conflict of interest: None Disclosed
Funding: Research relating to this abstract was funded by MO FVZ 0000502
117 HOW IS LIPID PROFILE ALTERED AFTER CARDIAC SURGERY
R. Alves, D. Aguiar, B. Andrade, K. Nakiri. Santa Cruz Hospital, São Paulo,
Brazil
Introduction: After acute myocardium infarction or cardiac surgery can occur
alterations in the lipid profile. However, exact values have not been well
evaluated.
Objective: To evaluate the alterations that occur in the lipid profile after coronary
artery bypass grafting (CABG) and/or valve replacement.
Methods: We evaluated 21 patients, 76% males, mean age 57 years, 32 to
78 years. The patients were evaluated in the period of one to thirty days
before surgery and four to six days post-operatively (75% CABG and 25%
valve replacement). Blood sample in fasted period. Statins were used by 35%
of the patients. There were 45% of patients with dyslipidemia, 70% with arterial
hypertension, 25% with diabetes and 25% smokers.
Results: The lipid profile suffered alterations after cardiac surgery, −23%,
−10% and −10%, in the LDL-c, HDL-c, and total cholesterol concentrations,
respectively. On the other side, tryglicerides concentrations were higher,
however 1% only. There were significative differences after cardiac surgery
between groups with and without statin in the reduction of LDL-c, −32% versus
−18%. Concentrations of total cholesterol, HDL-c and tryglicerides were −10%,
−11% e 0% versus −11%, −10% e +1%, respectively.
Conclusion: Our study demonstrated that the lipid profile was altered
after cardiac surgery. The alteration was higher reduction in the LDL-c
concentration when compared to that of total cholesterol and HDL-c. However,
without modification in the tryglicerides levels. The reduction in the LDL-c
concentrations were higher in the statin group.
28
118 STUDY ON CHANGE OF VALUES OF CHOLESTEROL AND
TRIGLYCERIDES IN HYPERLIPIDEMIC PATIENTS IN RELATION
TO THE SEASON OF THE YEAR
S.S. Patiakas1 , C.C. Charalampous2 . 1 Microbiological Laboratory, General
Hospital of Kastoria, Thessaloniki, 2 Microbiological Laboratory, General
Hospital of Kastoria, Thessalonı́ki, Greece
IL6 and high sensibility C-Reactive Protein – hsCRP. In a control group of
22 subjects the same serum tests were performed.
Results: see table.
Objective: To assess the possible existence of seasonal variations in
cholesterol and triglycerides values in plasma serum of patients under antilipidemic treatment.
Material and Methods: The material of our study were 185 patients, 63
men and 122 women, followed by outpatient clinics (a) pathological clinic
of the General Hospital of Kastoria and (b) the practice lipid disorders and
atherosclerosis pathological clinic of the Psychiatric Hospital of Thessaloniki.
Of all four samples were collected: two during the winter and two during the
summer, while physical exercise and dietary habits have not changed.
Results: Results are shown in Table 1.
CT (mg/dl)
TG (mg/dl)
CHDL (mg/dl)
CHDL 2 (mg/dl)
CHDL 3 (mg/dl)
LDLox (U/L)
Lpa (g/L)
IL6 (pg/dl)
hsCRP (mg/dl)
Table 1
mg/dl
Cholesterol
Triglycerides
Average winter value
Average summer value
Men
Women
Men
Women
212.6
156.8
236.7
146.3
208.5
138.9
225.8
137.2
Conclusions: From our study a small seasonal variation is confirmed in
average cholesterol values (by 4.1 mg/dl) in men, but higher in women (by
10.9 mg/dl). Similarly, for triglycerides fluctuations was 17.9 mg/dl for men and
9.1 mg/dl for women. Possibly, therefore, it should be taken into account in
designing management-patient treatment of hyperlipidemia’s seasonal variation
of these values.
119 ATHEROGENIC INDEX OF PLASMA
[=LOG10 (TRIGLYCERIDES/HDL-CHOLESTEROL)] AND CRP DURING
CHRONIC HEMODIALYSIS TREATMENT
V. Soska, D. Sobotova. Department of Internal Medicine, Masaryk University,
Brno, Czech Republic
Background and Aims: Major cause of mortality in hemodialysed patients is
coronary heart disease (CHD). Chronic inflammation, predominance of small
LDL particles and the presence of a subpopulation of small HDL particles
poses a higher risk for CHD. The measurement of the LDL and HDL particle
sizes can be substituted by calculating of atherogenic index of plasma (AIP)
[=log10 (triglycerides/HDL-cholesterol)], which reflects the presence of small
LDLs and HDLs. The aim of the study was to identify if a long-term hemodialysis
treatment influences AIP and C-reactive protein (hs-CRP) level in patients with
chronic renal failure.
Patients and Methods: A total of 48 patients on maintenance hemodialysis
were included to the study. Their chronic dialysis protocol consisted of 3−4 hours
sessions three times a week. Total cholesterol, HDL-cholesterol (HDL-C),
triglycerides, apolipoprotein-A1 (apoA1), apolipoprotein-B (apoB) and hs-CRP
were measured (ADVIA 1650 analyser, Siemense) at the start of the study
and then after 6, 12 and 18 months of regular hemodialysis treatment. AIP,
apo-B/apo A-1 and total cholesterol/HDL-C ratios were calculated.
Results: After 18 months of regular hemodialysis hs-CRP plasma level
(11.1±1.84 mg/L vs 10.2±2.69 mg/L), total cholesterol/HDL-cholesterol ratio
(4.45±1.72 vs 4.10±1.28; p = 0.076) and apoB/apoA1 ratio (0.72±0.29 vs
0.69±0.24; p = 0.301) remained unchanged. However, the AIP increased
significantly (0.10±0.16 vs 0.22±0.19; p = 0.046).
Conclusions: The increased AIP after 18 months of regular hemodialysis
treatment demonstrates that hemodialysis-induced dyslipidemia may have
a proatherogenic nature, even though the total cholesterol/HDL-C and
apoB/apoA1 ratios remain unchanged.
120 LIPIDS AND INFLAMMATORY MARKERS IN THE ACUTE PHASE
RESPONSE OF MYOCARDIAL INFARCTION
L. Bronze1,2 , J. Azevedo1,2 , M.J. Relvas1 , M.L. Andrade2 , I. Arroja1 ,
M. Mendes1 , M. Seabra2 , G. Morais2 , A. Aleixo1,2 . 1 Unicard, Serviço de
Cardiologia, Hospital de Sao Francisco Xavier, 2 Faculdade de Ciencias
Medicas, Lisbon, Portugal
The acute phase response (APR) is a defense mechanism associated to
inflammatory markers and metabolic surrogates (such as hyperglycemia).
Purpose: To study the lipid and inflammatory markers of a group of Acute
Myocardial Infarction (AMI) patients (pts).
Methods: Upon admittance the following were obtained through peripheral
venous blood sample in 57 AMI pts: total cholesterol – TC; triglycerides – TG;
HDL cholesterol – CHDL; HDL cholesterol subfractions 2 and 3 – CHDL2 and
CHDL3; oxidized LDL cholesterol – LDLox; lipoprotein a – Lpa; Interleucin 6 –
Control group
AMI group
P
212.6±36.7
136.0±59.4
51.2±11.6
9.1±2.9
42.0±9.9
72.0±20.7
0.6±0.6
9.9±18.8
0.29±0.32
171.5±41.4
141.0±103.3
43.0±12.7
10.7±5.0
32.5±9.5
48.0±18.5
0.54±0.35
36.1±50.8
2.0±3.5
<0.0001
ns
0.006
ns
<0.0001
<0.0001
ns
0.02
0.03
Conclusion: Paradoxically the overall lipid profile is better in the AMI group,
unlike the inflammatory values which are clearly connected to the acute event.
We believe that the lipid findings reflect the catabolic trend associated to the
APR.
121 DISORDERS OF LIPIDS METABOLISM IN PATIENTS WITH
RHEUMATOID ARTHRITIS
I. Klymas. National Medical Academy of Postgraduate Education Named after
P.L. Shupyk, Kyiv, Ukraine
Introduction: Patients with rheumatic disease characterized by pronounced
inflammation. Systemic chronic inflammation in patients with rheumatoid
arthritis (RA) defines a high risk of developing metabolic disorders.
Objective: To determine the frequency of metabolic disorders of lipids in
patients with rheumatoid arthritis.
Materials and Methods: 62 patients with RA (52 women, 10 men). The average
age of patients 49.7 years. Average disease duration − 7.6 years. 20 healthy
subjects appropriate for age and gender as a control group. All patients were
observed and treated in the Kiev Regional Hospital. In studies not included
patients with established markers of viral hepatitis B, C, patients who abuse
alcohol. All patients for the diagnosis of metabolic disorders were determined:
body mass index (BMI) (obese with BMI > 30 kg/m2 ), waist size (abdominal
obesity in OT >80 cm in women and >94 cm in men), lipid metabolism (total
cholesterol, high density lipoproteins (HDL cholesterol), low density lipoprotein
(LDL cholesterol) lipoproteins very low-density (VLDL cholesterol), triglycerides
(TG)), blood pressure (<130/85 mm Hg).
Results: In RA patients BMI > 30 kg/m2 met in 37.2% in the control group −
6.2% (p < 0.05), abdominal obesity defined by 58.3% and 29.3% in group control
(p < 0.05). Lipid metabolism: total cholesterol -6.43±1.44, HDL cholesterol
-1.62±0.36, LDL cholesterol -2.98±0.74, VLDL cholesterol -0.79±0.32,
triglycerides -2.35±0.48 in patients with RA and total cholesterol -4.82±0.33,
HDL cholesterol -1.66±0.20, LDL cholesterol -2.24±0.33, VLDL cholesterol
-0.42±0.15, triglycerides -1.12±0.22 in control group (p < 0.05).
Conclusions: Disorders metabolism of lipids have a high prevalence among
patients with RA. In 37.1% patients with RA met three or more characteristics
according to NCEP criteria can be estimated as the metabolic syndrome.
122 ACUTE EXERCISE INCREASES REMNANT LIPOPROTEIN
CHOLESTEROL IN TYPE 1 DIABETES (T1DM)
M. Gonzalez1 , L. Brugnara2 , S. Murillo2 , M. Guardiola1 , A. Novials2 ,
J. Ribalta1 . 1 Unitat de Recerca en Lı́pids i Arteriosclerosis, Universitat Rovira
i Virgili, Reus, 2 Institut d’Investigacions Biomèdiques August Pi I Sunyer
(IDIBAPS), Hospital Clinic de Barcelona, Barcelona, Spain
Introduction: Remnant lipoproteins (RLP) are a well-established cardiovascular
risk factor, which can be modified by physical activity, but to date no studies
have examined their role in T1DM. Thus our aim was to investigate the effect
of exercise on RLP cholesterol concentrations in T1DM patients.
Patients and Methods: Eleven healthy men (32.7±2.8 y) and ten T1DM
patients (35.1±2.7 y), all in a normal BMI range, were enrolled in the study.
Both groups had similar cardio-respiratory capacity. The day before the study,
participants did not practice physical exercise. T1DM patients presented
HbA1c=6.9±0.9%, were in use of bedtime insulin glargine and no one
presented chronic complications related to diabetes. Fasting serum samples
were withdrawn at t = 0, at t = 30minutes (after 30 min of acute exercise (bicycle,
80% VO2max)) and t = 48 hours.
Results: At baseline, control and T1DM subjects had normal concentrations of
total cholesterol and triglycerides.
Changes in RLPc levels after 30min of acute exercise were heterogeneous and
non significant in both groups. Mean HDLc and LDLc were increased in controls
(HDLc: 5.89±2.75%, LDLc: 2.62±9.08%) and in T1DM (HDLc: 5.77±6.06%,
LDLc: 4.95±5.08%).
79th EAS Congress
However, after 48hours, the effect of exercise on RLPc was homogeneous. In
control RLPc decreased in 6 out of 11 subjects by −30.05±50.25% and in
T1DM it increased in 4 out of 10 subjects by 5.24±31.92 (p =0.091).
Conclusion: The effect of the exercise in RLPc is not observed immediately
after the exercise, but after 48 hours it promotes a decrease in normal subjects,
which is not observed in T1DM patients.
123 EFFECT OF STANDARD COMBINATION OF VALSARTAN WITH
EITHER AMLODIPINE OR HYDROCHLOROTHIAZIDE ON LDL
SUBFRACTION PROFILE IN PATIENTS WITH HYPERTENSION
L.G. Christogiannis1 , M.S. Kostapanos1 , H.J. Milionis1 , Z. Mitrogianni1 ,
E. Moutzouri1 , A.D. Tselepis2 , M.S. Elisaf1 . 1 Department of Internal Medicine,
University of Ioannina School of Medicine, 2 Laboratory of Biochemistry,
Department of Chemistry, University of Ioannina, Ioánnina, Greece
To compare the effects of standard combination of valsartan with either
amlodipine (VA) or hydrochlorothiazide (VH) on low-density-lipoprotein (LDL)
subfraction profile in hypertensive patients.
Methods: Sixty drug-naı̈ve patients with stage II or III hypertension were
randomized to either VA (160/5 mg) or VH (160/12.5 mg) treatment [VA group
(N = 30) and VH group (N = 30), respectively]. At baseline as well as 16 weeks
post-treatment blood pressure (BP), serum lipid and apolipoprotein levels were
determined. The analysis of the LDL subfraction profile was conducted by the
LDL Lipoprint System.
Results: Both drug combinations effectively reduced BP levels; no difference
between groups was observed. No effect of VA on serum lipid and
apolipoprotein levels was noted. VH was associated with a significant increase
in triglyceride and apolipoprotein-E levels [by 9.0% (p < 0.05) and 8.9%
(p < 0.05), respectively] as well as with a significant decrease in high-densitylipoprotein (HDL)-cholesterol levels (by 5.0%, p < 0.05). A significant increase in
the cholesterol concentration of small-dense LDL subfractions [6.0 (0.0–44.6)
vs 10.5 (1.3–41.3) mg/dl, p < 0.05] was observed in the VH group, whereas
this parameter did not change from baseline in the VA group (p = 0.01 for the
comparison between groups). Consequently, mean LDL particle size decreased
in the VH group (from 267±5 to 266±5 Å, p < 0.05), whereas remained
unchanged in the VA group. No change in the cholesterol concentration of
both large-buoyant and intermediate LDL subfractions was recorded in either
group.
Conclusion: Despite similar reductions in BP, VH may adversely affect lipid
and LDL subfraction profile as compared to VA.
124 APOLIPOPROTEINS A1 AND B AND THE RISK OF
CARDIOVASCULAR RECURRENCES IN ISCHEMIC STROKE
PATIENTS
S. Vidale, A. Sampietro, L. Tancredi, M. Arnaboldi. Neurology Dept., Sant’Anna
Hospital, Como, Italy
Background and Purpose: Plasma apolipoproteins has been proposed as
risk factors for cardiovascular disease. No studies analysed the contribution of
those variables in the clinical follow up in stroke patients. Aim of this study was
to evaluate the association between plasma apolipoproteins and recurrences
of cardiovascular events (CVD) in patients with stroke.
Materials and Methods: We evaluated patients admitted in hospital for
ischemic stroke between July and December 2004. Vascular risk factors, clinical
features and blood variables (lipids and apolipoproteins) were registered during
hospitalization. Follow up data were obtained by ambulatory visits and hospital
registers. Statistical analysis was performed using chi-square and t-test.
Results: 129 patients were included (M/F: 1.4/1; mean age: 70 yrs).
Hypertension was the most frequent vascular risk factor (55.8%). Mean values
of apoA and apoB were 129 mg/dL and 98, respectively. Mean follow up time
was 4.6 yrs. CVD recurrences occurred in 27 patients (20.9%). A positive
association was observed between atrial fibrillation, apoB, apoB/A1 ratio and
CVD recurrences (p < 0.05).
Conclusions: Apolipoproteins could contribute to increase the risk of
cardiovascular accidents in ischemic stroke patients.
125 LDL SIZE DETERMINANTS DURING ANTIRETROVIRAL THERAPY
INCLUDING PROTEASE INHIBITORS IN HIV-1 INFECTED PATIENTS
R. Bittar. Unité Fonctionnelle de Biochimie des Maladies Métaboliques,
Service de Biochimie Métabolique, Groupe Hospitalier Pitié-Salpétrière,
Assistance Publique-Hôpitaux de Paris, Paris, France
Background: Lipid disorders are frequent in HIV1-infected patients taking
antiretroviral combinations that include protease inhibitors (PI). Hypertriglyceridemia in this population is frequently associated with increased small dense
LDL. The LDL concentration and particle size might be an important predictive
marker of cardiovascular disease in this setting.
Objective: To evaluate variables that influences the quantity and size of LDL
in a substudy of the ANRS 126 trial.
Methods: We studied 81 HIV1-infected patients with dyslipidemia (LDLcholesterol >4.1 mmol/l, triglycerides <8.8 mmol/l) who had been taking PI-
29
including cART for at least two months, and who were not taking lipidlowering drugs. Total cholesterol (TC), triglycerides (TG), LDL-cholesterol
(LDL-c), and HDL-cholesterol (HDL-c) were assayed in serum. LDL diameter
was assessed by gradient gel electrophoresis (GGE). Relationships between
the LDL diameter, and demographic metabolic and HIV-related variables, were
identified by using non parametric univariate tests and multiple linear regression
models.
Results: In univariate analyses, LDL diameter was related to gender, ethnic
origin, CDC stage, TG, HDL-c, duration of exposure to and numbers of
NRTIs and PIs. In a multivariable linear regression model, LDL diameter was
independently associated negatively with TG (P < 0.0001) and positively with
HDL-c (p < 0.0001). Per 1 mmol/L increase in TG, the LDL diameter decreased
by 0.281 nanometers. Conversely, per 1 mmol/L increase in HDL-c, the LDL
diameter increased by 1.175 nanometers.
Conclusion: Based on the LDL size, an atherogenic phenotype was
significantly related to TG and HDL-c concentrations. LDL diameter might help
predict and monitor the cardiovascular risk (CVD) in HIV1infected patients.
126 CARDIOVASCULAR RISK IN PATIENTS WITH TYPE 2 DIABETES
MELLITUS WITH OR WITHOUT METABOLIC SYNDROME
J. Millan1 , C. Garcia Calzado2 , C. Recarte3 . 1 Internal Medicine, Hospital GU
Gregorio Marañón, Madrid, 2 Endocrinology, Universidad de Cadiz, Cadiz,
3
Internal Medicine, Universidad Complutense, Madrid, Spain
Diabetes Mellitus is associated with a high cardiovascular morbi-mortality.
This feature is related with macrovascular and microvascular complications.
Metabolic syndrome, a clinical condition with high risk that is present frequently
in diabetic patients may be a factor that increase the risk.
This study was made to detect differences in the estimated risk in diabetic
patients depending of presence/absence of metabolic syndrome.
We have studied a group of patients with type 2 DM (n = 80) that were attended
in primary care. Diagnosis of metabolic syndrome was made according with
ATPIII criteria and IDF criteria. Cardiovascular risk was calculated according
with Framingham scale.
In patients with tipo 2 DM metabolic syndrome was found in 47.5% (ATPIII) and
49% (IDF). Cardiovascular risk (Framingham) up to 20% was present in 28.8%
of patients with and in 16.6% of patients without metabolic syndrome according
with ATPIII.
Men with diabetes show metabolic syndrome in 41.9% of cases with
Framingham >20%; but only 24.9% of diabetic men without metabolic syndrome
show Framingham >20%. In women, 21% of diabetics with metabolic syndrome
show Framingham >20%, and in diabetic women without metabolic syndrome
we have not found any patients with Framingham >20%.
We can conclude that the Framingham score is available to show the increased
risk attributable of metabolic syndrome in patients with diabetes mellitus.
127 CONSTITUTIVE INHIBITION OF PLASMA CHOLESTERYL ESTER
TRANSFER PROTEIN (CETP) BY APOLIPOPROTEIN C1 IN
NORMOLIPIDEMIC, BUT NOT IN HYPERLIPIDEMIC PATIENTS WITH
CORONARY ARTERY DISEASE. CONSEQUENCES ON PLASMA
CHOLESTEROL DISTRIBU
X. Pillois1 , T. Gautier2 , J.-P. Pais de Barros2 , A. Jeannin2 , J. Bonnet1,3 ,
L. Lagrost2,4 . 1 University Victor Segalen of Bordeaux II − INSERM Research
Center UMR828, Bordeaux, 2 University of Burgundy − INSERM Research
Center UMR866, Dijon, 3 University Hospital of Bordeaux, Bordeaux,
4
University Hospital of Dijon, Dijon, France
Objectives: Plasma cholesteryl ester transfer protein (CETP) promotes the
cholesterol enrichment of triglyceride-rich lipoproteins at the expense of HDL.
In earlier studies, HDL was found to inhibit CETP activity in a concentrationdependent manner. We were able to show that, among all the apolipoprotein
components of HDL, apolipoprotein (apo) C1 is a potent inhibitor of CETP
in vitro. In vivo studies in apoC1-knocked out/human CETP transgenic and
human apoC1 transgenic/human CETP transgenic mice came in direct support
of the inhibitory effect of human apoC1 that was initially observed in vitro.
Accordingly, we found a negative correlation between plasma concentrations
of apoC1 and levels of CETP activity in healthy, normolipidemic subjects. Our
goal was to establish whether modulation of cholesteryl ester transfer protein
(CETP) activity by apolipoprotein C1 (apoC1) can modify plasma cholesterol
transport in humans and whether it is influenced by hyperlipidemia in high-risk
patients.
Methods: Plasma CETP activity, apoC1 concentration, and lipoprotein profile
were determined in 240 patients with documented Coronary Heart Disease.
Results: ApoC1 levels correlated negatively with CETP activity in the total
population studied. However, this negative relationship was observed in
normolipidemic patients only, but not in those with hyperlipidemia (LDL
cholesterol >2.6 mmol/l and/or triglycerides >1.7 mmol/l). As a consequence
of CETP inhibition, and in the former group only, a high apoC1 level was
accompanied with higher HDL to LDL cholesterol ratio.
30
Conclusion: apoC1 is a constitutive inhibitor of CETP in normolipidemic
humans, but it no longer operates in hyperlipidemic patients with CAD.
128 ARAP2-INDUCED CHANGES IN SPHINGOLIPID BIOSYNTHESIS
PROMOTE LIPID DROPLET FORMATION BY INCREASING GLUT1
LEVELS IN THE PLASMA MEMBRANE
L. Li1 , R. Mobini1 , E. Lu1 , M. Rutberg1 , M. Ståhlman1 , L. Håversen1 , B. Liu1 ,
T. Larsson1 , R. Perkins1 , L. Andersson1 , K. Koistinen2 , K. Ekroos2 , J. Borén1 ,
S.-O. Olofsson1 . 1 Sahlgrenska Center for Cardiovascular and Metabolic
Research, Wallenberg Laboratory, University of Gothenburg, Gothenburg,
Sweden, 2 Zora Biosciences Oy, Espoo, Finland
Lipid droplets are organelles involved in the storage and processing of neutral
lipids such as triglycerides, and recent evidence suggests a close interaction
between lipid droplets and specialized domains of the plasma membrane known
as lipid rafts.
Here we identified ARAP2 as a lipid droplet-associated protein that increases
the formation of lipid droplets by promoting triglyceride biosynthesis. ARAP2
increased basal glucose uptake and GLUT1 levels in the lipid raft fraction
of the plasma membrane, which likely explains the increased triglyceride
biosynthesis. We also showed that ARAP2 influenced the lipid composition
of cells and the lipid raft fraction by promoting sphingomyelin biosynthesis
and reducing glucosylceramide biosynthesis. Furthermore, we showed that
ARAP2 could mediate these effects by inhibiting the activity of glucosylceramide
synthase. Inhibition of this enzyme promoted the activity of plasma membrane
sphingomyelin synthase 2 and the accumulation of GLUT1 in the lipid raft
fraction of the plasma membrane. We also showed that palmitic acid increased
the expression of ARAP2 and GLUT1 levels in the lipid raft fraction.
Our data indicate that GLUT1 levels in lipid rafts, and hence basal glucose
uptake and triglyceride biosynthesis, can be increased by an ARAP2-induced
reduction in GCS activity and in the glucosylceramide biosynthesis. We
therefore propose that ARAP2 is part of a mechanism that protects cells from
lipotoxic fatty acids by increasing basal glucose uptake to promote lipid droplet
formation.
129 A NOVEL THREE-PROTEIN COMPLEX REGULATES IN VITRO
VTV-GOLGI FUSION
S.A. Siddiqi, E. Nafi-Valencia, S. Siddiqi, A. Rahim. Burnett School of
Biomedical Sciences, University of Central Florida, Orlando, FL, USA
Elevated concentrations of circulating plasma very low-density lipoproteins
(VLDLs) play an important role in the pathogenesis of atherosclerosis. VLDLs
are synthesized in the liver and secreted into the blood. The rate-determining
step in the secretion of VLDLs from the liver is their transport from their
site of biogenesis, the endoplasmic reticulum (ER), to the Golgi. We reported
recently that this physiologically regulatable step is mediated by a specialized
ER-derived VLDL transport vesicle (VTV). VTV fuses with hepatic cis-Golgi to
facilitate the targeted delivery of VLDL to the Golgi lumen. In vitro fusion of VTV
with hepatic cis-Golgi requires cytosol, however, cytosolic factors regulating
fusion event remain unidentified. To identify cytosolic factors, we performed a
series of chromatographic steps including FPLC on liver cytosol and isolated
a complex of three cytosolic proteins, which we found is required for in
vitro VTV-Golgi fusion. Tandem mass spectrometry coupled with N-terminal
sequencing analyses identified the proteins as hsp72, p97 and hsp27. Western
blotting data using antibodies to hsc70, p97 and hsp27 confirmed their
identity. To examine the functionality of these proteins in VTV-Golgi fusion,
we immunodepleted hepatic cytosol of hsc70, p97 and hsp27 using specific
antibodies. Immunodepletion of each of the three proteins resulted in significant
reduction in VTV-Golgi fusion. Addition of recombinant hsc70, p97 and hsp27
proteins to the immunodepleted-cytosol completely restored the fusion activity.
We conclude that hsc70, p97 and hsp27 form a complex in cytosol, which
regulates the fusion VTV with hepatic cis-Golgi and thus VLDL-delivery to the
Golgi lumen.
130 MURINE BONE MARROW-DERIVED MACROPHAGES
DIFFERENTIATED WITH GM-CSF BECOME FOAM CELLS BY PI
3-KINASE GAMMA-DEPENDENT FLUID-PHASE PINOCYTOSIS
OF NATIVE LDL
J.J. Anzinger1 , J. Chang1 , Q. Xu1 , T. Bohnacker2 , M.P. Wymann2 , H.S. Kruth1 .
1
Experimental Atherosclerosis Section, NHLBI, NIH, Bethesda, MD, USA,
2
Department of Biomedicine, Institute of Biochemistry and Genetics, University
of Basel, Basel, Switzerland
Accumulation of cholesterol by macrophage LDL uptake is a key event in the
formation of atherosclerotic plaques. Previous research has shown that GMCSF differentiates macrophages, is present in atherosclerotic plaques, and
promotes plaque lipid accumulation. However, it has not been determined
if murine GM-CSF-differentiated macrophages take up LDL to become
cholesterol-rich foam cells. In this study, murine bone marrow-derived cells were
differentiated into macrophages with GM-CSF. These GM-CSF differentiated
macrophages showed massive, progressive accumulation of cholesterol during
incubation with LDL. Incubation of LDL receptor-null or wild-type macrophages
with increasing concentrations of 125 I-LDL showed non-saturable macrophage
LDL uptake that was linearly related to the LDL amount added, indicating
receptor-independent LDL uptake mediated by fluid-phase pinocytosis. Since
previous studies showed an important role for PI3K in atherosclerosis
development and macrophage fluid-phase pinocytosis, we investigated the role
of PI3K in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type
macrophages incubated with LDL and the PI3K-gamma inhibitor AS605240
showed a ~50% reduction in LDL uptake and net cholesterol accumulation
compared with macrophages incubated with LDL only. Incubation of PI3Kgamma-null macrophages with LDL showed a similar ~50% reduction in LDL
uptake and net cholesterol accumulation compared with wild-type macrophages
incubated only with LDL. These results show that GM-CSF-differentiated murine
macrophages become cholesterol-rich foam cells by fluid-phase pinocytosis
of native LDL and identify PI3K-gamma as contributing to this process.
PI3K-gamma-dependent macrophage foam cell formation mediated by fluidphase pinocytosis provides mechanistic insight for the decreased vascular lipid
accumulation previously observed during atherosclerosis development in mice
lacking PI3K-gamma.
131 LIPID DROPLETS: STRUCTURE, PROTEIN ORGANIZATION AND
BIOGENESIS
H. Robenek1 , N.J. Severs2 , G. Weissen-Plenz1 , A. Ruebel1 , I. Buers1 . 1 Cell
Biology and Ultrastructure Research, Leibniz-Institute for Arteriosclerosis
Research, Univ. Muenster, Münster, Germany, 2 Heart and Lung Institute,
Imperial College London, London, UK
An entirely new view of lipid droplets is emerging: lipid droplets are not merely
storage depots for superfluous intracellular lipids in times of hyperlipidic stress,
but metabolically active organelles involved in cellular homeostasis. Virtually
all recent advances in our concepts on the metabolic functions of lipid droplets
have come from studies on lipid droplet-associated proteins. This realization has
made the study of proteins, such as PAT family proteins, caveolins and several
others that are targeted to lipid droplets, an intriguing and rapidly developing
area of intensive inquiry. Despite the progress in lipid droplet research,
however, the lipid droplet is still a poorly understood organelle. Our existing
understanding of the structure, protein organization and biogenesis of the lipid
droplet has relied heavily on microscopical techniques that lack resolution and
the ability to preserve native cellular and protein composition. The electron
microscopic technique, freeze-fracture replica immunogold labeling, overcomes
these disadvantages and can be used to define at high resolution the precise
location of lipid droplet-associated proteins in specific membrane systems
and organelles of the cell. In this presentation illustrative examples of how
freeze-fracture immunocytochemistry has contributed to our understanding of
the identification, spatial organization in the membrane plane as well as the
dynamic cellular processes and function of PAT family proteins and caveolin-1
are shown. By revisiting the lipid droplet and its associated proteins with
freeze-fracture immunocytochemistry, new perspectives have emerged which
challenge prevailing concepts of lipid droplet biology and may hopefully provide
a timely impulse for many ongoing studies.
132 TRIACYLGLYCEROL-RICH LIPOPROTEINS INFLUENCE ON
THE INACTIVATION OF LIPOPROTEIN LIPASE BY ANGPTL 3 AND
ANGPTL 4
S. Nilsson1 , M. Larsson1 , E. Worrsjö1 , A. Lookene1,2 , V. Suokonina1,3 ,
E. Makoveichuk1 , J. Heeren4 , G. Olivecrona1 . 1 Physiological Chemistry, Umeå
University, Umeå, Sweden, 2 Tallinn University of Technology, Tallinn, Estonia,
3
Biomedicine, University of Gothenburg, Göteborg, Sweden, 4 IBMII: Molecular
Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg,
Germany
Objectives: Angiopoietin-like proteins (ANGPTL3 and -4) are present both
in tissues and in blood and have been found to modulate the activity of
LPL. We wanted to investigate if lipoproteins influence on this interaction
and if ANGPTL3 and -4 could influence LPL-dependent, receptor-mediated
endocytosis of lipoproteins by primary hepatocytes.
Methods: LPL activity was measured after pre-incubation in the presence
of ANGPTL3 or -4 with or without chylomicrons, VLDL or LDL. Binding of
ANGPTL3 and -4 to the lipoproteins was investigated by SPR and ELISA.
LPL-dependent uptake of 125 I-labeled triacylglycerol-rich lipoproteins (TRLs) in
hepatocytes was investigated in the presence or absence of ANGPTL3 and -4.
Results: TRLs significantly reduced the inactivation of LPL by ANGPTL3 and -4,
while LDL had less effect on the inactivation. LPL bound to the TRLs, while
ANGPTLs did not bind. The ANGPTLs were also found to decrease LPLdependent uptake of 125 I-labeled TRLs in primary hepatocytes.
Conclusions: Our data show that LPL is protected from inactivation by
ANGPTLs already at sub-physiological levels of TRLs, implying that circulating
ANGPTLs may have little effect on LPL at the luminal side of the endothelium.
This may explain why there is little correlation between plasma concentrations
of ANGPTLs and plasma TG levels or LPL activity in tissues. Our data
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support the view that ANGPTLs regulate LPL activity early during its secretion
process to the endothelium, probably in sub-endothelial compartments where
the concentration of TRLs is low.
133 THE GAIN OF FUNCTION D374Y-PCSK9 MUTANT CAN BE
FUNCTIONALLY INHIBITED IN VITRO BY AN EGF-A PEPTIDE
A.B. Cefalu’1 , D. Noto1 , E. Pinotti2 , V. Valenti1 , P. Tarugi2 , M.R. Averna1 .
1
Clinical Medicine and Emerging Diseases, University of Palermo, Palermo,
2
Department of Biomedical Sciences, University of Modena and Reggio
Emilia, Modena, Italy
Introduction: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to
low density lipoprotein receptor (LDLR) and induces its internalization and
degradation. PCSK9 binding to LDL-R is mediated through the LDL-R epidermal
growth factor-like repeat A (EGF-A) domain. It has been shown that a EGF-A
peptide inhibits PCSK9-mediated degradation of LDL-R in HepG2 cells.
Objectives: The aim of this study was to evaluate the effect of a synthetic
EGF-A peptide on the LDL-R protein levels in two different hepatic cell lines
(HepG2 and HuH7) transiently overexpressing a gain of function mutation of
PCSK9.
Methods and Results: A 40mer synthetic peptide of EGF-A domain customsynthesized was added to the medium of HepG2 and HuH7 cells transiently
overexpressing the gain-of-function D374Y PCSK9 mutant which has been
associated to an autosomal dominant hypercholesterolemia (ADH3) in humans.
D347Y-PCSK9-mediated LDL-R degradation was determined by Western
blotting of cell lysates, showing a >85% LDL-R protein levels reduction.
In HepG2 and HuH7 cells the treatment with media containing increasing
concentrations of the EGF-A peptide (in the range of 12.5−50 mM) for 18 h
restored the LDL-R protein levels.
Conclusions: In summary, these data demonstrate that a synthetic EGF-A
peptide is able to efficiently inhibit in vitro the degradation of LDL-R mediated
by a gain of function PCSK9 mutant. This data confirm that the inhibition of
the extracellular interaction between PCSK9 and the LDLR by using a decoy
strategy could represent a valid therapeutic approach for the treatment of
Hypercholesterolemia in humans.
134 PCSK9 IN FAMILIAL HYPERCHOLESTEROLEMIA (FH)
G. Lambert1,2 , K.-A. Rye2 , J. Burnett3 , P. Barter2 , D. Marais4 . 1 Inserm U957,
Nantes, France, 2 Heart Research Institute, Sydney, NSW, 3 Royal Perth
Hospital, Perth, WA, Australia, 4 Groote Schuur Hospital, Capetown, South
Africa
Introduction: In normolipaemic individuals, circulating PCSK9 and LDL-C
levels are positively correlated. It is not known whether elevated PCSK9 levels
constitute a greater risk in people who already have reduced LDLR levels such
as patients with FH.
Methods and Results: PCSK9 levels were measured by ELISA in samples
from normolipaemic controls (n = 152) and in FH patients carrying either a
D206E (n = 237) or a V408M (n = 117) mutation on their LDLR. We also
transfected pCMV-LDLR either wild type, D206E, or V408M in HEK-293 cells
expressing PCSK9 or not.
Plasma PCSK9 levels ranged from 25 to 700 ng/mL in all groups. We found
a positive correlation between plasma PCSK9 and LDLC levels in D206E and
control individuals but not in V408M patients. This correlation was significantly
(p < 0.02) more pronounced in D206E (B = 0.6) vs. controls (B = 0.3). We also
demonstrated by western blot and flow cytometry that unlike LDLR wild type
or D206E, the V408M mutant is absent from the plasma membrane and that
its expression is not modulated by PCSK9. Accordingly, cells expressing the
V408M mutant were the least able to bind 125I-labelled LDL compared with cells
expressing D206E or wild-type LDLR, and primary lymphocytes isolated from
V408M heterozygous carriers were insensitive to rPCSK9, unlike lymphocytes
from control or D206E heterozygous patients.
Conclusion: This study demonstrates that circulating PCSK9 levels account
more for the variation in LDLC levels that is observed in some but not all
heterozygous FH subjects vs. normolipaemics and that elevated PCSK9 is
highly detrimental in those patients.
135 RIP2 LINKS IMMUNE SIGNALING AND SUBENDOTHELIAL LIPID
ACCUMULATION BY REGULATING TLR4-DEPENDENT LIPID
UPTAKE IN MACROPHAGES
M. Levin1 , P. Jirholt1 , M. Johansson1 , A. Wramstedt1 , A. Lundberg2 ,
M. Ståhlman1 , P. Fogelstrand1 , M. Brisslert1 , Z.-Q. Yan2 , G. Hansson2 ,
H. Björkbacka3 , S.-O. Olofsson1 , J. Borén1 . 1 Gothenburg University,
Gothenburg, 2 Karolinska Institute, Stockholm, 3 Lund University, Lund,
Sweden
Lipid accumulation and inflammation are closely linked in atherogenesis.
However, the molecular mechanisms that link retention of atherogenic
lipoproteins and activation of the inflammatory response are still unclear. Recent
evidence implicates a key role for the innate immune system and pathogen
pattern-recognition receptors, in particular the Toll-like receptors (TLRs) and the
31
nucleotide-binding oligomerization domain (Nod)-like receptors. Ligand binding
to these receptors results in activation of the proinflammatory transcription
factor NF-kB and expression of pro-inflammatory molecules. The receptorinteracting protein 2 (Rip2) is a serine/threonine kinase that is involved multiple
NFkB activation pathways, including TLRs and Nod-like receptors. It was
recently demonstrated that the regulation of Rip2 involves a novel feed-forward
regulatory mechanism. These studies thus suggest a therapeutic potential of
inhibiting Rip2 to inhibit inflammation and thus protect against the development
of atherosclerosis.
In this study we investigated the role of Rip2 in mice prone to developing
atherosclerosis. Unexpectedly, we observed that mice transplanted with Rip2−/−
bone marrow displayed increased subendothelial lipid accumulation in the
aorta despite impaired immune signaling. We also showed that lipid uptake
was increased while immune signaling was reduced in Rip2−/− macrophages.
Further analysis in Rip2−/− macrophages showed that the lipid accumulation
scavenger-receptor independent and instead mediated by TLR4-dependent
cytoskeletal rearrangements and macropinocytosis.
In conclusion, our work identifies Rip2 as a novel player in modulating
macrophage lipid metabolism and cardiovascular disease. Our results yield
important insights into the underlying association between lipid accumulation
and inflammation in atherogenesis.
136 REDUCED ATHEROSCLEROTIC PLAQUE FORMATION IN APOE−/−
MICE LACKING PERILIPIN
P.K. Doddapattar1 , E. Aflaki1 , B. Radovic1 , N. Rathke1 , R.B. Gruenberger2 ,
D. Kratky1 . 1 Institute of Molecular Biology and Biochemistry, 2 Center for
Medical Research, Graz, Austria
Intracellular lipid droplet (LD) formation, neutral lipid hydrolysis and lipid release
are processes tightly regulated by intracellular hydrolases and regulatory
proteins. Foam cells store cholesterol and fatty acids within cholesteryl
esters (CE) and triacylglycerol (TG). Compared to adipocytes, in which LD
formation and degradation has been studied in more detail, only few data are
available regarding macrophage and foam cell-derived LDs. Here, we aimed
at elucidating the consequences of Plin deficiency in murine macrophages on
foam cell formation and atherosclerotic plaque development.
Conflicting data have been published on perilipin expression in human
macrophages and foam cells. Utilizing Plin−/− and wild-type (Wt) mice, we
investigated the differences in macrophage and foam cell morphology and
function between both genotypes. Interestingly, the number of LDs in Plin−/−
foam cells after incubation with acetylated (ac)LDL was found to be lower
compared to foam cells from Wt mice indicating a regulatory role of Plin in
LD formation of macrophages. To examine whether observed differences in
LDs could affect atherosclerosis, we challenged apolipoprotein E (apoE)−/−
and Plin−/− apoE−/− mice with western type diet and performed athero-assays.
Plin−/− apoE−/− mice had reduced plaque formation compared to apoE−/−
mice. Compared to foam cells from Wt mice, Plin−/− foam cells showed
decreased expression of ADRP, which was reported to result in reduced foam
cell formation.
Conclusion: Together these data suggest that Plin has a regulatory role
not only in adipocytes but also in macrophages. Further work is needed to
understand the underlying mechanism.
137 DYNASORE, A DYNAMIN GTPASE INHIBITOR, BLOCKS LATE
ENDOSOMAL/LYSOSOMAL CHOLESTEROL TRAFFICKING IN
HUMAN MACROPHAGES
E. Girard1 , J.-L. Paul1,2 , P. Beaune2 , C. Lamaze3 , B. Védie2 . 1 Laboratoire
de Biochimie Appliquée EA 4529, UFR de Pharmacie, Université Paris-Sud
11, Châtenay-Malabry, 2 Service de Biochimie, Hopital Europeen Georges
Pompidou, 3 Laboratoire ‘Trafic, Signalisation et Ciblages Intracellulaires’ UMR
144 Curie/CNRS, Institut Curie, Paris, France
Dynamin is a GTPase responsible of the spin off of vesicles and clathrin coated
pits from the plasma membrane. We have previously shown that dynamin
is involved in cholesterol egress from the endolysosomal compartment to
the endoplasmic reticulum (ER). We used the small drug, dynasore, which
inhibits the dynamin GTPase activity, to better understand the role of late
endosomal cholesterol trafficking in macrophages, which play a central role
in the pathogenesis of atherosclerosis. The endolysosomal network is the
intracellular compartment where endocytosed low-density lipoproteins (LDL) are
degraded. Free cholesterol must leave this compartment to reach the ER, where
transcriptional control of sterol-sensitive genes and cholesterol esterification
occur. In HMDM (human monocyte derived-macrophages), we observed by
immunofluorescence that dynasore induced the accumulation of acetylated
LDL (AcLDL) in enlarged late endosomes/lysosomes. This accumulation results
in the complete block of sterol-sensitive genes expression downregulation as
monitored by real time quantitative RT-PCR. There was also a partial inhibition
of cholesterol esterification. We showed that the increase in mRNA levels of
both cholesterol transporters ABCA1 and ABCG1 following cholesterol loading
with AcLDL was abolished by dynasore treatment with a decreased capacity of
32
macrophages to promote mass efflux of cellular cholesterol to lipid-free apoA1
and HDL, respectively. Our data therefore indicate that dynasore represents a
new interesting drug for studying cholesterol intracellular trafficking.
138 CHOLESTEROL LOADING OF HUMAN MACROPHAGES REDUCES
GROWTH FACTOR-INDUCED HETEROGENEITY OF GENE
EXPRESSION INVOLVED IN FOAM CELL FORMATION
J. Lappalainen1 , N. Yeung1 , M. Jauhiainen2 , P.T. Kovanen1 , M. Lee-Rueckert1 .
1
Wihuri Research Institute, 2 National Institute of Health and Welfare, Helsinki,
Finland
Background: Distinct subpopulations of macrophages have been identified
in the human arterial intima, with varying effects on the development
of atherosclerosis. Granulocyte-macrophage colony-stimulating factor (GMCSF) and macrophage colony-stimulating factor (M-CSF) are hematopoietic
growth factors that regulate the generation and function of monocyte-derived
macrophages.
Methods: A gene expression profile related to cholesterol uptake and efflux,
and the cholesterol contents were analyzed in cultured human macrophages
differentiated with either GM- or M-CSF, and after pre-exposure to acetylated
LDL in the absence or presence of cholesterol acceptors.
Results: Macrophages differentiated with M-CSF expressed higher mRNA
levels of CD36 and SR-AI, the two scavenger receptors for modified
LDL. This correlated with a greater ability of macrophages to accumulate
cholesteryl esters in response to incubation with acetylated LDL. Macrophages
differentiated with GM-CSF, again, showed higher expression of SR-BI and
ABCG1 mRNA, two receptors capable of mediating cholesterol efflux. However,
such divergent gene expression profile favoring a pro-atherogenic role for
M-CSF, rather than for GM-CSF, was attenuated in the foam cell phenotype.
Furthermore, HDL-2 and human blood plasma induced similar degrees of
cholesterol efflux in both types of macrophages.
Conclusions: A protective compensatory upregulation of some components
of the cholesterol efflux machinery was observed in the M-CSF-subtype of
macrophages upon foam cell formation. Thus, the gene profile of human
macrophages is influenced, in addition to growth factors, also by the relative
availability of modified LDL particles and of HDL particles. Ultimately, the
composite effect of growth factors and lipoproteins determine cholesterol
balance of the macrophages.
139 LOW DENSITY LIPOPROTEIN ENHANCES C-REACTIVE
PROTEIN-MEDIATED FCg RECEPTOR SIGNAL TRANSDUCTION
IN MACROPHAGES BUT NOT IN MONOCYTES
K. Li1,2 , M. Radermacher1 , M. Kächele1 , C. Röcker1 , A. Pott1 , D. Manolov1 ,
W. Ito3 , U. Nienhaus1,4 , J. Torzewski1,3 , O. Zimmermann1 , J. Wiehe1 ,
M. Bienek-Ziolkowski1 , H. Ruland1 , K. Vogt1 . 1 University of Ulm, Ulm,
Germany, 2 Sino-German Heart Center, Shanghai, China, 3 Cardiovascular
Center Oberallgäu, Immenstadt, 4 Institue of Applied Physics (KIT) Karlsruhe,
Karlsruhe, Germany
Background and Aims: C-reactive protein (CRP) and low density lipoprotein
(LDL) are risk markers for cardiovascular disease. Both of them deposit in the
arterial wall during atherogenesis. CRP binds to LDL and has been implicated
to play a pivotal role in cardiovascular disease. Previous reports proposed that
FcgRI and FcgRIIa are the major receptors for CRP. Syk kinase is essential
for FcgR signaling. Our objectives is to examine whether CRP can trigger Syk
kinase phosphorylation in human monocytes and macrophages and whether
this effect can be enhanced by the involvement of LDL.
Methods: CRP-LDL complex formation was evaluated by fluorescence
correlation spectroscopy. CRP interacts with LDL in solution with a equilibrium
dissociation constant of 8.0 mM. Peripheral human blood monocytes (PBMC)
and mature macrophages were exposed to CRP and LDL. Equal amounts
of protein were assayed by immunoblotting in order to investigate Syk kinase
phosphorylation.
Results: Induction of Syk kinase phosphorylation was not observed in
monocytes. In macrophages, however, Syk kinase phosphorylation in response
to CRP (100 mg/mL) is equal to that induced by human IgG. Significant, and
CRP dose-dependent, induction of the signal was observed in response to the
CRP-LDL complex (50 mg/mL LDL). LDL by itself (50 mg/mL) only induced trivial
Syk kinase signaling.
Conclusions: Our experiments suggest that human CRP-mediated tyrosine
phosphorylation of the immunoreceptor-tyrosine based activation motif (ITAM)
of Syk kinase in human macrophages is enhanced by LDL. CRP may recognize
human tissue-deposited LDL as a foreign antigen.
140 IMPACT OF OMEGA 3 AND OMEGA 6 FATTY ACIDS MEMBRANE
INCORPORATION ON CHOLESTEROL EFFLUX FROM CULTURED
RAT CARDIOMYOCYTES
N. Fournier1,2 , A. Reboulleau1 , V. Robert1 , B. Vedie2 , J.-L. Paul1,2 ,
A. Grynberg1 . 1 Laboratoire de Biochimie, UMR INRA 1154, Faculte des
Sciences Pharmaceutiques, Universite Paris-Sud 11, Chatenay-Malabry,
2
Laboratoire de Biochimie, Hopital Europeen Georges Pompidou, Paris,
France
Although cholesterol-rich microdomains are highly involved in the functions of
cardiomyocytes, the cholesterol (C) homeostasis is largely unknown in these
cells. We previously reported that C efflux to apolipoprotein AI (apo AI) or to HDL
is primarily mediated by ATP Binding Cassette ABCA1 and ABCG1 transporters,
respectively, both being upregulated by LXR/RXR activation (unpublished data).
We investigated the impact of omega 3 (docosahexaenoate) and omega
6 (arachidonate) polyunsaturated fatty acids (PUFAs) membrane enrichment
(80 mM, 72 h) on isotopic C efflux from cultured rat cardiomyocytes. The C
efflux was appreciated by measuring the cellular [3 H] free C release from
primary cultures of cardiac myocytes obtained from 2−4 days-old Wistar rats
to the extracellular acceptors after 4 h of incubation. Under basal conditions,
docosahexaenoate membrane incorporation induced a significant increase of
C efflux to apo AI (25%) whereas arachidonate induced a significant decrease
of C efflux to both apo AI (25%) and HDL (14%). Under activated conditions,
only the ABCA1 activity remained slightly although significantly increased by
docosahexaenoate (8%) and decreased by arachidonate (10%). Moreover, the
ABCA1 and ABCG1 mRNA expression was not affected by PUFA treatments.
These results show that omega 3 supply to cardiomyocytes may be beneficial
on C homeostasis by enhancing C efflux active processes whereas omega 6
supply may be deleterious, likely by modulating the chemo-physical properties
of the cellular membrane. We are currently quantitating ABCA1 and ABCG1
protein levels as well as their cellular localization to investigate the potential
mechanism(s) by which PUFA affect the ABC functions.
141 NEW LMF1 VARIANTS IDENTIFIED IN HYPERTRIGLYCERIDEMIC
PATIENTS
I. De Castro-Orós1,2 , E. Ros3 , F. Civeira2,4 , M. Pocovı́1,5 . 1 Dpto. Bioquı́mica,
Biologı́a Molecular y Celular, University of Zaragoza, 2 RECAVA, Instituto
Aragonés de Ciencias de la Salud, Zaragoza, 3 Unitat de Lipids, Servei
d’Endocrinologia i Nutrició. Institut d’Investigacions Biomèdiques August Pi
Sunyer (IDIBAPS), Hospital Clı́nic de Barcelona and Ciber Fisiopatologı́a de la
Obesidad y Nutrición (CIBERobn), Barcelona, 4 Unidad de Lipidos. Laboratorio
de Investigación Molecular, Hospital Universitario Miguel Servet, 5 CIBERER,
Zaragoza, Spain
Lipase maturing factor 1 (LMF1) plays an essential role in the formation of
catalytically active Lipoprotein Lipase (LpL). Mutations in LMF1 gene have been
described as cause of combined deficiency lipase affecting the enzyme activity
for both LpL and Hepatic Lipase and leading to severe hypertriglyceridemia
(HTG). The aim of our study was to identify the contribution of LMF1 gene
in severe HTG. For this purpose, we have selected 37 unrelated subjects
with severe forms of HTG, previously analysed for LPL, APOA5, APOC2
and GPIHBP1 genes. The LMF1 11 exons and the boundaries exon-intron
were sequenced. We have identified 9 heterozygote variants not previously
described. The R450W (exon 9) variant was found in a 48 y.o. male with TG:
3,757 mg/dl affected of a pancreatitis episode. The L44L (exon 2) was found
in a 31 y.o. male with TG: 3,584 mg/dl with pancreatitis and diabetes. The
R350Q was found in a 7 y.o. girl with TG: 346 mg/dl. Two subjects with TG
levels >3,000 mg/dl showed combined variants in heterozygosis: K520X and
L552R; R100T and A286A, respectively. Other two variants, A251A (exon 6)
and R353W (exon 7), were found in HTG subjects and also in normolipaemic
individuals, indicating these variants such plausible polymorphisms.
In conclusion, we have identified 9 new LMF1 variants in HTG patients which
must be further studied in order to determine its contribution to severe HTG.
142 REGULATION OF LIVER AND ADIPOSE TISSUE LIPOGENESIS
IN HUMAN OBESITY
J. Heeren1 , M. Vossen1 , T. Scherer2 , L. Eissing1 , U. Knippschield3 ,
K. Toedter1 , C. Buettner2 , A. Wolf3 , L. Scheja1 . 1 IBMII: Molecular Cell Biology,
Hamburg, Germany, 2 The Mount Sinai Medical Center, New York, NY, USA,
3
University Hospital of Ulm, Ulm, Germany
Objective: De novo lipogenesis (DNL) and DNL-associated fatty acids derived
from adipose tissue have been shown to improve systemic insulin sensitivity
in rodents while increased hepatic DNL is generally associated with insulin
resistant states. DNL is down-regulated in adipose tissue of obese rodents,
thereby contributing to systemic insulin resistance. Here we investigated
whether human obesity is associated with impaired DNL in adipose tissue
and how adipose tissue and hepatic DNL correlates with liver steatosis, insulin
resistance and inflammation.
Methods: Liver and visceral adipose tissue (VAT) samples from more than
60 surgery patients, comprising lean, overweight and obese subjects, were
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analyzed for fatty acids, key DNL enzymes (FASN, ACLY, ACACA) and insulin
resistance (HOMA-IR).
Results: DNL enzymes were significantly down-regulated in VAT of obese
subjects at both mRNA and protein level. Despite this suppression in lipogenic
capacity VAT concentrations of DNL-associated fatty acids such as palmitoleic
and vaccenic acid were not decreased but rather increased in obese subjects. In
contrast to VAT, in livers of obese subjects FASN was markedly induced. Liver
FASN expression correlated strongly with HOMA-IR (R = 0.72, p < 10-9 ) and
liver steatosis (R = 0.57, p < 10-5 ). On the other hand, adipose FASN expression
showed no or much weaker correlation with clinical parameters.
Conclusion: In human obesity DNL-associated fatty acids are increased in
adipose tissue and appear to be derived from liver but not adipose DNL.
Obesity-induced induction of liver FASN is likely to play a critical role in the
development of liver steatosis and insulin resistance.
143 CHOLESTEROL LOWERING DRUGS INHIBIT THE ABSORPTION
OF CARDIOPROTECTIVE OMEGA-3 FATTY ACIDS
D. Blackwood, A. Al-Busaidi, R. La Vallee, D. Jassal, G. Pierce. Institute of
Cardiovascular Science, St. Boniface Hospital Research Centre, Winnipeg,
MB, Canada
Dietary flaxseed has anti-atherogenic properties largely through its content of
the omega-3 fatty acid, alpha linolenic acid (ALA). Therefore, the presence
of elevated ALA in circulation may be an important benefit to cardiovascular
health. Animal studies have demonstrated that ALA bioavailability is enhanced
by a cholesterol mediated micelle formation in the intestines. However, if the
presence of intestinal cholesterol increases fatty acid absorption, cholesterollowering drugs may conversely inhibit fatty acid absorption. The purpose of
the study was to determine if a cholesterol absorption inhibitor (ezetimibe)
will decrease circulating levels of ALA. Ezetimibe acts by selectively inhibiting
the Niemann-Pick C1-Like 1 receptor in the intestinal brush border. Patients
between 44−80 years old, requiring statin therapy to regulate blood cholesterol
levels were randomly assigned to one of four groups for a 6 week trial:
1. placebo;
2. ezetimibe therapy;
3. a supplement of flaxseed oil (containing 1.0 g ALA); or
4. ezetimibe and flaxseed oil supplementation.
There were no significant differences amongst the groups in terms of circulating
total cholesterol, LDL, HDL, triglyceride, DHA or EPA levels. As expected,
flaxseed oil treatment resulted in a significant increase in circulating ALA
levels. However, patients treated with ezetimibe did not increase their circulating
ALA levels even in the presence of flax oil supplementation. Our data
demonstrates that cholesterol lowering therapy can interfere with omega-3 fatty
acid absorption. In view of the cardiovascular benefits that these patients may
receive from high circulating levels of ALA, this represents a potentially serious
negative drug-diet interaction. Supported by CIHR.
144 GROUP V SECRETORY PHOSPHOLIPASE A2 PROTECTS AGAINST
CARDIAC FIBROSIS − A POSSIBLE TARGET FOR HEART FAILURE
TREATMENT
B. Boyanovsky. Internal Medicine, University of Kentucky, Lexington, KY,
USA
Objective: The goal of this study is to identify the mechanisms underlying our
newly discovered anti-fibrotic activity of group V secretory phospholipase A2
(GV sPLA2 ).
Background: Heart failure (HF) is the leading cause of hospitalization in people
older than 65 and more than 50% of the patients are re-admitted within 6 months
after treatment. HF is the end-stage complication of heart fibrosis caused by
various pathologic conditions, such as hypertension, diabetes and myocardial
infarction.
Results: Our studies in apoE−/− and apoE−/− /GV sPLA2 −/− double knockout (GV
DKO) mice showed that:
1. GV DKO mice have approximately 3.5-fold higher heart collagen content
upon Ang II infusion compared to apoE−/− mice.
2. GV DKO mice respond to Ang II infusion similarly to apoE−/− mice with
comparable elevation in blood pressure and systemic aldosterone and PG
levels. Therefore, the increased heart fibrosis observed is apparently not due
to a systemic effect of GV sPLA2 .
3. GV sPLA2 is present in the heart and it is associated with the fibrotic tissue.
4. PGI2 and GV sPLA2 are elevated upon Ang II infusion in apoE−/− mice.
5. Heart expression of COX-2 is lower in GV DKO mice compared to apoE−/−
mice, pointing that cardioprotective COX-2 derived prostaglandins may be
lower in GV DKO mice.
Conclusion: These studies hold the potential to identify new mechanisms by
which GV sPLA2 protects against heart collagen deposition and may provide
new targets for development of therapies for adequately treating heart fibrosis.
33
145 LACK OF INVARIANT NATURAL KILLER T CELLS DOES NOT
AFFECT ADIPOSE TISSUE INFLAMMATION AND METABOLIC
DYSREGULATION IN DIET-INDUCED OBESE MICE
D. Strodthoff1 , H.E. Agardh1 , A.M. Lundberg1 , G. Paulsson-Berne1 ,
G.K. Hansson1 , N. Gerdes1,2 . 1 Karolinska Institute, Stockholm, Sweden,
2
Ludwig-Maximilians-University, München, Germany
Objective: Obesity is associated with a chronic inflammatory condition in
adipose tissue, characterized by infiltration of macrophages and, as recently
discovered, T cells. Kinetics of diet-induced obesity in mice demonstrated
impairment of insulin sensitivity coinciding with infiltration of T cells into adipose
tissue in early stages of obesity. The present study determines the role
of invariant natural killer T (iNKT) cells in adipose tissue inflammation and
development of glucose intolerance.
Methods and Results: Ja18−/− mice, which specifically lack NKT cells, and
C57BL/6 (control) mice consumed a high fat diet (HFD) for 10 weeks. iNKT cells
were detected in adipose tissue of control but not Ja18−/− mice. Intraperitoneal
glucose administration revealed modestly impaired glucose tolerance in mice on
chow diet, that lack iNKT cells. However, no difference between the groups was
observed after 10 weeks of HFD, although both developed pronounced glucose
intolerance compared to those on chow diet. Flow cytometry indicated similar
levels of CD4+ and CD8+ T cells as well as CD68+ macrophages in adipose
tissue of Ja18−/− and control mice. Quantitative PCR analysis demonstrated
no difference in the visceral fat of mRNA expression of several inflammatory
mediators usually implicated in adipose tissue inflammation.
Conclusion: iNKT cells do not affect adipose tissue inflammation or glucose
control in diet-induced obese mice.
146 LIPID DROPLETS IN HUMAN MACROPHAGES RECRUIT
CYTOSOLIC PHOSPHOLIPASE A2 AND PROMOTE SECRETION
OF INFLAMMATORY MEDIATORS
P. Boström1 , Y. Wickström1 , L. Mattsson Hultén1 , R. Perkins1 , M. Ståhlman1 ,
O. Wiklund2 , S.-O. Olofsson1 , J. Borén1 . 1 Sahlgrenska Center for
Cardiovascular and Metabolic Research, 2 Wallenberg Laboratory, University
Lipid-loaded macrophages and inflammation are key features of atherosclerosis
and important in other metabolic disease such as type 2 diabetes. It is well
established that cholesteryl esters and triglycerides are stored in cytosolic lipid
droplets. Here we investigated the relationship between lipid droplet formation
and the induction of inflammation in human macrophages. We demonstrate
that increase in the amount of lipid droplet in macrophages by two methods,
incubation with oleic acid and exposure to hypoxia, resulted in increased
secretion of the eicosanoids leukotriene B4 (LTB4 ) and prostacylin (PGI2 )
and chemokines RANTES and interferon-inducible protein 10 (IP-10). We also
showed that exposure of macrophages to hypoxia promoted translocation of
cytosolic phospholipase A2 (cPLA2 ) to the lipid droplet fraction. Furthermore,
we used a novel experimental system to increase the surface area of lipid
droplets without affecting the total amount of core lipids in the cell, and showed
that increased droplet surface area was associated with an increase in the
association between cPLA2 and the lipid droplets, as well as in an increased
secretion of inflammatory mediators. We also showed that the chemokine
secretion was inhibited by a PLA2 inhibitor and by LTB4 receptor antagonists
and was stimulated by LTB4 and PGI2, indicating that the secretion of the
eicosanoids promote the chemokine secretion. We thus propose a mechanism
by which an increased lipid droplet surface gives rise to increased cPLA2
recruitment to lipid droplets. This promotes the formation of eicosanoids that,
though an autocrine/paracrine mechanism, induce the formation and secretion
of chemokines.
147 REDUCED SYNTAXIN-5 IN SKELETAL MUSCLE OF PATIENTS
WITH TYPE 2 DIABETES. A LINK BETWEEN LIPID STORAGE AND
INSULIN RESISTANCE
L. Andersson1 , S. Myhre1 , P. Bostrom1 , M. Ståhlman1 , B. Vind2 , L. Håversen1 ,
J. Borén1 , K. Hojlund2 , S.-O. Olofsson1 . 1 Wallenberg Laboratory, Sahlgrenska
Center for Cardiovascular and Metabolic Research, Göteborg, Sweden,
2
Diabetes Research Centre, Department of Endocrinology, Odense University
Hospital, Odense, Denmark
Insulin resistance in patients with type 2 diabetes (T2D) is characterized by
impaired insulin signaling and accumulation of triglycerides (TG) in skeletal
muscle. We have previously shown that syntaxin-5 is required for normal
intramyocellular TG storage by promoting fusion of lipid droplets (LD).
We examined syntaxin-5 level in skeletal muscle biopsies from patients
with T2D, and matched lean and obese, glucose tolerant control subjects
metabolically characterized by euglycemic-hyperinsulinemic clamp. Cultured
human and rat skeletal muscle cells were used to dissect possible mechanism
by which syntaxin-5 levels affect storage of lipids and insulin signaling.
Here, we report that reduced syntaxin-5 protein levels in skeletal muscle
of patients with T2D parallels insulin resistance, increased lipid content and
impaired insulin action on Akt Ser473 phosphorylation. Exposure of human
34
myotubes to high levels of fatty acids alone, or high fatty acids in combination
with high insulin and/or glucose decreased syntaxin-5 levels. Syntaxin-5 siRNA
in L6 myoblast inhibited the formation of large LD and caused increased
levels of diacylglycerol (DAG) when exposed to oleic acids. The storage of
TG was, however, unaffected. In addition, syntaxin-5 knock-down in human
myotubes and L6 myoblasts caused impaired insulin action on Akt Ser473
phosphorylation.
In conclusion, syntaxin-5 levels are down-regulated in vivo infatty acids of
patients with T2D. Our results suggest a role for increased fatty acids, and
provide evidence for a link between reduced levels of syntaxin-5 levels and
the increased formation of DAG and impaired insulin action observed in insulin
resistant muscle.
148 SALVIANOLIC ACID B CAN INHIBIT MACROPHAGE UPTAKE OF
MODIFIED-LDL BY DIRECTLY BINDING TO TYPE B SCAVENGER
RECEPTOR CD36
L. Wang1 , Y. Bao1,2 , Y. Xu1 , Y. Yang1 , L. Wang1 , S. Si1 , S. Cho2,3 , B. Hong1 .
1
The Institute of Medicinal Biotechnology, Chinese Academy of Medical
Sciences & PUMC, Beijing, China, 2 Burke-Cornell Medical Research Institute,
White Plains, 3 Department of Neurology and Neuroscience, Weill Medical
College of Cornell University, New York, NY, USA
Implicated in the pathogenesis of an array of inflammatory diseases, targeting
CD36 was suggested for the treatment of cardio- and cerebrovascular diseases.
Through a highthroughput screening (HTS) assay, we previously identified
salvianolic acid B (SAB), a hydrophilic component derived from the herb
Danshen, as a potential CD36 antagonist. Owing to its antioxidant activity
to attenuate LDL oxidation, SAB has been widely used for the prevention
and treatment of atherosclerosis-related disorders in China. To validate the
mechanism of action, the present study investigates the specificity and efficacy
of SAB in attenuating CD36 expression and function. SAB reduced lipid uptake
in a dosedependent manner in PMA-stimulated THP-1 cells and RAW 264.7
cells. In the cells transfected with CD36-siRNA, SAB had no effect in reducing
lipid uptake, whereas overexpression of CD36 reinstates the effect, indicating
a specific involvement of CD36 in the process. Surface plasmon resonance
(SPR) analysis revealed a direct binding of SAB to CD36 with a high affinity
(KD =3.74 mM), confirming physical interactions of SAB with the receptor.
Additionally, SAB reduced oxLDL-induced CD36 gene expression in the cultured
cell lines and primary macrophages. In hyperlipidemic ApoE KO mice, we found
that SAB reduced CD36 gene expression and lipid uptake in both macrophages
and the liver, and also attenuated lipid content in the vessels, showing its ability
to antagonize CD36 function. The specificity and efficacy of SAB in cells and
the hyperlipidemic ApoE KO mice validate SAB as a potential therapeutic agent
to treat inflammatory vascular diseases including atherosclerosis and stroke.
149 INHIBITION OF ENDOPLASMIC RETICULUM STRESS RECOVERS
ABCA-1 EXPRESSION AND APO A-I-MEDIATED CHOLESTEROL
EFFLUX IN MACROPHAGES SUBMITTED TO GLYCOXIDATION
G. Castilho1 , L.S. Okuda1 , E.R. Nakandakare1 , C.X.C. Santos2 ,
F.R.M. Laurindo2 , M. Passarelli1 . 1 Lipids Laboratory (LIM 10), Faculty of
Medical Science, University of São Paulo, 2 Vascular Biology Laboratory, Heart
Institute, São Paulo, Brazil
Advanced glycated albumin (AGE-albumin) induces macrophage cholesterol
accumulation and reactive oxygen species generation which are conditions
that elicit endoplasmic reticulum (ER) stress. We evaluated ER stress and
unfolded protein response (UPR) markers in macrophages treated with AGEalbumin and its interference in ABCA-1 protein levels and cholesterol efflux.
AGE-albumin was made by incubating fatty acid free albumin with 10 mM
glycolaldehyde (4 days, 37ºC) and control albumin (C-albumin) with PBS alone.
Mouse peritoneal macrophages were incubated during 8 h with AGE-albumin
(2 mg/mL) in the absence or presence of the ER stress inhibitor, 4-phenylbutiric
acid (PBA; 2.5 or 5 mM) or the proteasomal inhibitor MG132 (1 mM). ER
stress and UPR markers were determined by immunoblot and ABCA-1
by flow citometry. Apo-AI-mediated 14 C-cholesterol efflux was measured in
macrophages overloaded with acetylated LDL. As compared to C-albumin,
AGE-albumin induced a time-dependent increase in Grp78, Grp94, eIf2a,
ATF6 and ubiquitin. ABCA-1 protein content and cholesterol efflux to apo
A-I were, respectively, 33% (p < 0.0001) and 47% (p = 0.002) reduced in
macrophages treated with AGE-albumin in comparison to C-albumin. MG132
did not recover ABCA-1 or cholesterol efflux, although PBA reduced Grp78 and
totally normalized ABCA-1 levels and cholesterol efflux. ER stress inhibition
prevents the AGE-induced ABCA-1 loss and recovers the apo A-I mediated
cholesterol efflux. ER stress inhibitors represent a promising tool to prevent
atherogenesis in diabetes mellitus and other carbonyl stress conditions.
Funding: FAPESP # 2010/50108−4 (Brazil)
150 TG INTERACTING FACTOR ACTS AS A TRANSCRIPTIONAL
REPRESSOR OF ACAT2 IN HUMAN
C. Pramfalk1,2 , M. Eriksson2,3 , P. Parini1,2 . 1 Laboratory Medicine, Karolinska
Institutet, 2 Biosciences and Nutrition, Molecular Nutrition Unit, Centre for
Nutrition and Toxicology, Karolinska Institutet, 3 Medicine, Karolinska Institutet,
Stockholm, Sweden
Objective: Acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2), confined
to enterocytes and hepatocytes, is considered to be responsible for cholesteryl
ester production for storage and secretion in the lipid core of very low
density lipoprotein and chylomicrons. ACAT2 deficient mice do not develop
atherosclerosis, are protected against diet-induced hypercholesterolemia and
cholesterol gallstone formation, and also have reduced hepatic steatosis.
Hence, ACAT2 is considered as a possible therapeutic target in the prevention
and treatment of dyslipidemia. We previously found evidence of potential
positive and negative elements in the human ACAT2 promoter. We then chose
to characterize the positive regulatory element in detail. Thus, this study aims to
identify the potential repressor element and evaluate its relevance in humans.
Methods: Screening of the ACAT2 promoter sequence, mutagenesis, cotransfections, and chromatin immunoprecipitation (ChIP) assays and qPCR in
human liver were performed.
Results: We identified a putative binding site for the transcription factor TG
interacting factor (TGIF). Mutagenesis of this site increased the human ACAT2
promoter activity ~2-fold in human hepatoma HuH7cells. TGIF decreased dosedependently the promoter activity. Since women have ~70% lower hepatic
ACAT2 activity than men, we are currently investigating whether levels of TGIF
may be of importance for this sex-related difference.
Conclusions: TGIF decreases dose-dependently the human ACAT2 promoter
activity and its expression levels may be of importance for the sex-related
difference in hepatic ACAT2 activity. TGIF may thus have an important role
in the lipid metabolism in humans.
151 DIETARY SUGAR INCREASES LIVER CHOLESTEROL PARTLY
THROUGH REDUCED BILIARY SECRETION OF CHOLESTEROL
AND BY REDUCED SYNTHESIS OF BILE ACIDS
J. Lundberg, L. Persson, C. Gälman, B. Angelin, M. Rudling. Department of
Medicine, Huddinge, Karolinska Institutet at Karolinska University Hospital −
Huddinge, Stockholm, Sweden
Introduction and Aim: Cholesterol is eliminated from the body via conversion
into bile acids (BAs) or by direct secretion into the bile. Transport of cholesterol
from the liver into the bile is mediated by ATP-binding cassette subfamily
G, member 5 and 8 (ABCG5 and ABCG8). The rate limiting enzyme in the
synthesis of BAs is cholesterol 7a-hydroxylase (CYP7A1), a protein that is under
feed-back regulation by BAs. When BAs bind the farnesoid X receptor (FXR) in
liver or intestine, transcription of CYP7A1 is reduced. The expression of FXR
has been reported to be up-regulated by glucose, in vitro.
Material and Methods: Twelve SD rats received chow or a carbohydrate-rich
diet (CRD) (chow with 60% sucrose +10% fructose in the water) for 10 d.
Results: Liver cholesterol increased by 70% by the CRD. The CRD reduced
the BA synthesis, indicated by a 60% reduction of the serum marker for BA
synthesis, 7a-hydroxy-4-cholesten-3-one (C4), and by a 70% reduction of liver
mRNA levels of CYP7A1. The cholesterol transporters ABCG5 and ABCG8
were both reduced by more than 85%. Protein and mRNA levels of liver FXR
and mRNA level of FXR in distal intestine were unaltered by the CRD.
Conclusion: Dietary sugar increases liver cholesterol partly by reduced
expression of the biliary cholesterol transporters ABCG5 and ABCG8 and by
reduced expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis.
The reduction of CYP7A1 appeared independent of FXR.
152 INHIBITION OF NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE
INCREASES LIPID ACCUMULATION IN MACROPHAGES
T. Boerresdatter Dahl, T. Ranheim, S. Holm, P. Aukrust, B. Halvorsen. Oslo
University Hospital, Rikshospitalet, Oslo, Norway
Objective: Accumulation of modified LDL in macrophages, leading to foam
cell formation, is a key event in atherogenesis. We have previously shown
an increased level of nicotinamide phosphoribosyltransferase (nampt) also
termed Visfatin, in symptomatic atherosclerotic carotid lesions, primarily located
to macrophages. In the present study we sought to investigate the role of
intracellular nampt in lipid accumulation in macrophages.
Methods: We used siRNA and stimulation of the monocytic cell-line THP-1 cells
to investigate the role of nampt on lipid accumulation. RNA and protein levels
were measured by qPCR and Western analysis. Lipid contents were determent
by colourimetric analysis.
Results and Conclusion:
1. Nampt is markedly increased during macrophage differentiation.
2. Tumor necrosis factor (TNF)a, oxidized (ox)LDL, and hypoxia increased
nampt expression in macrophages, with further enhancing effect of TNFa
in combination with oxLDL and hypoxia.
79th EAS Congress
3. Silencing of nampt markedly increased lipid accumulation in macrophages
as shown by increased protein levels of the lipid droplet marker adipose
differentiation-related protein (ADRP). A specific inhibitor of nampt enzymatic
activity, increased ADRP and cholesterol levels in oxLDL stimulated
macrophages, and enhanced the binding of acetylated LDL in these cells.
4. Inhibition of nampt down-regulated the relative amount of cholesterol efflux
in oxLDL exposed macrophages.
Our data suggest that the regulation of nampt in macrophages is closely linked
to inflammatory and pro-atherogenic stimuli, potentially mediating counteracting
effects on lipid accumulation and foam cell formation. These findings suggest
a rather complex role of nampt in atherogenesis, potentially mediating both
adaptive and maladaptive responses.
153 ANTI-ARRHYTHMIC EFFECTS OF VERY LOW DENSITY
LIPOPROTEINS
Y. Shao1 , A. Mijatovic1 , S. Gizurarson2 , T. Råmunddal2 , J. Borén1 ,
E. Omerovic2 . 1 Wallenberg Laboratory at Sahlgrenska Academy, Göteborg
University, 2 Department of Molecular and Clinical Medicine, Sahlgrenska
Academy at University of Gothenburg, Gothenburg, Sweden
Objective: The heart has ability to synthesize low density-like lipoproteins
and to internalize, very low density lipoproteins (VLDL). The physiological
and/or pathophysiological significance of these biochemical properties of heart
muscle are not well understood. Lysophophatidylcholine (LPC), a hydrolysis
product of (membrane) phospholipid degradation, is one of the most potent
pro-arrhythmic substances that accumulate in the human heart in the setting
of myocardial ischemia. We hypothesize that exogenous VLDL (possibly as
a donor of exogenous apoB lipoproteins) may have anti-arrhythmic effects
mediated through neutralization of pro-arrhythmic electrophysiological events
elicited by LPC.
Methods and Results: Spontaneously beating mouse HL-1 cardiomyocytes
were plated onto MEA micro-chips and cultured for 3 days for electrical
recording in the form of field potentials (FP). Ischemic conditions were
mimicked with LPC application at different concentrations. We found that VLDL
acts as an anti-arrhythmic substance at rather low concentration of 1mM.
VLDL attenuated electrophysiological abnormalities caused by LPC such as
reduction in beating rate and FP duration by 36.24±0.15% and 56.80±0.24%
respectively, (n = 25, P < 0.05). LPC effects was completely blocked at VLDL
concentration of 2.5 mM. No significant alterations in electrophysiological
properties of beating cardiomyocytes were observed in the presence of VLDL
particles only. Cardiomyocytes exposed to VLDL (10mM) over a period of 24
hours have shown the prolonged resistance toward pro-arrhythmic effects of
LPC even removing VLDL from media.
Conclusions: Exogenous VLDL effectively protects cardiomyocytes from proarrhythmic effects caused by LPC. Our study points toward a novel, intriguing
and potentially important aspects of lipoprotein biology.
154 FATTY ACID BINDING PROTEIN 4 INDUCES ENDOTHELIAL
DYSFUNCTION THROUGHT INSULIN-SIGNALING PATHWAY IN
HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS
G. Aragonès, J. Girona, I. Lázaro, A. Cabré, L. Masana. Vascular Medicine
and Metabolism Unit, Research Unit on Lipids and Atherosclerosis, Universitat
Rovira i Virgili, Internal Medicine Department, Sant Joan University Hospital,
IISPV. CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM),
Reus, Spain
Background: Circulating FABP-4 has been shown to play an important role in
atherosclerosis in animal models and plasma concentrations are increased in
MS and T2DM. Under pathological conditions, proinflammatory factors cause
impairment in insulin-signaling pathway in vascular endothelium and leads
endothelial dysfunction. The aim of this work was to study the effect of FABP4
on the insulin-signaling pathway in endothelial cells.
Methods: Experiments were performed using cultured Human Umbilical Vein
Endothelial Cells (HUVEC). Endothelial NO synthase (eNOS), Akt, p44/p42
MAPK (Erk1/2) and FABP4 were assessed by Western blotting. eNOS, VCAM1
and E-selectin mRNA were measured by RT-PCR. Leukocyte adhesion to
endothelial cells was assessed by the adhesion assay.
Results: FABP4 inhibited insulin-induced serine phosphorilation of Akt
activation in human umbilical vein endothelial cells (HUVEC). These effects
of FABP4 led to reduced endothelial nitric oxide synthase (eNOS) activation.
FABP4 inhibited insulin-stimulated p42/p44 ERK1/2 activation. Long time
exposure of FABP4 resulted in an inhibition of insulin-stimulated eNOS mRNA
expression and increased of VCAM and E-selectin expression. Adhesion cell
based studies, showed that FABP4 stimulated monocyte adhesion to endothelial
cells.
Conclusions: FABP4 induces endothelial dysfunction by inhibiting the
activation of eNOS. FABP4 could be a good therapeutic target in patients with
metabolic syndrome and insulin resistance. These finding provide mechanistic
linkage among FABP4, impaired insulin signaling in endothelium, and greater
cardiovascular risk in diabetes.
35
155 SERUM LEVELS OF ANGIOPOIETIN-LIKE PROTEIN 4 (ANGPTL4)
ARE INVERSELY CORRELATED WITH OBESITY IN HEALTHY
YOUNG ADULT TWINS
M.R. Robciuc1 , J. Naukkarinen2 , H. Tyynismaa3 , T. Raivio3 , A. Ortega-Alonso3 ,
A. Suomalainen3 , J. Kaprio1 , A. Rissanen4 , M. Jauhiainen1 , C. Ehnholm1 ,
K. Pietiläinen4 . 1 National Institute for Health and Welfare, 2 Institute for
Molecular Medicine, 3 University of Helsinki, 4 Helsinki University Central
Hospital, Helsinki, Finland
Animal studies suggest a regulatory role for Angptl4 in obesity. We aimed
to investigate associations between serum Angptl4 levels and obesity related
parameters in humans.
Serum Angptl4 was measured by ELISA in 75 dizygotic (DZ) twin and 46
monozygotic (MZ) twin pairs, of which 21 MZ pairs are discordant for BMI (BMIdifference >2.5 kg/m2 units, age 22−33 yr), from the FinnTwin12 and FinnTwin16
cohorts.
In MZ pairs discordant for BMI, Angptl4 levels were significantly decreased
(p = 0.04) in obese twins (14.8±3.48 ng/ml) compared to their non-obese cotwins (43.3±19.7 ng/ml) in contrast, Angptl3 levels that did not differ between
the two groups (p = 0.832). Angptl4 was not different between concordant
MZ co-twins (p = 0.555). Furthermore, in all twins, intra-pair differences of
Angptl4 serum levels were inversely correlated with intra-pair differences in
BMI (r = −0.27, p = 0.003), waist (r = −0.21, p = 0.019) and body fat percentage
(r = −0.19, p = 0.039). Angptl4 was strongly correlated with FGF21 serum levels
(r = 0.486, p < 0.001) and both factors were modulated by common genetic and
environmental factors. The heritability was only 19% for ANGPTL4, with the
respective 81% remaining being attributable to environmental effects. In MZ
twins, adipose tissue Angptl4 mRNA levels were inversely correlated with BMI
but not with Angptl4 serum levels.
Our results show that Angptl4 serum levels are highly modulated by
environmental factors and suggest a role for Angptl4 in acquired human
obesity.
156 THE REDUCTION IN HDL RECEPTOR LEVELS IN MACROPHAGES
IS RELATED TO ROS GENERATION INDUCED BY ADVANCED
GLYCATED ALBUMIN
R. Souza Pintio1 , B.A. Paim2 , G. Castilho1 , E.R. Nakandakare1 , A.E. Vercesi2 ,
M. Passarelli1 . 1 Lipids Laboratory (LIM 10), Faculty of Medical Sciences,
University of São Paulo., São Paulo, 2 Pathology Department, University of
Campinas − UNICAMP, Campinas, Brazil
Introduction: Advanced glycation impairs the reverse cholesterol transport in
diabetes mellitus (DM). We analyzed the role of AGE-albumin (AGE-alb) in
the ABCA-1 and ABCG-1 expression in J774 macrophages (J774-macs) and
related to the production of reactive oxygen species (ROS).
Methods: AGE-alb was made by incubating albumin with 10mM glycolaldehyde
and control albumin (C-alb) with PBS alone (4 days at 37ºC). J774-macs
were incubated along time with C-alb or AGE-alb in the absence or presence
of 10 mM aminoguanidine (AMG) or 350 mM benfotiamine (BF). ABCA-1
and ABCG-1 protein levels and ROS generation were determined by flow
citometry.
Results: ROS generation increased and ABCA-1 and ABCG-1 expression
reduced in macs treated along time with AGE-alb. After the 8 hour-treatment
with AGE-alb on J774-macs, ROS generation was increased 155% as
compared to macs treated with C-alb, which was prevented by AMG but not BF.
The reduced expression of ABCA-1 (27%) and ABCG-1 (16%) in cells treated
with AGE-alb in comparison to C-alb was recovered by AMG but not by BF.
Conclusion: ROS production induced by AGE-alb in macrophages relates to
the reduced expression of HDL receptors, contributing to the development of
atherosclerosis in DM.
Funding: FAPESP (Brazil).
157 AGE-ALBUMIN INDUCES INTRACELLULAR LIPID ACCUMULATION
BY MODIFYING THE EXPRESSION OF GENES INVOLVED IN
MACROPHAGE REVERSE CHOLESTEROL TRANSPORT AND
CHOLESTEROL HOMEOSTASIS
A. Machado-Lima1 , R.T. Iborra1 , R.S. Pinto1 , C.H. Sartori1 , E.R. Oliveira2 ,
J.T. Stefano3 , E.R. Nakandakare1 , D. Giannella-Neto3 , M.L. CorrêaGiannella2 , M. Passarelli1 . 1 Lipids Laboratory (LIM-10), 2 Cellular and
Molecular Endocrinology Laboratory (LIM 25), 3 Clinical and Experimental
Gastroenterology (LIM-07), Faculty of Medical Sciences, University of São
Paulo, São Paulo, Brazil
Objective: In diabetes mellitus (DM), advanced glycated albumin (AGEalbumin) reduces the apo A-I and HDL-mediated cholesterol efflux, inducing
lipid accumulation in macrophages. We analyzed the role of AGE-albumin
isolated from uncontrolled type 1 (DM1) and type 2 (DM2) DM subjects in the
expression of genes associated with cholesterol removal in macrophages.
Methods: Serum albumin from control (C; n = 12) and uncontrolled DM1
(n = 13) and DM2 (n = 11) individuals was isolated by FPLC and purified by
alcoholic extraction. Macrophages were treated for 18 h with C, DM1 or DM2
36
albumin (1 mg/mL) and total RNA extracted in order to perform microarray
analysis (RNeasy Mini Kit; Agilent arrays; 44,000 probes). Data were normalized
and probes differentially expressed >2.0-fold were selected (Student t test
+ Benjamini-Hochberg with FDR correction, p < 0.05). Gene expression was
validated by real-time RT-PCR (TaqMan Two Step RT-PCR).
Results: DM1 and DM2 albumin presented a higher amount of carboxymethyllysine as compared to C and induced differential expression of respectively, 907
and 1854 genes in macrophages. Janus kinase 2 was reduced in DM1 and DM2
albumin-treated macrophages. In addition, stearoyl-Coenzyme A desaturase 1,
NADPH oxidase (NOX-4) and scavenger receptor AI were enhanced and LDL
receptor reduced in macrophages treated with DM1 albumin.
Conclusion: In DM, AGE-albumin induces intracellular lipid accumulation by
selectively modifying the expression of genes involved in the modulation of
reverse cholesterol transport and cholesterol homeostasis.
Funding: FAPESP, Brazil
158 INCREASED EXPRESSION OF LDLR AND ACCUMULATION OF
CHOLESTEROL ESTERS IN RISK AND INFARCT AREA OF PIG
HEARTS AFTER MYOCARDIAL INFARCTION
L. Karlsson, C. Olsson, M. Ståhlman, T. Larsson, J. Perman, L. Grip,
J. Borén, S.-O. Olofsson. Department of Molecular and Clinical Medicine and
Sahlgrenska Center for Cardiovascular and Metabolic Research, Wallenberg
Laboratory, University of Gothenburg, Göteborg, Sweden
Myocardial infarction is a major cause of death and disability worldwide. It
has been demonstrated in different research animals that hypoxia results in
increased accumulation of lipids. In this report we have compared the normal
myocardium with area at risk (AAR) and the infarct area (IA) of pigs in which
an acute myocardial infarction had been induced. Seven anesthetized pigs
underwent 40 min of coronary occlusion followed by 4 hours of reperfusion. Oil
Red O staining revealed an increased accumulation of lipid droplets in the AAR
and IA compared to normal hearts. Lipidomic analyses showed that cholesterol
esters (CE) increased significantly in these areas (p < 0.0001) while there were
no changes in triglycerides. Phosphatidylcholine, phosphatidylethanolamine
and ceramides decreased in the AAR and IA most likely due to increased
apoptosis/cell death in these areas. In agreement with the increased CE, we
observed a 10-fold increase of the expression of LDL receptor mRNA levels in
AAR and IA. Results from proteomics data indicated an increased production of
lipoproteins in AAR and IA. In summary our results indicate that ischemia induce
expression of the LDL receptor in the pig heart. This results in increased storage
of lipid droplets containing cholesterol esters and an increased lipoprotein
production.
159 EFFECTS OF THE COMBINATION OF SIMVASTATIN AND
EZETIMIBE IN CHYLOMICRON KINETICS IN PATIENTS WITH
STABLE CORONARY ARTERY DISEASE
O.C. Mangili, R.D. dos Santos, R.C. Maranhão, A.C.M. Gagliardi, L.C. Mangili.
InCor, São Paulo, Brazil
Introduction: The plasma clearance of chylomicrons has been correlated
with the incidence and severity of atherosclerosis. Several studies showed
lower plasma clearance of chylomicrons in patients with coronary artery
disease (CAD). The effects of ezetimibe 10 mg in the kinetics of chylomicrons
were initially compared to simvastatin 20 mg and subsequently evaluated the
progression of treatment for high dose statin and combination of statin and
ezetimibe.
Methods: Patients with stable CAD (25 pacients) were randomized for
treatment with ezetimibe (group 1) or simvastatin (group 2) with progression to
10 mg ezetimibe + simvastatin 20 mg or simvastatin 80 mg, respectively. Kinetic
studies were made before the start of drug therapy and after each treatment
period of 6 weeks. The lipid emulsion labeled with 14 C-CE and 3 H-TG was
injected and blood samples were collected during 60 minutes to determine
the fractional removal of 14 C-CE (CE-FCR) and 3 H-TG (TG-FCR). Statistical
analysis were made by ANOVA.
Results: The CE-FCR in group 1 was 0.005±0.004, 0.011±0.007 and
0.018±0.004 min-1 and group 2 was 0.004±0.002, 0.011±0.008 and
0.019±0.007 min-1 in the first, second and third assessments (p < 0.05). The
TG-FCR in group 1 was 0.017±0.01, 0.024±0.011 and 0.042±0.013 min-1
and group 2 was 0.01±0.016, 0.022±0.009 and 0.037±0.011min-1 in the first,
second and third assessments (p < 0.05).
Conclusion: In conclusion, both treatments increased the removal of 14 C-CE
and 3 H-TG significantly both in low-dose or optimized-dose, this increase being
similar between groups, primary in the slow component of plasma removal of
chylomicrons remnants.
160 PHOSPHOLIPIDOSIS IN STRESSED BY PRESENCE OF TNFA AND
FATTY ACIDS (MIMICKING METABOLIC SYNDROMECONDITION)
ENDOTHELIAL CELLS
B. Kiec-Wilk1 , U. Czech1 , A. Knapp1 , M. Awsiuk1 , J. Goralska1 , A. Sliwa1 ,
A. Gruca1 , H. Robenek2 , G. Schmitz3 , A. Dembinska-Kiec1 . 1 Clinical
Biochemistry, Jagiellonian University, Collegium Medicum, Cracow, Poland,
2
Institute for Arteriosclerosis Research, Muenster, Muenster, 3 Clinical
Biochemistry, University Cardiology Clinic Hospital Regensburg, Regensburg,
Germany
Nutritional overload induces endoplasmic reticulum (ER)-stress, changes
in mitochondrial membrane potential (MMP), lipid droplet (LD) formation
accompanied by autophagy important for cell survival. Excess of free fatty
acids and insulin resistance (mediated in between by TNFa) are responsible
for lipotoxicity.
Study is aimed to find the sequence of the gene expression/protein biosynthesis
and the lipid content changes characteristic for the LD formation in the not
devoted to lipid storage endothelial cells.
Methods: HUVEC were cultured with PA, AA, EPA (30mM) for 24h with addition
of TNFa (5ng/ml) for 4 last hours. Gene expression was analyzed by microarray.
ATP production (ROCHE) and oxygen requirement (Oxygraf 2-K (Oroboros)
were followed. Changes in MMP, PAT proteins and ER chaperones were
followed by the fluorescence microscopy imaging in vivid cells (BioimagerBD ).
The freeze-fracture technique combined with replica immunolabeling was
applied for high-resolution imaging of chaperones.
Results: FFA did not significantly change oxygen consumption, but ATP
generation was decreased by PA and OA. Microarray analysis revealed an
induction of substrate transporters genes, involved in substrate metabolism,
angiogenesis as well as ER-shock chaperones by FFA. The freeze-fracture
replica immunolabeling illustrates the localization of the ER-shock proteins
p61ER, GRP78, and GRP94. on the E-face of the ER membrane, the E-face
of the inner nuclear membrane and in the lumen of the ER, accompatied with
the increased amount of PAT proteins (adipophylin, perilipin) and decrease in
calreticulin (immunostaining, confocal microscopy BioimagerBM ).
The TNFa with PA and OA induces ER shock and formation of the LD-like
structures (phospholipidosis) in endothelial cells.
161 PANCREATITIS DUE TO MASSIVE HYPERTRIGLYCERIDAEMIA
IN ALSTRÖM’S SYNDROME − A CASE REPORT
N. Gauer, E. Giannakidou-Jordan, E. Steinhagen-Thiessen, U. Kassner.
Ambulatory Lipid Clinic, Charite Berlin, Campus Virchow Klinikum, Berlin,
Germany
Acute pancreatitis is a severe consequence of massive hypertriglyceridaemia.
Hypertriglyceridaemia is a hallmark of Alström‘s syndrome, a rare autosomal
recessive, single gene disorder caused by mutations in ALMS1, a gene
of currently unknown function. We report about a 25 year old woman
with undectected hypertriglyceridaemia in Alström‘s syndrome who developed
severe complications of a necrotizing pancreatitis. Only after hospitalization
hypertriglyceridaemia as well as low HDL-cholesterol, insulin resistance and
hyperglycemia were discovered. The diagnosis Alström‘s syndrome could also
be verified by genetic testing for mutations in ALMS1. The patient is compound
heterozygote with two different mutations (pGlu3773TrpfsX18 and IVS16-G>A).
Hypertriglyceridaemia was effectively treated with a combination of fenofibrate
and omega-3-fatty-acids. Hyperglycaemia was well controlled with metformin.
Previous clinical findings included retinal dystrophy with juvenile blindness,
sensorineural hearing loss, obesity, short stature, bone-skeletal disturbances,
chronic bronchitis, hypertension, hypothyroidism and mild hepatic and renal
impairment. Cardiomyopathy, also often present in Alström‘s syndrome, was
absent. As reported, the life span of patients with Alström‘s syndrome is
restricted due to the lack of a specific therapy. Early diagnosis and adequate
symptomatic treatment can moderate progression and prevent complications
as seen in the reported patient.
Conclusion: Our case report shows that diagnostic elaborateness in patients
with a set of conspicuous findings is necessary to avoid severe consequences
like severe pancreatitis. Especially metabolic parameters like serum lipids and
glucose should be monitored routinely.
162 ADIPOSE TISSUE GENE EXPRESSION OF FACTORS RELATED
TO LIPID PROCESSING IN OBESITY
M. Clemente-Postigo1 , F.J. Tinahones2 , F. Cardona3 . 1 Fundacion IMABIS,
2
Hospital Clinico Universitario Virgen de la Victoria. CIBEROBN, 3 Fundacion
IMABIS. CIBEROBN, Málaga, Spain
Adipose tissue lipid storage and processing capacity can be a key factor for
obesity-related metabolic disorders. Tthe aim of this study was to analyze
the gene expression of factors implied in the clearance of circulating lipids
and processing in subcutaneous (SAT) and visceral (VAT) adipose tissue. It
was studied the gene expression of the VLDL receptor (VLDLR), lipoprotein
lipase (LPL), acylation stimulating protein (ASP), LDL receptor-related protein
1 (LRP1) and fatty acid binding protein 4 (FABP4) in the VAT and the SAT of 28
79th EAS Congress
37
morbidly obese patients with Type 2 Diabetes Mellitus (T2DM) or high IR, 10
morbidly obese patients with low IR, 10 obese with low IR and 12 lean healthy
controls. LPL, FABP4, LRP1 and ASP in the VAT was higher in lean controls, in
the SAT, LPL and FABP4 were significantly higher. LPL and LRP1 were greater
in the SAT than in the VAT. ASP mRNA levels were lower in the SAT than in
the VAT. BMI, plasma insulin levels and HOMA-IR were negatively correlated
with LPL and FABP4 in the VAT, while SAT LPL was inversely correlated with
the BMI and triglyceride levels and FABP4 in the SAT was inversely correlated
with the BMI. The multiple regression analysis showed that the BMI was the
main variable which contributes to LPL and FABP4 as in the VAT as in the
SAT. These results suggest that adipose tissue of obese patients could have
a decreased lipid up-take and processing capacity compared to lean subjects
above all in the VAT.
PPARg, and the phosphorylation of ERK1/2 were significantly increased.
However, when cells were treated with ERK1/2 inhibitor or PPARg inhibitor,
they were decreased. When treated with PPARg agonists, they were increased.
Compared with control group, the accumulation of lipids significantly increased
in adipophilin gene overexpression cells, and the expression of PPARg and
the phosphorylation of ERK1/2 were reduced in adipophilin gene knockdown
cells.
Conclusion: These results indicated that adipophilin involves in the cellular
lipid accumulation and Ox-LDL induces adipophilin via ERK1/2 activation.
Grant: Project supported by the National Natural Science Foundation of China
(30971268), the Scientific Research Foundation for the Returned Overseas
Chinese Scholars from State Education Ministry. ([2008]890).
163 OLD MYTHS AND NEW INSIGHTS: INTERACTION OF
LDL-SUBCLASSES WITH HUMAN FIBROBLASTS
G. Putz, K. Winkler. Clinical Chemistry, University Freiburg Medical Center,
Freiburg im Breisgau, Germany
166 WHAT CAN TEACH US CORRELATIONS BETWEEN MRNAS
CONTENTS IN MACROPHAGES FROM MEN AND WOMEN?
A. Smirnov1 , T.A. Shchelkunova1 , I.A. Morozov2 , P.M. Rubtsov2 , I.A. Sobenin3 ,
A.N. Orekhov3 , I.V. Andrianova4 , E.G. Rudymov1 . 1 School of Biology, M.V.
Lomonosov Moscow State University, 2 V.A. Engelhardt Institute of Molecular
Biology, 3 Institute of General Pathology and Pathophysiology, 4 Institute of
Experimental Cardiology, Moscow, Russia
Background: Classification and preparation of LDL-subspecies is usually
done by ultracentrifugation (UCF). So far, interaction of LDL subspecies
with lipoprotein receptor (LDLR) was investigated by UCF derived LDL, with
conflicting results. In this study, gel permeation chromatography (GPC) was
used as alternative method to prepare LDL-subclasses.
Methods: LDL-subspecies as low density (ldLDL), medium (mLDL) and small,
dense (sdLDL) fractions were prepared in parallel by standard GPC and UCF
methods. LDL-subspecies were labelled individually with 125 I or [3 H]cholesterol.
Binding, uptake and degradation were measured on human skin fibroblasts.
Results: LDL-subspecies discriminated by UCF or GPC were comparable
in size and density, but differed in apoprotein content due to stripping of
apoproteins during UCF. When LDL protein was labelled, ldLDL and mLDL
showed little differences in interaction. GPC derived sdLDL showed significant
lower interaction than ldLDL- and mLDL, in striking contrast to UCF derived
sdLDL, wich showed much higher degradation than all other species. When
[3 H]cholesterol labelled subspecies were used, again ldLDL and mLDL showed
little differences and GPC derived sdLDL showed less interaction with LDLR
than larger subspecies. In contrast to protein labelled LDL, [3 H]cholesterol
labelled sdLDL derived by UCF showed markedly diminished interaction.
Interpretation: LDL associated apoproteins are stripped during UCF. Stripping
of apoproteins does not markedly influence interaction of ldLDL and mLDL with
LDLR. But stripped apoproteins may lead to artificial high interaction rates for
sdLDL when LDL protein is labelled. In contrast to other studies, this study
indicates significantly decreased interaction of sdLDL with LDLR.
164 ELEVATED APOLIPOPROTEIN C-III CONTENT IN RECONSTITUTED
LOW-DENSITY LIPOPROTEIN EXACERBATED CELLULAR
ATHEROSCLEROSIS AND NONENZYMATIC PROTEIN GLYCATION
S.-M. Kim, K.-H. Cho. School of Biotechnology, Yeungnam University,
Gyeongsan, Republic of Korea
Although its biological role is still debated, apolipoprotein C-III (apoC-III)
is minor component of very low-density lipoprotein (VLDL) and low-density
lipoprotein (LDL) with unknown biological function. In the current study, to
investigate physiological role of apoC-III in LDL, we purified human apoC-III
and reconstituted into LDL containing apoB and apoC-III from molar ratio 1:0
to 1:1 (apoB:apoC-III). With increase of apoC-III content, protein glycation in
LDL was more occurred with time-dependent manner. More increase of apoCIII content in LDL, more rapid production of oxidized species in fragmentation
of apo-B. Increased apoC-III content induced more incorporation of cholesterol
in oxidized LDL particle and LDL phagocytosis into THP-I cell with cytotoxicity.
In conclusion, elevation of apoC-III content in LDL caused to change more
atherogenic properties in vitro and cell models.
165 ADIPOPHILIN ACCUMULATED CELLULAR LIPID THROUGH
ERK1/2-PPARg PATHWAY IN RAW264.7 CELLS
Z. Yuan, Q. Liu, Z. Liu, C. Tang, Z. Wang, G. Yi, Y. Yang. Institute of
Cardiovascular Disease, Key Laboratory for Arteriosclerology of Hunan
Province, University of South China, Hengyang, China
Objective: To explore whether adipophilin promoted intracellular lipids
accumulation through ERK1/2-PPARg pathway.
Method: RAW264.7 cells were incubated with 50 mg/ml Ox-LDL for different
time, or were preincubated with ERK1/2 inhibitors or PPARg inhibitors/agonists
for 2 h following with 50 mg/ml Ox-LDL incubated for 24 h. Constructing retroviral
vectors pQCXIP-HA-Adipophilin and pSuper-retro-adipophilin siRNA, we got
adipophilin gene overexpression and knockdown cells. Then, the cells were
incubated with 50 mg/ml Ox-LDL for 24 h. Cellular lipid was stained with
Oil red O, the expression of mRNA and proteins of adipophilin, PPARg and
phosphorylation of ERK1/2 were detected by RT-PCR and western blot.
Result: When RAW264.7 cells were incubated with Ox-LDL, the amount of
cytoplasmic lipid droplets, the expression of mRNA and proteins of adipophilin,
Higher risks of cardio-vascular disorders in men versus women raise a question
concerning endocrine mechanisms responsible for this gender difference.
Given a role of macrophages in local inflammation and foam cell formation
at atherogenesis, we decided to assess the influence of sex hormones and
naturally modified low density lipoproteins (mLDL) on cholesterol accumulation
and expression of 28 mRNAs species in peripheral blood monocyte-derived
macrophages from male and female donors. Both estrogen and androgen
inhibited mLDL-induced cholesterol accumulation in cells. A number of mRNA
species was found to be regulated by mLDL and hormones, while the direction
and magnitude of regulation of some of the mRNAs was gender-dependent.
Multiple correlations between the expression levels of mRNAs encoding for sex
hormone receptors and metabolizing enzymes, adhesion molecules, factors of
cholesterol uptake, (de)esterification and reverse transport, lipid sensors, and
cytokines were found. Remarkably, the number of such correlations in male
macrophages was 2−4-fold higher than in female cells. The treatment of cells
with estradiol significantly diminished this difference due to an increase in the
number of correlations in female cells and a decrease in male cells. These
data let us speculate that the coupling between expression of certain mRNAs
species in macrophages may be gender- and sex hormones-dependent. In this
case a better coordinated response of male macrophages to proatherogenic
stimuli as compared with female cells, may contribute to higher susceptibility of
men to atherosclerosis.
Supported by the Russian Foundation for Basic Research (grant No. 09−04–
00329a) and Russian Ministry of Education and Science
167 ESTIMATION OF THE CONTRIBUTION OF MACROPHAGE MRNAS
TO THE OBSERVED CHANGES IN MRNAS LEVELS IN THE VESSEL
INTIMA AT ATHEROGENESIS
A. Smirnov1 , T.A. Shchelkunova1 , I.A. Morozov2 , P.M. Rubtsov2 , I.A. Sobenin3 ,
A.N. Orekhov3 , I.V. Andrianova4 . 1 School of Biology, M.V. Lomonosov Moscow
State University, 2 V.A. Engelhardt Institute of Molecular Biology, 3 Institute of
General Pathology and Pathophysiology, 4 Institute of Experimental Cardiology,
Moscow, Russia
Results of any biochemical measurement (content of polypeptides and/or
respective mRNAs) should be corresponded to specific cellular elements for
relevant interpretation. We validated normalization to house-keeping gene
(GAPDH) performing qPCR on mixed cDNAs (from human monocyte-derived
macrophages loaded with modified low density lipoproteins and from the intima
of intact human aorta); a ratio of expected/measured levels for 15 mRNA
species was 99±19%. Based on this result, we performed a rough estimation
of the contribution of macrophage mRNAs to the observed changes in the
levels of these mRNAs in a vessel wall at atherogenesis. The estimation was
performed for pairs of vessel fragments with and without atherosclerotic lesions
from the same donors. For calculations, mean values of the mRNA levels
in mLDL-loaded macrophages from 9 donors were used. Expected values
were calculated using ratios of macrophages in the atherosclerotic lesions of
stages II and Va (0−4% and 8−12%, respectively) measured previously using
marker antigens. The comparison of expected and observed values for lesions
revealed several variants of gene expression rise in atheroma tissue vs normal
vessel wall: associated with macrophages infiltration and simultaneous downregulation in other cells; mainly due to macrophages infiltration; and associated
exclusively with up-regulation in non-macrophage cells. The results suggest
that the observed changes in mRNA content in vessels at atherogenesis are
associated with both the infiltration of monocytes/macrophages and changes in
expression in non-macrophage cells.
Supported by the Russian Foundation for Basic Research (grant No. 09−04–
00329a) and Russian Ministry of Education and Science.
38
168 ENHANCED BIOSYNTHESIS OF GANGLIOSIDE GM3 IN
ATHEROSCLEROSIS
N.N. Samovilova1 , E.V. Gracheva1 , N.K. Golovanova1 , O. Kaliya2 ,
N.V. Prokazova1 . 1 Research Institute of Experimental Cardiology, Russian
Cardiology Research Centre, 2 Organic Intermediates and Dyes Institute,
Moscow, Russia
Our previous immunohistochemical studies in human atherosclerotic aorta
revealed high expression levels of ganglioside GM3 and GM3 synthase in
cells of monocyte/macrophage origin abundantly infiltrating aortic intima in
atherosclerosis.
The aim of our study was to assess ganglioside GM3 biosynthesis in monocytes
of atherosclerotic patients and differentiated monocyte-derived macrophages.
We found that GM3 levels and GM3 synthase activity in human monocytederived macrophages were 5- and 10-fold higher, respectively, compared to
freshly isolated human monocytes. GM3 synthase mRNA levels were also
significantly higher in differentiated monocyte-derived macrophages compared
to monocytes and in atherosclerotic aorta compared to normal aorta. GM3
synthase activity in monocytes from atherosclerotic patients was about 3.5fold higher compared to monocytes from healthy subjects. Monocytes treated
with GM3 (40 uM) showed an increase in cellular GM3 levels from 1.5 to
5.2 mg per cell protein and a 1.1−4.4-fold enhancement of their adhesion to
HUVEC depending on the subject. Our data suggest a role for biosynthesis
of ganglioside GM3 in monocyte activation and monocyte/macrophage
differentiation in atherosclerosis.
The authors would like to acknowledge financial support from Russian
Foundation for Basic Research (grant No. 10−04–00888a).
169 POSSIBLE ROLE OF SPHINGOLIPIDS IN BETA-CAROTENEINDUCED ENDOTHELIA PROANGIOGENIC ACTIVITY
A. Polus1 , B. Kiec-Wilk1 , M. Mikołajczyk1 , J. Hartwich1 , R. Goralczyk2 ,
A. Dembinska-Kiec1 . 1 Clinical Biochemistry, Jagiellonian University, Medical
College, Krakow, Poland, 2 DSM Nutritional Products Ltd., R&D, Human
Nutrition and Health, Basel, Switzerland
Different type of carotenoids function as: free radical scavengers, immunomodulators, regulators of gap junction communication and apoptosis, are claimed
in the cardiovascular diseases and cancer prevention. Beta-carotene is the
most efficient source of retinol. It has been demonstrated that the activity of
fenretinide a synthetic retinoid could be mediated by sphingolipids: ceramide
and its derivatives.
HUVEC cells were incubated with BC (3mM) for 24 hrs. The effect on cell
proliferation was measured by BrdU incorporation. BC uptake was measured by
HPLC. The effect on cell proliferation was measured by the BrdU incorporation.
Chemotaxis was performed in the Boyden’s chamber. Changes of gene
expression were analyzed using microarray hybridization method and confirmed
using the real-time PCR method. To measure the activity of following kinases:
AKT, p38 Fast Activated Cell-based ELISA Kits were used. BC activated
chemotaxis of HUVECs.
The microarray data analysis revealed that the genes, involved in proliferation,
cell/cell; cell/matrix adhesion; matrix reorganization, and activation of
chemotaxis were the most group of affected by BC genes in HUVEC. This effect
was also confirmed by the in vitro and in vivo angiogenesis models. Inhibition
of AKT kinase and activation of p38 kinase phosphorylation was observed.
BC in the physiological concentration range stimulates early steps of
angiogeniesis. Observed effects of BC influence could confirmed participation
of ceramide or its derivatives.
Partially supported by FP7 EU 202272 LIPIDOMICNETproject
170 MITOCHONDRIAL INHIBITORY FACTOR 1 (IF1) IS PRESENT IN
HUMAN SERUM AND ITS LEVEL IS POSITIVELY ASSOCIATED
TO HDL-C
A. Genoux1 , V. Pons1 , M. Laffargue1 , G. Combes1 , J.-B. Ruidavets2 ,
J. Ferrières2 , B. Perret1 , L.O. Martinez1 . 1 Institute of Metabolic and
Cardiovascular Diseases, INSERM U1048, University of Toulouse III,
2
Epidemiology and Analysis in Public Health, INSERM U1027, University of
Toulouse III, Toulouse, France
Specific object of study: Mitochondrial ATP synthase is expressed as a
plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein
component in high-density lipoproteins (HDL). On hepatocytes, apoA-I binding
to cell surface ATP synthase (namely ecto-F1 -ATPase) stimulates the ATP
hydrolytic activity of this ecto-enzyme and this production of extracellular
ADP subsequently activates a P2Y13 -mediated HDL endocytosis pathway.
Conversely, exogenous IF1 (Inhibitory Factor 1), classically known as a natural
mitochondrial specific inhibitor of F1 -ATPase activity, inhibits ecto-F1 -ATPase
activity and decreases HDL uptake by human hepatocytes and perfused rat
liver. Since recent reports also described the presence of IF1 at the plasma
membrane of different cell types, we investigated whether IF1 was present in
the systemic circulation in human.
Methodology and Results: We first unambiguously detected IF1 in human
serum by mass spectrometry and then set up a competitive Elisa assay
in order to quantify its level in human serum. Analyse of IF1 levels in 50
normolipemic male subjects evidenced a normal distribution, with a median
value at 0.49 mg/mL and a 95% confidence interval, 0.44–0.54 mg/L. Correlations
between IF1 levels and plasma lipid level parameters were investigated and we
observed that plasma IF1 levels were positively correlated with HDL-cholesterol
and negatively with triglycerides.
Conclusions: Altogether, these data support the view that, in human,
circulating IF1 might impact HDL levels by inhibiting hepatic HDL uptake through
its inhibitory activity on ecto-F1 -ATPase.
171 HDL FUNCTION IS IMPAIRED IN PATIENTS WITH ACUTE
MYOCARDIAL INFARCTION INDEPENDENT OF PLASMA HDL
CHOLESTEROL LEVELS
W. Annema1 , H.M. Willemsen2 , R.A. Tio2 , U.J.F. Tietge1 . 1 Department of
Pediatrics, 2 Department of Cardiology, University Medical Center Groningen,
Groningen, The Netherlands
Objective: To determine the impact of acute myocardial infarction on three key
anti-atherogenic functions of HDL.
Patients and Methods: Patients with either acute non-ST segment elevation
myocardial infarction (non-STEMI, n = 41) or STEMI (n = 37) were compared
with controls experiencing non-cardiac acute chest pain (n = 33). Groups were
matched for age, sex, CRP, multiple cardiovascular risk factors, and importantly
plasma HDL cholesterol. The in vitro potential of HDL to (i) mediate cholesterol
efflux, (ii) prevent LDL oxidation, and (iii) inhibit cytokine-induced adhesion
molecule expression in endothelial cells was measured.
Results: HDL from STEMI patients displayed reduced cholesterol efflux
versus controls (P < 0.001) and non-STEMI patients (P < 0.001). Anti-oxidative
properties of HDL to inhibit LDL oxidation did not differ among groups. Relative
to controls, adhesion molecule expression remained 1.5-fold higher with HDL
from non-STEMI and 3.2-fold higher with HDL from STEMI patients (P < 0.001),
indicating impaired anti-inflammatory HDL functionality in the STEMI group.
Cholesterol efflux correlated with anti-inflammatory activity in MI patients
(P < 0.001) but not in controls. CRP levels, a systemic inflammation marker,
were not linked to HDL function. Nevertheless, higher plasma myeloperoxidase
levels were associated with impaired HDL function in MI patients (efflux:
P < 0.05; anti-inflammation: P < 0.05) but not in controls.
Conclusions: Independent of plasma HDL cholesterol, two out of three
anti-atherogenic HDL functions tested were significantly impaired in STEMI
patients, namely cholesterol efflux and anti-inflammatory properties. Specifically
myeloperoxidase might contribute causally to decreased HDL functionality.
Assessment of HDL function might reveal important clinical information over
mere HDL cholesterol mass measurements.
172 NIACIN, ATORVASTATIN AND FENOFIBRATE DECREASE PLASMA
CETP BY REDUCTION OF THE HEPATIC MACROPHAGE CONTENT
IN APOE*3-LEIDEN.CETP MICE
Y. Wang1 , Z. Li2 , M. Hoekstra2 , V. Bieghs3 , R. Shiri-Sverdlov3 , T.J.C. Van
Berkel2 , J.W.A. Smit1 , H.M.G. Princen4 , J.A. Romijn1 , L.M. Havekes1,4 ,
P.C.N. Rensen1 . 1 Leiden University Medical Center, 2 Leiden Amsterdam
Center for Drug Research, Leiden, 3 Maastricht University, Maastricht,
4
Netherlands Organization for Applied Scientific Research, Leiden, The
Netherlands
Introduction: Hypolipidemic drugs decrease (V)LDL-C and increase HDL-C.
We previously demonstrated that niacin, atorvastatin and fenofibrate increase
HDL-C by reducing hepatic CETP expression and consequently plasma CETP
levels. Since hepatic CETP is expressed by both hepatocytes and liver
macrophages, the aim of the present study was to evaluate the contribution
of hepatic macrophages to the CETP-lowering effect of these hypolipidemic
drugs by using APOE*3-Leiden (E3L).CETP mice, a unique mouse model for
human-like lipoprotein metabolism.
Methods and Results: Female mice (n = 8 per group) were fed a Western-type
diet containing 0.15% cholesterol without or with niacin (1% w/w), atorvastatin
(0.01% w/w) or fenofibrate (0.04% w/w) for 3 weeks. As compared to vehicle,
niacin, atorvastatin and fenofibrate reduced plasma (V)LDL-C (−72%, −58%
and −80%; all P < 0.01) and elevated HDL-C (+56%, +48% and +131%) as
related to reduced plasma CETP mass (−51%, −45% and −49%; all P < 0.001).
Concomitantly, they all reduced hepatic CD68 gene expression, and reduced the
number of F4/80-positive macrophages (−28%, −29% and −54%; all P < 0.05)
as well as Mac1-positive macrophages (−36%, P < 0.001; −7%, n.s; and −22%;
P < 0.05). The hepatic mRNA expression of CETP strongly correlated with
that of CD68 (R2 = 0.444; P < 0.001), which strongly suggests that niacin,
atorvastatin and fenofibrate decrease hepatic CETP expression by reducing
the hepatic macrophage content.
Conclusion: These data suggest that atorvastatin and fenofibrate decrease
the macrophage content of the liver, leading to a decreased hepatic CETP
expression, thereby reducing plasma CETP and increasing HDL-C.
79th EAS Congress
173 PROTEOMIC ALTERATIONS OF HDL FROM HEMODIALYSIS
PATIENTS ARE ASSOCIATED WITH IMPAIRED CHOLESTEROL
EFFLUX CAPACITY
M. Holzer1 , R. Birner-Gruenberger2 , V. Binder1 , D. El-Gamal1 , G. Marsche1 .
1
Institute of Experimental and Clinical Pharmacology, 2 Centre for Medical
Research, Medical University of Graz, Graz, Austria
Rationale: Functional impairment of high-density lipoprotein (HDL) during renal
disease may contribute to the excess cardiovascular mortality in these patients.
Cholesterol efflux capacity from macrophages, a metric of HDL function, has
a strong inverse association with both carotid intima-media thickness and the
likelihood of coronary artery disease. However, the data available regarding the
impact of advanced renal disease on HDL composition and functionality are
limited.
Objective: Here, we investigate whether renal disease remodels the proteome
and functionality of HDL.
Methods and Results: Mass spectrometry and biochemical analyses showed
specific alterations in the proteome and lipid composition of HDL isolated
from patients on maintenance hemodialysis. Proteomic and immunoblot studies
revealed a significant increase in serum albumin, acute phase protein serum
amyloid A1, lipoprotein associated phospholipase A2 and apoC-III content of
uremic HDL. These proteomic alterations were accompanied by a decreased
phospholipid and increased triglyceride content of HDL. Most importantly, the
changes in the proteome and lipid composition impaired the ability of uremic
HDL to promote cholesterol efflux from macrophages.
Conclusions: HDL from renal patients carries a unique cargo of proteins and
lipids associated with a decreased cholesterol efflux capacity.
174 PROTECTION OF HDL AGAINST SPHINGOMYELINASE-MODIFIED
LDL AGGREGATION AT ACIDIC PH
S.D. Nguyen, K. Öörni, P.T. Kovanen. Wihuri Research Institute, Helsinki,
Finland
Aggregation and fusion of lipoproteins induce subendothelial retention of
cholesterol, initiating the progression of atherosclerosis. It has been shown that
the extracellular pH of atherosclerotic lesions decreases in atherogenesis. Here,
we have examined the effect of pH and HDL on sphingomyelinase-induced LDL
aggregation. LDL was modified by Smase and the aggregation was monitored
in microtiter well assays at pH 5.5−7.5. We found that when pH decreased,
the Smase-induced LDL aggregation remarkably enhanced. At lowest pH 5.5
tested, Smase-modified LDL aggregation occurred at a much higher rate than
at neutral pH (7.4). While HDL effectively blocked LDL aggregation, lipid-free
Apo AI only partially inhibited LDL aggregation at acidic pH. Among HDL
subfractions, HDL3 showed the highest protection against Smase-induced LDL
aggregation. Our results suggest that in areas of atherosclerotic arterial intima
where the extracellular pH decreases, modification LDL by Smase might lead
to formation of large extracellular LDL aggregates, so resulting in the lipid
accumulation. Furthermore, we have identified a novel beneficial role of HDL in
blocking Smase-induced LDL aggregation, adding to the list of anti-atherogenic
properties of HDL.
175 THE INHIBITION OF HEPATIC ACYL COENZYME A:CHOLESTEROL
ACYLTRANSFERASE (ACAT) 2 POSITIVELY AFFECTS HDL
METABOLISM AND FUNCTIONALITY IN MICE
M. Pedrelli1,2 , P. Davoodpour1 , C. Degirolamo3 , M. Gomaraschi4 , L. Larsson1,2 ,
L.L. Rudel3 , L. Calabresi4 , J.Å. Gustafsson2 , K. Steffensen2 , M. Eriksson1,5 ,
P. Parini1,2 . 1 Dept. of Laboratory Medicine, 2 Dept. of Biosciences and
Nutrition, Karolinska Institutet, Stockholm, Sweden, 3 Dept. of Pathology, Wake
Forest University School of Medicine, Winston-Salem, NC, USA, 4 Dept. of
Pharmacological Sciences, University of Milan, Milan, Italy, 5 Dept. of Medicine,
Karolinska Institutet, Stockholm, Sweden
Introduction: Being the unique cholesterol esterifying enzyme in hepatocytes,
ACAT2 may reduce the amount of free cholesterol (FC) available for secretion
into apoAI by ABCA1, affecting plasma HDL.
Aim: To investigate the effect of hepatic ACAT2 activity on HDL functionality.
Methods: Wild type and LXRa/b knockout (DOKO) mice, on western type diet,
were injected 4 weeks with an antisense oligonucleotide targeted to knockdown
hepatic ACAT2 (ASO6) or with a non-sense ASO.
Results: ASO6 reduced hepatic TC and CE, but ASO6 treated mice did
not show hepatic FC accumulation. ASO6 strongly increased ABCA1 protein
expression in liver membranes of both WT and DOKO mice. This effect
was paralleled with a slight increase in plasma HDL-TC but with a great
increase in HDL-FC. In WT mice ASO6 did not affect preb-HDL, but
generated larger a-particles. Conversely in DOKO mice, ACAT2 downregulation
increased preb-HDL content and induced the appearance of small a-HDL. HDL
modifications resulted in positive effects on the capacity of sera from ASO6
treated mice to promote cellular cholesterol efflux. Reflecting their HDL particles
distribution, sera from ASO6 treated WT mice showed increased efflux potential
by passive diffusion and SR-BI, while sera from DOKO treated mice showed
higher capacity to accept cholesterol by the ABCA1-mediated efflux.
39
Conclusion: Hepatic ACAT2 seems to play a key-role in HDL metabolism in
mice. When hepatic ACAT2 activity is reduced, FC seems to be preferentially
channelled into apoAI, by the upregulated ABCA1. This seems to result in a
positive modification of HDL particle distribution and functionality.
176 ANTI-INFLAMMATORY EFFECTS OF A NOVEL INDUCER OF
APOLIPOPROTEIN A-I SYNTHESIS: INSIGHTS FROM THE ASSERT
STUDY
S.J. Nicholls1 , A. Gordon2 , J. Johansson2 , C.M. Ballantyne3 , J.J. Kastelein4 ,
N.C. Wong5 , M. Borgman6 , K. Wolski6 , S.E. Nissen6 . 1 Cardiovascular
Medicine, Cleveland Clinic, Cleveland, OH, 2 Resverlogix Corporation, San
Francisco, CA, 3 Baylor College of Medicine, Houston, TX, USA, 4 Academic
Medical Center, Amsterdam, The Netherlands, 5 Resverlogix Corporation,
Calgary, AB, Canada, 6 Cleveland Clinic, Cleveland, OH, USA
Background: Induction of apolipoprotein (apo) A-I synthesis represents a novel
therapeutic strategy to promote the functionality of high-density lipoproteins
(HDL). ApoA-I induction may have a beneficial effect on inflammatory pathways
implicated in atherosclerosis.
Methods: The ASSERT study evaluated early biochemical changes in
association with increasing doses of an apoA-I inducer (RVX-208 100–300 mg
daily) for 12 weeks in statin-treated patients with stable coronary artery disease.
Factors associated with changes in CRP levels with RVX were investigated.
Results: Administration of RVX-208 was associated with dose-dependent
increases in levels of apoA-I by 0.1−5.6%, HDL cholesterol by 3.2−8.3% and
large HDL particles by 11.1–21.1%. A non-significant dose-dependent reduction
in CRP by 13.0–22.0% was observed. A significant inverse relationship was
observed between changes in HDL cholesterol and CRP in RVX treated patients
(r = −0.19, p = 0.004). When RVX treated patients were stratified according to
tertiles of percent change in CRP, those with the greatest reduction in levels
demonstrated the greatest elevation in apoA-I (+5.6 vs +0.1%, p = 0.004), HDL
cholesterol (+7.7 vs +2.8%, p = 0.02), and concentration of large HDL particles
(+27.5 vs +3.9%, p = 0.003).
Conclusion: The greatest reduction in systemic CRP in association with
RVX-208 administration was observed in patients with greatest increases in
quantitative measures of HDL. This may reflect an anti-inflammatory effect
arising from newly generated functional HDL particles with apoA-I induction. The
impact of these changes on atherosclerotic plaque remains to be determined.
177 PROTEIN CARBAMYLATION RENDERS HIGH-DENSITY
LIPOPROTEIN DYSFUNCTIONAL
G. Marsche1 , M. Gauster2 , M. Holzer1 . 1 Institute of Experimental and Clinical
Pharmacology, 2 Institute of Cell Biology, Histology and Embryology, Medical
University of Graz, Graz, Austria
Objective: Post-translational modification of proteins through reactive cyanate
(protein carbamylation) has been demonstrated to predict an increased
cardiovascular risk. Cyanate is formed in vivo by break-down of urea and
at sites of inflammation by the heme protein myeloperoxidase of activated
phagocytes. Since myeloperoxidase is a component of high-density lipoprotein
(HDL) in human atherosclerotic intima, we examined in the present study
whether cyanate specifically targets HDL in the vessel wall.
Results: Immunohistochemical analysis of human atheroma sections showed
marked colocalization of carbamyllysine with apoA-I, the major HDL
apolipoprotein and macrophages expressing the HDL receptor scavenger
receptor B1 (SR-BI). Mass spectrometry analysis revealed that carbamylation
is a major protein modification of HDL. The carbamyllysine content of
lesion derived HDL was markedly increased compared to circulating HDL
and correlated with levels of 3-chlorotyrosine, a specific oxidation product
of MPO. A very important finding of the present study was that one
carbamyllysine residue per HDL-associated apoA-I was sufficient to render HDL
pro-atherogenic. Carbamyllysine present on HDL destabilized the HDL/SR-BI
mediated balance between cholesterol-uptake versus efflux, thereby promoting
cholesterol accumulation and lipid droplet formation in macrophages.
Conclusion: The present results provide first evidence that carbamylation is a
major modification of HDL that renders HDL pro-atherogenic.
178 GENE PROFILING ON THE EFFECTS OF HDL ON ACTIVATED
HUMAN MONOCYTES
X.S.L. Moore1 , Y. Fu1 , I. Haviv2 , D. Sviridov3 , J. Chin-Dusting1 . 1 Vascular
Pharmacology, 2 Bioinformatics & Systems Biology, 3 Lipoproteins &
Atherosclerosis Laboratory, Baker IDI Heart & Diabetes Institute, Melbourne,
VIC, Australia
HDL induces multiple anti-atherosclerotic effects beyond reverse cholesterol
transport. Previous work from our laboratory demonstrated that HDL inhibits
monocyte activation. To gain some understanding of the potential mechanisms
underlying the effects of HDL and monocytic activation, we performed a wholegenome gene expression analysis on activated monocytes in the presence or
absence of HDL.
40
To minimize variability related to primary monocyte sources, THP-1 cells
(human acute monocytic leukemia cell line) were selected for the study.
Following 15hr-incubation with HDL (400 ug/ml) or vehicle, half of the
HDL-treated and half of the vehicle-treated cells were subjected to 2 hrLPS stimulation (1 ug/ml). Total RNA were expression-profiled on Illumina
HumanWG-6 3.0 Expression BeadChip. A total of 12 chips were hybridized
allowing the analysis of triplicate cell samples for each of the four treatment
conditions (vehicle, HDL, LPS and LPS+HDL). The microarray analysis was
performed using BRB-Array tool interface with Bioconductor package at R.
Genes deemed significantly regulated were those with 2-fold change between
treatment conditions in comparison (p < 0.05).
LPS treatment increased the expression levels of 438 genes while reducing the
levels of 227 genes. HDL was capable of blocking 30% of these changes in both
directions respectively. Genes affected were analysed further for ontology, using
Gene Set Analysis and Ingenuity Pathway Analysis, both of which identified
genes reduced by HDL and increased by LPS to be associated with NF-kB
signalling. This result confirms inhibitory effects of HDL on monocyte activation
and it provides new candidate genes for further deciphering antiatherosclerotic
effects of HDL.
179 A NOVEL APOA-I MIMETICS, FAMP CONSTRUCTS PRE-BETA
HDL IN VITRO AND IN VIVO
Y. Uehara, K. Oniki, H. Tanigawa, S. Abe, S. Ando, E. Yahiro, S.-I. Miura,
K. Saku. Department of Cardiology, Fukuoka University, Fukuoka, Japan
Apolipoprotein (apo)-AI stimulates cholesterol efflux via the ABCA1, thus
generating HDL and reversing the macrophage foam-cell phenotype. Despite
these anti-atherogenic roles, the impact of specifically promoting ABCA1
cholesterol efflux on the development of atherosclerosis is not well known.
In this report, we describe FAMP, a novel apoA-I mimetic peptide of 24
amino acids without phospholipids. In cholesterol efflux, lipid-free apoA-I
from human plasma and FAMP could take up cholesterol from A172 cells.
Interestingly, after stimulation with LXR/RXR agonists, both plasma apoAI- and FAMP-mediated cholesterol efflux were drastically increased in A172
cells. Furthermore, stimulated cholesterol efflux by FAMP was significantly
suppressed by probucol (6.09±0.24% to 3.53±0.19%, p < 0.001). One week of
i.p. infusion of FAMP in C57BL6-mice resulted in an increase on the subfraction
of pre-beta HDL which was measured by agarose gel electrophoresis. In
addition, FAMP significantly reduced plasma triglyceride level (−35.7%), and
significantly increased total cholesterol (+13.8%) and free cholesterol (+62.3%)
in small subfraction of HDL by HPLC analysis. Finally, we evaluate an activity
for macrophage-specific reverse cholesterol transport on human CETP Tgmice. Five days of i.p. infusion of FAMP on CETP Tg-mice resulted in 42.3%
increasing on the cholesterol level in the feces from macrophages despite of
the reduced plasma HDL level.
In summary, FAMP markedly increases pre-beta HDL in vitro and in vivo,
as well as overall cholesterol efflux from peripheral tissues, particularly from
macrophages, and this depends on ABCA1. It might have tremendous atheroprotective potential and might be new therapeutic target for cardiovascular
disease.
180 CORONARY ANGIOPLASTY CHANGES THE HDL PROTEOME
OF PATIENTS WITH CORONARY DISEASE
E. Burillo1 , I. Jorge2 , E. Nuñez2 , R. Mateo-Gallego1 , P. Martin-Fuentes1 ,
A. Cenarro1 , J. Vazquez2 , F. Civeira1 . 1 Laboratorio de Investigacion Molecular,
Instituto Aragones de Ciencias de la Salud, Zaragoza, 2 Centro de Biologia
Molecular Severo Ochoa, Universidad Autonoma de Madrid-CSIC, Madrid,
Spain
Proteins related to inflammation, complement regulation, protease inhibition and
acute phase have been found in HDL, suggesting unsuspected roles not related
to lipid metabolism. We have investigate whether HDL could be implicated in
the reverse transport of proteins and degradation products of the atheroma
plaque to its removal from peripheral tissues. Coronary angioplasty (PTCA), a
model involving rupture of the atheroma plaque, could change the pattern of
HDL proteome.
Methods: Plasma before and after intervention was collected from 9 stable
ischemic cardiopathy patients undergoing PTCA. ApoAI-containing HDL was
purified by affinity chromatography. Proteins from HDL were solubilized,
digested with trypsin and labeled with O16 /O18 in a pool study or with
iTRAQ in a subject-to-subject study. Labeled peptides were fractionated and
identified/quantified by HPLC-MS/MS using a linear ion trap.
Results: We were able to quantify 295 HDL-associated proteins, whose
amounts were highly variable between individuals, indicating that HDL protein
composition was highly patient-specific. However, inter-individual differences
were reproducible and the majority of HDL-bound proteins remained at constant
levels within the same individual. Around 10% of HDL-bound proteins changed
expression levels after angioplasty in each individual. Among a total of 43
changing proteins, we detected complement, extracelular matrix, coagulation
factors, apolipoproteins, and cytoskeletal proteins.
Conclusion: The HDL protein composition is highly variable among patients
and shows significant changes after PTCA. This has important implications for
the use of HDL proteome as a model in search of biomarkers, and confirm
that HDL is also involved in non-cholesterol reverse transport from peripheral
tissues.
181 ADDITIVE EFFECTS OF NIACIN ON TOP OF SIMVASTATIN
TREATMENT IN REDUCING ATHEROSCLEROSIS IN
APOE*3LEIDEN.CETP MICE
M. Louwe1 , S. Kühnast1,2 , J. Smit1 , L. Havekes1,2 , P. Rensen1 , J. van der
Hoorn1,2 , H. Princen1,2 , W. Jukema1 . 1 Leiden University Medical Center,
2
TNO-Biosciences, Leiden, The Netherlands
Objective: Niacin (nicotinic acid) is the most potent HDL-cholesterol raising
drug clinically used, but its effect on atherosclerosis development is unclear.
The aim of this study was to evaluate the effect of niacin, with and without simvastatin, on atherosclerosis development in APOE*3Leiden.CETP transgenic
mice, a well-established model for human-like lipoprotein metabolism.
Methods: APOE*3Leiden.CETP mice were fed a western-type diet (containing
0.1% cholesterol) without or with niacin (120 mg/kg/day), simvastatin
(36 mg/kg/day) or their combination for 18 weeks.
Results: Total cholesterol levels were decreased by niacin (−39%, p < 0.001),
simvastatin (−30%, p < 0.001) and their combination (−55%, p < 0.001), the
latter was more potent than simvastatin alone (−36%, p < 0.001). HDLcholesterol levels were significantly increased by niacin (+28%, p < 0.01).
Niacin, simvastatin and their combination reduced atherosclerotic lesion area
by −78%, −49% and −87% (all p < 0.001), respectively. A decrease in
severe lesions as percentage of all lesions was revealed with all treatments.
Niacin alone and in combination with simvastatin reduced the number
of lesions (−23%; p = 0.056; −48%, p < 0.001) and increased percentage
undiseased segments (+141%, p < 0.001; +210%, p < 0.001). The combination
was significantly more potent than simvastatin treament alone, evidenced by
less lesions (−44%, p < 0.01) and lesion area (−74%, p < 0.001) and more
undiseased segments (+110%, p < 0.01). Results of lesion composition are
pending.
Conclusion: Niacin, simvastatin and their combination all improve plasma lipid
levels and reduce atherosclerotic lesion area and severity. However, niacin
significantly added to the effect of simvastatin alone, regarding lipid levels and
atherosclerosis parameters.
182 DETERMINATION OF PARAOXONASE-3 CONCENTRATION IN
HUMAN SERUM: METHOD EVALUATION, REFERENCE VALUES AND
INFLUENCE OF GENOTYPES IN A POPULATION-BASED STUDY
J. Camps1 , G. Aragonès1 , A. Garcı́a1 , M. Barreda1 , J. Marsillach2 ,
R. Beltrán-Debón1 , A. Rull1 , B. Mackness1 , M. Mackness1 , J. Joven1 . 1 Centre
de Recerca Biomèdica, Hospital Universitari de Sant Joan, Reus, Spain,
2
Department of Genome Sciences, University of Washington, Seattle, WA,
USA
Background: Experimental studies showed that paraoxonase-3 (PON3) retards
lipoprotein oxidation and may play an anti-atherogenic role. Our objective was
to describe a new assay to measure serum PON3 concentrations, and report
their reference values, the influence of PON3 promoter polymorphisms, and the
relationships with PON1 and lipid profile in a population-based study.
Methods: We generated an anti-PON3 antibody by inoculating rabbits with a
synthetic peptide specific of mature PON3. This antibody was used to develop
an enzyme-linked immunosorbent assay for serum PON3 concentration. This
parameter was analyzed in 356 healthy subjects of Caucasian ethnic origin.
PON3 promoter polymorphisms were analyzed by the Iplex Gold MassArray™
method.
Results: The evaluated range of concentration was from 0.1 to 3.0 mg/L
and the average regression line (N = 8) was y = 0.9587 (0.3392) log10 x
+ 1.9466 (0.0861) [r 2 = 0.924 (0.0131); P < 0.001]. Cross-reactivity studies
showed that there was no cross reaction with PON1. Detection limit was
0.24 mg/L. Imprecision was 13.2%. Reference interval (median and 95% CI)
in our normal population was 1.78 (1.00–2.47) mg/L. PON3 was observed in
HDL particles containing apolipoprotein A-I and PON1, but not apolipoprotein
A-II and apolipoprotein E. Serum PON3 concentrations showed a moderate
influence (about 10% variation) by PON3 promoter polymorphisms.
Conclusions: The present study describes for the first time a method to
measure serum PON3 concentrations. This offers new opportunities in the
investigation of the properties and role of PON3 in cardiovascular disease with
possible implications in clinical practice.
79th EAS Congress
183 REGULAR EXERCISE INCREASED PLASMA PRE-BETA
1-HDL CONCENTRATION IN ADULT WOMEN WITH METABOLIC
SYNDROME
M. Rosety-Rodriguez1 , F.J. Ordoñez2 , I. Rosety2 , A. Diaz-Ordoñez3 ,
M.A. Rosety3 , A. Camacho3 , N. Garcia2 , J. Rosety4 , G. Fornieles-Gonzalez1 .
1
Internal Medicina, 2 Medicina, 3 School of Sport Medicine, University of Cadiz,
4
Urology, Hospital Universitario Puerta del Mar, Cadiz, Spain
Background and Aim: Recent studies have shown that the prebeta-1 subclass
of high-density lipoprotein particles (pre-beta 1-HDL) may play an important
role in the reverse cholesterol transport pathway as the initial acceptors of
cellular cholesterol. This study was designed to assess the influence of a 12week aerobic training program in plasma pre-beta 1-HDL concentration in adult
women with metabolic syndrome.
Methods: Sixty young women (39.1±3.5years; 28.7±1.9kg/m2 ) with metabolic
syndrome according to the criteria reported by the National Cholesterol
Education Program Adult Treatment Panel III volunteered for this study. Fortyfive were randomly included in experimental group to perform a 12-week aerobic
training program, 3 days/week, consisting of warm up (10-min), main part in a
treadmill (20−35-min [increasing 5 min each 3 weeks]) at a moderate work
intensity of 60−75% of peak heart rate (increasing 5% each 3 weeks) and cooldown (10-min). Control group included 15 age, sex and BMI-matched women
with metabolic syndrome that did not perform any training program. Plasma
pre-beta 1-HDL levels were measured using ELISA (Daiichi-Pure-Chemicals,
Tokyo, Japan)
Results: When compared to baseline, plasma pre-beta 1-HDL was increased
significantly after a 12-week aerobic training protocol (131.3±11.7 vs.
166.4±13.0 mg/ml serum;p = 0.02). In contrast, no changes were reported in
controls.
Conclusion: Aerobic training at moderate intensity increased plasma prebeta 1-HDL concentration in women with metabolic syndrome. Further longterm well-conducted studies are required in order to highlight potential clinical
benefits of this improvement in these patients.
184 HDL2 LEVELS REDUCE THE ASSOCIATION OF LDL WITH
ARTERIAL PROTEOGLYCANS: A NOVEL ANTI-ATHEROSCLEROTIC
ROLE OF HDL SUBCLASSES
G. Camejo, B. Rosengren, M. Umaerus, E. Hurt-Camejo. Cardiovascular R&D,
AstraZeneca Pharmaceuticals, Mölndal, Sweden
Aims: Participation in reverse cholesterol transport and anti-oxidative and antiinflammatory properties appear responsible for the atheroprotective properties
of ing HDL. In addition, HDL and apoAI have been shown to reduce in vitro the
association of LDL with arterial proteoglycans (Camejo et al Acta Med Scand
642:159;1980). Intimal entrapment of LDL by iproteoglycans is a key step in
atherogenesis (Tabas et al Circulation 116:1832; 2007), therefore is important
to elucidate mechanism by which HDL can interfere with this entrapment.
Methods:
1. Serum and plasma from subjects with insulin resistance (IR), obesity or not,
and type 2 diabetes (T2D) and controls were used to evaluate the affinity
of LDL for isolated arterial proteoglycans and their HDL2 and HDL3 content
using ultracentrifugation in D2 O/sucrose isotonic buffers (Stahlman et al J
Lipid Res 49: 481, 2008).
2. Isolated HDL2 or HDL3 was added to HDL-free plasma and the amount
of LDL complex to proteoglycan was evaluated by measurements of the
insolubilized LDL after centrifugation and electrophoresis.
Results:
1. In plasma of subjects with the dyslipidemia of IR there is a sinificant linear
correlation (r2 = 0.64, p < 0.02) between the absolute content of HDL2 and
the amout of LDL/proteoglycan complex formed.
2. Addition of autologous HDL2 at physiological levels, but not HDL3, to
LDL-containing plasma inhibit the formation LDL/proteoglycan insoluble
complex.
Conclusions: HDL2 appears responsible for reducing the association of LDL
with arterial proteoglycans. If present in vivo this phenomenon may contribute to
the atherogenicity of conditions with low circulating lavels of the HDL2 subclass
as in insulin resistance.
185 HDL MODULATES INSULIN SENSITIVITY AND SECRETION DURING
ACUTE PHASE OF MYOCARDIAL INFARCTION
L.S. Fernandes De Carvalho1 , R.M.R. Cintra1 , A.L.R. Araújo1 , F.A. Moura1 ,
J.C.Q.E. Silva1 , O.R. Coelho2 , A.C. Sposito2 . 1 Universidade de Brası́lia,
Brasilia, 2 Cardiology Division, Faculty of Medical Sciences, Unicamp,
Campinas, Brazil
During myocardial infarction (MI), a transient decrease of both insulin sensitivity
and secretion triggers stress hyperglycemia which is followed by a substantial
increase in mortality. Recent data indicate that HDL modulates insulin sensitivity
and secretion. To date, however, the HDL role on stress hyperglycemia remains
unknown. We explored the influence of HDL on stress hyperglycemia during
acute phase of MI. Consecutive non-diabetic patients with ST-elevation MI
41
(n = 183) were selected from the Brasilia Heart Study for this investigation.
Plasma insulin, glucose and C-peptide were measured in the first 24 hours
and at the fifth day after MI onset. Patients were divided into HDL-C quartiles
for the analyses (Q1: <31, Q2: 31−38, Q3: 38−47 and Q4: >47 mg/dL).
On admission, no difference was found between the quartiles in plasma
glucose (p = 0.6), insulin (p = 0.6), C-peptide (p = 0.5), HOMA2S (p = 0.9) and
HOMA2B (p = 1.0). On the fifth day there was a reduction in blood glucose
whose intensity was directly proportional to HDL-C quartile (−1±32, −13±46,
−19±25, −27±21; respectively, p < 0.001). In parallel, there was a reduction in
plasma insulin [−7.1 (−25, +1), −5.5 (−22, −1), −9.9 (−26, −1), −17.7 (−32, −9);
respectively, p < 0.001] and C-peptide [0 (−3, +1), −1.2 (−3, +1), −1.7 (−3.0),
−2.4 (−4.0); respectively, p < 0.001] whose magnitude was inversely proportional
to HDL-C quartile. Consistently, the change in HOMA2S [+1 (−11, +15),
+7 (−15, +17), +10 (−5, +30), +15 (−8, +28); respectively, p < 0.001] and
HOMA2B [−11 (−43, +31), +16 (−45, +48), +22 (−8, +44), +24 (−23, +61);
respectively, p = 0.01] were also proportional to HDL-C quartiles. This study
provides the first evidence that elevated HDL plasma levels may attenuate
stress hyperglycemia during MI via acceleration of the improvement in insulin
secretion and sensitivity.
186 NOVEL ABCA1 MUTATIONS IN L PATIENTS WITH SEVERE HDL
DEFICIENCY WITH OR WITHOUT CLASSIC MANIFESTATIONS
OF TANGIER DISEASE
P. Zanoni1 , T. Fasano1 , P.B. Deegan2 , A. Park3 , M.D. Feher4 , F. Gurakan5 ,
E. Favari6 , F. Bernini6 , S. Calandra1 . 1 Dpt of Biomedical Sciences, University
of Modena and Reggio Emilia, Modena, Italy, 2 Dpt of Medicine, 3 Dpt of
Clinical Biochemistry, Addenbrooke’s Hospital, Cambridge, 4 Dpt of Beta Cell
Diabetes Centre, Chelsea and Westminster Hospital, London, UK, 5 Unit of
Pediatric Gastroenterology, Hepatology and Nutrition, Hacettepe University
Medical Faculty, Ankara, Turkey, 6 Dpt of Pharmacological and Biological
Sciences and Applied Chemistries, University of Parma, Parma, Italy
Biallelic ABCA1 mutations are the cause of Tangier Disease (TD), a rare
disorder characterized by extremely low HDL-C levels and accumulation of
cholesteryl esters in tissues. We investigated 6 patients with low HDL-C
suspected to have TD.
Two identical twins presented undetectable HDL-C and facial dysmorphic
features. The first twin had died at 36 from CHD, while the second was a
41 yrs male with thrombocytopenia, and splenomegaly. Both were compound
heterozygotes for novel ABCA1 mutations (c.1758 ins G, M586>Fs629X and
H1600R) that abolished ABCA1 mediated cholesterol efflux in fibroblasts.
Two sisters (6 and 37 yrs old) presented with undetectable HDL-C but only
the younger had splenomegaly and anemia. They were homozygous for a
novel splicing mutation (IVS31−34a>g) that caused the formation of a transcript
retaining part of intron 31 and encoding a truncated protein.
A 74 yrs old female had very low HDL-C, mild thrombocytopenia, splenomegaly,
Type 2 diabetes and angina. She was a simple heterozygous for a known
mutation (D1099Y); however, the ABCA1 mediated cholesterol efflux in her
fibroblasts was abolished.
A 37 yrs old male presented with very low HDL-C, high triglycerides but no
clinical manifestations of TD. He was heterozygous for a novel splicing mutation
(IVS10−27 g>a), resulting in a mRNA containing part of intron 10 and encoding
a truncated protein.
We report four novel ABCA1 mutations. The classic signs of TD were not
constant among patients with severe HDL deficiency. Intronic mutations, that are
not uncommon in patients carrying ABCA1 mutations, represent a diagnostic
challenge.
187 SPHINGOSINE KINASE INHIBITION INDUCES BOTH PRO- AND
ANTI-ATHEROGENIC EFFECTS IN LOW-DENSITY LIPOPROTEIN
RECEPTOR-DEFICIENT MICE
S. Costa1 , F. Poti1 , M. Bot2 , V. Bergonzini1 , M. Galletti1 , L. Maynes3 ,
G. Varga4 , M. Simoni1 , J.-R. Nofer1,5 . 1 Dept. of Medicine, Endocrinology,
Metabolism and Geriatrics, University of Modena and Reggio Emilia, Modena,
Italy, 2 Division of Biopharmaceutics, Gorlaeus Leiden/Amsterdam Center
for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden, The
Netherlands, 3 Apogee Biotechnology Corporation Hershey, Hershey, PW,
USA, 4 Institute of Immunology, University of Münster, 5 Center for Laboratory
Medicine, University Hospital Münster, Münster, Germany
Background: Sphingosine 1-phosphate (S1P), a lysosphingolipid associated
with high-density lipoprotein (HDL), contributes to the anti-atherogenic potential
attributed to this lipoprotein. This study examined whether a reduction of S1P
plasma levels affects atherosclerosis in a murine model of disease.
Methods and Results: LDL-R−/− mice on Western diet were given ABC294640,
an inhibitor of sphingosine kinases 1 and 2 for 16 weeks at a dose of 50 mg/kg/d.
ABC294640 decreased plasma S1P by approx. 30−40%. However, ABC294640
failed to affect atherosclerotic lesion formation. Plasma triglycerides were
reduced whereas total and HDL-cholesterol remained unchanged in course of
ABC294640 treatment. ABC294640 increased plasma IL-12p70 and IL-12p70
42
and IFN-gamma production by peritoneal cells and this was paralleled by
enhanced activity of peritoneal and spleen dendritic cells as evidenced by upregulation of CD68 and MHC-II on CD11c+ cells. As a consequence, increased
T-cell activation was noted in ABC294640-treated mice as evidenced by
enhanced CD4+ splenocyte proliferation, IFNgamma and IL-2 production, and
CD69 expression. Concomitantly, however, ABC294640 treatment redistributed
CD4+ and CD8+ cells from blood to lymphatic organs and reduced T-cell
number within atherosclerotic lesions. In addition, plasma sVCAM-1, sICAM1, and MCP-1 levels as well as in vivo leukocyte adhesion and CCL19induced T-cell penetration into peritoneum were lower in ABC294640-treated
animals. In vitro experiments demonstrated reduced VCAM-1 and ICAM-1
expression and lymphocyte adhesion to TNFalpha stimulated endothelial
cells.
Conclusions: Treatment with SK inhibitor leads to both pro- and antiatherogenic effects in LDL-R−/− mice. As a consequence, SK inhibition fails
to affect atherosclerosis despite significant S1P reduction in plasma.
188 DIABETIC DYSLIPIDEMIA IS RESPONSIBLE FOR QUALITATIVE
AND QUANTITATIVE CHANGES IN HDL LIPID COMPOSITION
M. Ståhlman1 , A. Kontush2 , B. Fagerberg1 , K. Ekroos3 , J. Chapman2 ,
J. Borén1 . 1 Wallenberg Laboratory, University of Gothenburg, Gothenburg,
Sweden, 2 INSERM UMR S939; Pitié Salpêtrière Hospital, Paris, France, 3 Zora
Biosciences Oy, Espoo, Finland
In this study we wanted to investigate whether dyslipidemia is required on top
of insulin resistance to induce alterations in HDL lipid composition. We also
wanted to identify possible biomarkers of HDL dysfunction.
Using a combination of robot-assisted lipid extraction, straight phase HPLC
and shotgun lipidomics, the lipid composition of HDL isolated by sequential
ultracentrifugation was investigated. More than 200 individual lipids from 11
lipid classes were quantified.
The results revealed major alterations in HDL isolated from dyslipidemic
T2D subjects. These included increased amount of triacylglycerols and
diacylglycerols, which is probably a consequence of the increased CETP
activity that we observed in these subjects. Other changes in lipid
class composition included reduced amount of membrane lipids (e.g.
sphingomyelin, ceramide and free cholesterol). The level of membrane
lipids correlated well with the particle size as measured by native gel
electrophoresis. Other changes in the dyslipidemic group included reduced
amount of ether-linked phosphatidylcholine and increased amount of lysophosphatidylcholine. Both of these changes might be involved in the
reduced anti-oxidative ability and reduced atheroprotectivity of HDL in
T2D subjects. No changes were observed in the lipid class composition
of HDL isolated from normolipidemic T2D subjects when compared to
controls. The analysis of molecular lipids revealed an increased amount
of saturated fatty acids in the triacylglycerols and diacylglycerols. This
indicates an increased de novo lipogenesis in the liver. Finally, in the
cholesteryl ester and phosphatidylcholine lipid class, lipid species containing
C16:1 and C20:3 was increased indicative of increased hepatic desaturase
activity.
189 APOLIPOPROTEIN A-I STIMULATES BOTH APOLIPOPROTEIN
E AND CHOLESTERYL ESTER TRANSFER PROTEIN SECRETION
IN LIPID LOADED MACROPHAGES
L.S. Niculescu1 , C.S. Stancu1 , M. Robciuc2 , A.V. Sima1 . 1 Lipoproteins and
Atherosclerosis, Institute of Cellular Biology and Pathology ‘N. Simionescu’,
Bucharest, Romania, 2 Public Health Genomics Research Unit, National
Institute for Health and Welfare, Helsinki, Finland
Apolipoprotein E (apoE) and cholesteryl ester transfer protein (CETP) are
secreted by macrophages and macrophage-derived foam cells. Human apoA-I
is a strong inducer for apoE secretion in lipid loaded macrophages, but the
underling mechanism is not completely understood. We aimed to identify the
signaling pathways involved in apoA-I and HDL regulation of the secretion of
apoE and CETP in lipid loaded macrophages. To this purpose, we used human
macrophages (THP-1) loaded with lipids by incubation with oxidized human LDL
(oxLDL). Cells were incubated with human HDL3 or apoA-I purified from human
HDL in the presence of NF-úB (TPCK) or PKA (H89) inhibitors. Real-time PCR
and western blot analysis were used to measure cellular and secreted apoE
and CETP. Results showed that:
i. gene expression of apoE and CETP are increased in the presence of apoA-I
and after inhibition of NF-kB pathway;
ii. apoE secretion depends of the presence of apoA-I and HDL3 and is
stimulated by TPCK;
iii. CETP secretion is stimulated by apoA-I, independently of NF-úB inhibition;
iv. apoA-I inhibits p65 NF-úB subunit;
v. gene expression and secretion of apoE is reduced after inhibition of PKA
signaling pathway, independent of a cholesterol acceptor;
vi. PKA-gamma subunit is activated by oxLDL and apoA-I.
In conclusion, our results provide new insights into the mechanism by which
apoA-I induces apoE secretion and show that CETP secretion from lipid loaded
macrophages is also stimulated by apoA-I.
This study was supported by the Ministry for Education, Research, Youth and
Sport, Romania, grant 1220/2009−11.
190 MAST CELL ACTIVATION IN VIVO IMPAIRS THE MACROPHAGE
REVERSE CHOLESTEROL TRANSPORT PATHWAY IN THE MOUSE
R. Silvennoinen1 , M. Lee-Rueckert1 , N. Rotllan2 , I. Judstrom1 , F. BlancoVaca2 , M. Jauhiainen3 , J. Metso3 , J.C. Escola-Gil2 , P.T. Kovanen1 . 1 Wihuri
Research Institute, Helsinki, Finland, 2 Institut d’Investigacio Biomedica Sant
Pau, Barcelona, Spain, 3 National Institute for Health and Welfare and FIMM,
Biomedicum, Helsinki, Finland
Chymase released by activated mast cells degrades HDL particles. We
evaluated whether local activation of mast cells would attenuate cholesterol
efflux from neighboring macrophage foam cells, thereby disrupting the entire
macrophage-specific reverse cholesterol transport (RCT). C57Bl/6J mice
received intraperitoneal (i.p.) injections of the mast cell-degranulating compound
48/80 to activate peritoneal mast cells, human apoA-I to stimulate RCT, and
[3 H]cholesterol-labeled J774 macrophages for measurement of the RCT rate.
After 3 h, 3 H-radioactivity was measured in the intestinal lumen contents.
Activation of peritoneal mast cells depleted human apoA-I prebeta-migrating
species so impairing the ability of peritoneal fluid to promote efficient cholesterol
efflux from cultured macrophage foam cells. Moreover, intact, but not chymasetreated (proteolyzed) apoA-I, accelerated the transfer of the macrophagederived 3 H-radioactivity to the intestinal contents. Importantly, RCT stimulation
by human apoA-I was fully blocked by 48/80 in mast cell-competent, but not
in mast cell-deficient W-sash c-kit mutant mice. The ability of i.p. administered
phospholipid vesicles to promote RCT in C57Bl/6J mice was unaffected by
48/80, supporting the notion that mast cell-dependent proteolysis of apoA-I
inhibited RCT. Overall, our results suggest that tissue-specific release of mast
cell chymase is able to proteolytically inactivate apoA-I in the microenvironment
of the activated mast cells, so locally impairing the initiation of macrophage
RCT in vivo.
191 CELLULAR CHOLESTEROL EFFLUX CAPACITY AND
ANTIOXIDATIVE ACTIVITY OF HDL SUBPOPULATIONS SHARE
MAJOR COMPOSITIONAL AND STRUCTURAL DETERMINANTS:
ARE COMMON MECHANISMS INVOLVED?
L. Camont1 , M. Lhomme1 , S. Chantepie1 , F. Rached1,2 , R. Frigola1 , W. Le
Goff1 , M.J. Chapman1 , A. Kontush1 . 1 INSERM UMRS 939, Paris, France,
2
Heart Institute (InCor) HCFMUSP − University of São Paulo Medical School,
São Paulo, Brazil
Plasma HDL consist of multiple, highly dynamic particle subpopulations which
are structurally and functionally heterogeneous. The proteome and lipidome
of HDL particles are critical determinants of their biological activities. The
major structural determinants of the diverse atheroprotective functions of HDL
and the molecular mechanisms which underlie individual biological activities
remain to be identified. Lipidomic and proteomic characterisation of five
major HDL subpopulations (HDL2b, 2a, 3a, 3b and 3c) isolated from healthy
normolipidemic subjects by isopycnic density gradient ultracentrifugation
was performed by LC-MS; cholesterol efflux capacities were determined in
macrophage-like human THP-1 cells and anti-oxidative activity characterised
as HDL-mediated protection of LDL by AAPH-induced free radical oxidation.
The cellular cholesterol efflux capacity of HDL subpopulations, assessed
on a unit phospholipid (PL) mass basis, was directly correlated with HDL
protein/PL ratio, wt% of apoA-I and wt% of phosphatidylcholine, and inversely
correlated with wt% of sphingomyelin. Similarly, HDL antioxidative activity
was directly correlated with wt% of apoA-I and negatively correlated with
wt% of sphingomyelin and of free cholesterol; the latter components are
critical in regulating the surface lipid rigidity of HDL particles. Importantly,
cholesterol efflux capacity and antioxidative activity of HDL subpopulations were
significantly correlated (r = −0.82, p < 0.001). These data indicate that two major
biological activities of HDL particles can be driven by common mechanisms
which involve similar structural determinants; transfer of non-oxidized and/or
oxidized lipids to HDL particles modulated by apoA-I content together with the
physicochemical properties of surface phospholipids and free sterols constitutes
a central feature of such mechanisms.
192 AGE-ASSOCIATED DECREASE IN HDL AND PON-1
ANTI-INFLAMMATORY ACTIVITY AND EFFECT OF OLIVE OIL
SUPPLEMENTATION
S. Loued, O. Helal, T. Saiid, A.J. Rouleau, H. Berrougui, A. Khalil. Research
Centre on Aging, University of Sherbrooke, Sherbrooke, QC, Canada
Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated enzyme,
and is one of the most studied genes regarding atherosclerosis risk.
79th EAS Congress
Objectives: This study was aimed to investigate the effect of aging on the antiinflammatory effect of HDL and of PON1. Also to elucidated the effect of 12
weeks of extra-virgin olive oil (EVOO) consumption on this activity.
Methods: HDL and PON1 were isolated from healthy young (29±5.0 years)
and elderly (71±4.0 years) volunteers before and after 12 weeks of EVOO
consumption (25 ml/day). The ICAM in Eahy926 cells was measured by using
FACScan technique. THP1 monocytes chemotaxis assay was measured using
Boyden chamber.
Results: As assessed by the ICAM expression, Y-HDL exhibited the highest
anti-inflammatory activity when compared to E-HDL (28.72% versus 44.22%).
Moreover, we showed an age impact on PON1 anti-inflammatory effect. Indeed,
Y-PON1 incubated in presence of HDL, has a highest anti-inflammatory action
compared to E-PON1 subjects (67.36% versus 87.31% respectively). These
results are confirmed with those obtained following the measurement of THP-1
macrophages chemotaxis. In addition, we showed that twelve weeks of EVOO
consumption increased significantly the anti-inflammatory effect of HDL (ICAM
expression −23%) and particularly in Elderly (−29% versus 17% in Young).
Conclusions: The anti-inflammatory effect of HDL and PON1 decreases with
aging which may explain the increase of the incidence of cardio-vascular risk
in elderly. Supplementation with EVOO may be a good strategy for preventing
the reduction of the anti-inflammatory activity of HDL in the elderly.
193 COMPARISON OF THEORETICAL AND EXPERIMENTAL
RELATIONSHIPS BETWEEN HDL SIZE AND THE RATIO OF HDL
CHOLESTEROL (HDL-C) TO APOLIPOPROTEIN A-I (APOA-I)
N.A. Mazer1 , S. Mora2 . 1 Translational Research Sciences, F. HoffmannLa Roche, Basel, Switzerland, 2 Divisions of Preventative and Cardiovascular
Medicine, Brigham and Women’s Hospital/Harvard Medical School, Boston,
MA, USA
Introduction: Shen (PNAS 1977:74 (3); 837–841) proposed a theoretical model
relating the size, structure and composition of lipoproteins. For HDL, the model
predicts that particle size is correlated with the HDL-C to ApoA-I ratio.
Objective: To compare predictions of Shen’s model with measured data on
HDL size and HDL-C/ApoA-I ratio from the Women’s Health Study (WHS).
Theoretical aspects: Shen’s model assumes that cholesterol esters (CE)
and triglycerides (TG) form a spherical core of radius rc. Phospholipids (PL),
unesterified cholesterol (C) and apolipoproteins (70% being ApoA-I) form a
surface monolayer of thickness t (2.02 nm). The corresponding numbers n_CE.
n_TG, n_PL, n_C and n_ApoA-I per particle are determined by geometric,
compositional and thermodynamic relationships as a function of rc. The HDL-C/
ApoA-I ratio equals (n_CE + n_C)/n_ApoA-I multiplied by the molecular weight
ratio (387/28,000). Particle diameter equals 2(rc + t).
Experimental methods: 26,766 WHS subjects had mean HDL diameter
measured by nuclear magnetic resonance (NMR); HDL-C was measured
enzymatically and ApoA-I by immunoturbidometric assay.
Figure 1
Results: The predicted relationship between HDL diameter and HDL-C/ApoA-I
(shown for different molar ratios of TG/CE) is in reasonable agreement with
experimental data from the WHS study (Pearson r = 0.574) (Figure 1). The
model further predicts that n_ApoA-I varies from 2.9 to 4.7 as HDL diameter
43
increases from 8 to 11 nm, consistent with Kontush’s data (Pharmacol Rev
2006).
Conclusions: As predicted by Shen’s model, HDL size is significantly correlated
with the HDL-C/ApoA-I ratio.
194 HISTONE DEACETYLASE (HDAC) INHIBITORS UPREGULATE
CLUSTERIN/APOLIPOPROTEIN J EXPRESSION IN VASCULAR
SMOOTH MUSCLE CELLS (VSMCS)
F.M. Yatsu1 , K. Ranganna2 , O.P. Mathew2 . 1 Neurology, University of Texas
Health Science Center at Houston, 2 Texas Southern University, Houston, TX,
USA
Objective: Clusterin/Apolipoprotein J (CLU), a secreted heterodimeric
glycoprotein, composed of alpha and beta subunits is induced in VSMCs
during atherosclerosis and injury-induced neointimal hyperplasia, although its
functional roles in VSMCs remain obscure and mysterious. Here our goal is to
confirm and evaluate the significance of CLU upregulation in proliferation arrest
of VSMCs by HDAC inhibitors, butyrate and trichostatin A (TSA).
Methods: VSMC proliferation is assessed by cell counting. CLU, p21Cip1 and
p15INK4B expression is evaluated by western analysis and immunostaining.
CLU knockout is performed with siRNA. Involvement of PI3/AKT pathway is
determined by western blotting.
Results: Contrast to untreated proliferating VSMCs, butyrate and TSA treated
VSMCs exhibit proliferation arrest and upregulation of both cellular and
secretory CLU along with increased levels of cdk/cell cycle inhibitors, p21Cip1
and p15INK4B. Moreover, CLU-specific siRNA inhibits butyrate and TSAinduced upregulation of both cellular and secretory CLU confirming butyrate
and TSA indeed induce clusterin expression and secretion. Furthermore, based
on the phosphorylation state of Akt and impact of PI3/Akt pathway inhibitor,
Ly294002, it appears butyrate- and TSA-induced CLU expression involves
PI3/Akt signaling pathway.
Conclusion: Taken together HDAC inhibitors appear to have antiatherogenic
potential by arresting VSMC proliferation that may be associated with
CLU upregulation, which is suggested to have multiple protective roles in
atherosclerosis and restenosis.
Support: G12RR003045−21 and C06RR012537 from NIH/NCRR and Adriana
Blood Endowment
195 CHOLESTEROL EFFLUX POTENTIAL OF SERA FROM PATIENTS
BEFORE AND AFTER LDL-APHERESIS
E. Favari1 , M.P. Adorni1 , F. Sbrana2 , M. Puntoni2 , F. Bigazzi2 , F. Zimetti1 ,
F. Bernini1 , T. Sampietro2 . 1 Pharmacological and Biological Sciences and
Applied Chemistries, University of Parma, Parma, 2 CNR Institute of Clinical
Physiology, Pisa, Italy
Efflux of free cholesterol from peripheral cells to serum is the first step in reverse
cholesterol transport. Serum cholesterol efflux potential depends on the ability
of individual lipoproteins to interact with specific transporters. LDL-apheresis
may dramatically affect lipoprotein profile by lowering, in addition to LDL,
triglycerides and HDL.
Aims: To study the impact of LDL-apheresis on
1. efflux potential of sera
2. serum ability to induce cholesterol accumulation in macrophages.
Sera were obtained from 15 patients with FH receiving bi-weekly
LDL-apheresis.
Methods: Serum cholesterol efflux potential was tested in pathway-specific,
[3 H]-cholesterol cell models. Cholesterol mass was determined in THP-1 human
macrophages by fluorimetric method.
Results: LDL-apheresis reduced total cholesterol and triglycerides by 70%
and HDL by 30%. Cholesterol efflux by SR-BI, passive diffusion and ABCA1
were reduced by 18.2% (±1.3%; p < 0.001), 23% (±1.3%; p < 0.0001), and
24% (±1.8%; p < 0.0001) respectively. Within 48h LDL increased about two
times compare to the values reached soon after LDL-apheresis treatment, while
HDL and sera efflux potential returned to pre-treatment levels. LDL-apheresis
reduced about 50% serum-induced cholesterol accumulation in human
macrophages. This effect was maintained in the 48h after treatment.
Conclusions: LDL-apheresis:
1. reduces serum cholesterol efflux potential and HDL are the only responsible
for this effect;
2. reduces serum ability to induce macrophage cholesterol accumulation by
both affecting LDL and HDL levels.
44
196 INCREASING CIRCULATING CONCENTRATION OF ALL
HDL PARTICLE SUBCLASSES IN HYPER-RESPONDERS TO
APOLIPOPROTEIN A-I INDUCTION: INSIGHTS FROM THE ASSERT
STUDY
S.J. Nicholls1 , A. Gordon2 , J. Johansson2 , C.M. Ballantyne3 , J.J. Kastelein4 ,
N.C. Wong5 , M. Borgman1 , K. Wolski1 , S.E. Nissen1 . 1 Cleveland Clinic,
Cleveland, OH, 2 Resverlogix Corporation, San Francisco, CA, 3 Baylor College
of Medicine, Houston, TX, USA, 4 Academic Medical Center, Amsterdam, The
Netherlands, 5 Resverlogix Corporation, Calgary, AB, Canada
Background: Apolipoprotein A-I (apoA-I) induction represents a novel
therapeutic approach to promoting high-density lipoprotein (HDL) functionality.
The relationship between changes in conventional HDL measures and particle
markers with apoA-I induction has not been investigated.
Methods: The ASSERT study evaluated early biochemical changes with
increasing doses of an apoA-I inducer (RVX-208 100–300 mg daily) for 12
weeks in statin-treated patients with stable coronary artery disease. Changes
in HDL particle markers on 2D gel electrophoresis and NMR spectroscopy were
characterized.
Results: Administration of RVX-208 was associated with dose-dependent
increases in levels of apoA-I (0.1−5.6%), HDL cholesterol (3.2−8.3%), large
HDL particles (11.1–21.1%) on NMR and larger a1 particles (3.7−8.8%), but
no change in small, lipid-deplete preb1 particles on 2D gel analysis. Significant
correlations were observed between changes in HDL cholesterol and changes
in total HDL particles (r = 0.44, p < 0.0001), large HDL (r = 0.55, p < 0.0001),
small HDL (r = 0.22, p = 0.0002), preb1 (r = 0.16, p = 0.009) and a1 (r = 0.58,
p < 0.0001) particles. Patients in the highest quartile increase in HDL cholesterol
(>14%) demonstrated greater increases in HDL particle concentration (+10.1 vs
−3.1%, p < 0.001) and size (+2.3 vs 0%, p < 0.001), large HDL particles (+46.9
vs −9.1%, p < 0.001), preb1 particles (+6.6 vs −5.1%, p = 0.04) and a1 particles
(+29.5 vs −20.9%, p < 0.001) compared with lowest quartile.
Conclusion: While changes in larger HDL particles predominate with apoA-I
induction, patients with the most robust lipid response demonstrated increases
in both small and large HDL particles. The functional consequences of these
changes on atherosclerotic plaque remain to be determined.
197 LYCOPENE REDUCES INFLAMMATION AND IMPROVES HDL
FUNCTIONALITY IN OVERWEIGHT INDIVIDUALS
J. McEneny1 , L. Wade1 , A. Mcginty1 , I. Young1 , F. Thies2 . 1 Centre for Public
Health, Queen’s University Belfast, Belfast, 2 School of Medicine and Dentistry,
Aberdeen University, Aberdeen, UK
Background: Cardiovascular disease (CVD) is the leading cause of morbidity
and mortality in the developed world, and its incidence increases with increasing
BMI. Dietary factors, such as increased lycopene intake, may lower CVD risk.
Objectives: To assess if increasing lycopene intake reduces the inflammatory
marker serum amyloid-A (SAA) and its incorporation into high density lipoprotein
(HDL), helping to restore HDL’s anti-atherogenic functionality.
Design: Following a 4-week washout, serum was collected before and
after an 8-week intervention from 54-overweight (25.6±0.47Kgm2 ) middleaged (50.4±1.0years) individuals. Subjects were randomised to one of three
groups, Group-1) lycopene-rich diet (equivalent to 70 mg/week); Group-2)
lycopene supplement (10 mg/day); Group-3) lycopene-deplete diet (18 per
group). HDL was subfractionated into HDL2&3 by rapid ultracentrifugation.
Serum and HDL2&3 were assessed for lycopene incorporation (HPLC) and
SAA levels (ELISA). The arylesterase activity of HDL2&3 (PON-1) was assessed
spectrophotometrically.
Results: Within group analyses found that lycopene was significantly increased
in serum and HDL2&3 in both treatment groups, but was unaltered in the control
group. Furthermore, SAA decreased in HDL3 in the diet group (p = 0.026) and in
serum and HDL2&3 in the supplement group (all p < 0.05). Additionally, PON-1
activity increased in HDL2&3 in both treatment groups (Group-1, p = 0.000 &
0.035: Group-2, p = 0.018 & 0.000, respectively). Between group analyses found
that, compared to the control group, SAA was lower in HDL3 (p < 0.05), while
PON-1 activity was higher in HDL2 (p < 0.006) in both treatment groups.
Discussion: These results demonstrate that increasing lycopene intake
produced beneficial changes within HDL which helped restore its antiatherogenic functionality.
198 EFFECT OF DALCETRAPIB ON NON-CHOLESTEROL MARKERS
OF CHOLESTEROL HOMEOSTASIS IN HEALTHY SUBJECTS
E.J. Niesor1 , E. Chaput1 , A. Staempfli2 , M. Derks3 , D. Kallend4 .
1
Discovery Research Metabolic Disease, 2 Discovery Technologies, 3 Clinical
Pharmacology, 4 Pharma Development Metabolism, F. Hoffmann-La Roche
Ltd, Basel, Switzerland
Background and Aims: Markers of cholesterol homeostasis correlate with
plasma HDL-C in different populations devoid of intervention (cholesterol
absorption markers correlate positively, while synthesis markers correlate
negatively, with HDL-C). We measured the effect of dalcetrapib (CETP
modulator that raises HDL-C) on markers of cholesterol homeostasis.
Methods: Randomised, open-label, crossover study in 22 healthy subjects.
Three 7-day treatment periods (6 sequences): dalcetrapib 900 mg OD,
ezetimibe 10 mg OD, dalcetrapib 900 mg OD + ezetimibe 10 mg OD with
between-treatment washout 10−14 days. Blood samples were collected pretreatment (Day 1) and post-treatment (Day 7). Plasma lipids and non-cholesterol
sterol markers of cholesterol absorption (campesterol, b-sitosterol, cholestanol)
and synthesis (lathosterol, desmosterol) were determined and expressed as
percent change in non-cholesterol:cholesterol ratio.
Results: HDL-C increased with dalcetrapib (+27%, p < 0.0001) and when
co-administered with ezetimibe (+31%, p < 0.0001) but not with ezetimibe
alone (−5%, p = 0.03). Respective changes in LDL-C were −16, −36%
and −21% (each p < 0.0001). Absorption markers: Dalcetrapib increased
campesterol, b-sitosterol, and cholestanol by +27 (p = 0.001), +32 (p < 0.001),
and +12% (p = 0.03), respectively. In contrast co-administration with ezetimibe
reduced campesterol marginally by −11% (p = 0.02) while b-sitosterol and
cholestanol were unaffected. Synthesis markers: lathosterol and desmosterol
were unchanged by dalcetrapib treatment but increased with ezetimibe alone
(48−56%, p < 0.001) or combined with dalcetrapib (32−38%, p < 0.001).
Conclusions: Dalcetrapib increases HDL-C and alters cholesterol homeostasis
towards increasing markers of absorption as observed in the general population.
This correlated with HDL-C levels suggesting dalcetrapib treatment preserves
HDL functions involved in maintaining total cholesterol balance.
199 CERAMIDE AS A POTENTIAL LIPIDOME BIOMARKER OF HDL
FUNCTIONALITY: RELEVANCE TO POTENT ANTI-APOPTOTIC
ACTIVITY IN SMALL, DENSE HDL3
A. Kontush1,2,3 , M. Stahlman4 , M. Lhomme1,2,3 , J. de Souza1,2,3 , J. Boren4 ,
M.J. Chapman1,2,3 . 1 INSERM UMRS 939, 2 Université Pierre et Marie Curie −
Paris 6, 3 Hospital Pitié-Salpêtrière, Paris, France, 4 Sahlgrenska Center for
Cardiovascular and Metabolic Research/Wallenberg Laboratory, Goteborg
University, Goteborg, Sweden
Atheroprotective HDL particles are heterogeneous in structure, metabolism and
function. Knowledge of the lipidome of HDL particle subpopulations at the
molecular level may provide insight into their metabolic interrelationships and
distinct biological properties.
Five major HDL subpopulations (HDL2b, 2a, 3a, 3b, 3c) were isolated
from plasmas of healthy normolipidemic subjects (n = 6) by isopycnic density
gradient ultracentrifugation. Some 200 individual lipid molecules representing
11 lipid classes were determined by LC/MS/MS analysis, in order of
decreasing abundance: cholesteryl esters ≈ phosphatidylcholine > free cholesterol ≈ triacylglycerides > sphingomyelins > phosphatidylethanolamine ≈
ester phosphatidylcholine > phosphatidylinositol > lysophosphatidylcholine >
diacylglycerides > ceramides. Anti-apoptotic activity of HDL particles was
evaluated as their capacity to protect human endothelial cells from apoptosis
induced by oxidised LDL and expressed as %inhibition of cellular annexin V
binding.
As HDL particle size and molecular weight decreased with progressive
increment in density, particle lipid content diminished concomitantly. On a wt%
basis, free cholesterol, sphingomyelin and ceramide abundance diminished
in parallel with increase in HDL density from HDL2b to HDL3c; in contrast,
the abundance of lysophosphatidylcholine increased. The anti-apoptotic activity
of HDL particle subpopulations was elevated in small dense HDL3, and was
inversely related to content of ceramide, consistent with the potent proapoptotic
actions of the latter.
These data suggest that lipidomic analysis can identify compositional
determinants of HDL functionality. Specifically, diminished abundance of
ceramide in small HDL3 may contribute to elevated anti-apoptotic activity;
furthermore, enhanced content of lysoPC and diminished content of free
cholesterol suggests that small, dense HDL3 are a major substrate of
cholesterol trans-esterification and LCAT activity.
200 ARE HDL PARTICLES DYSFUNCTIONAL IN ACUTE CORONARY
SYNDROME PATIENTS WITH STEMI AND LOW HDL-CHOLESTEROL
LEVELS: IMPACT OF STATIN?
F.H. Rached1,2 , C.V. Serrano Jr.2 , L. Camont1 , A.L. Pinto2 , M. Barros2 ,
W. Goff1 , J. Chapman1 , A. Kontush1 . 1 INSERM UMR S939; Pitié Salpêtrière
hospital, Paris, France, 2 Heart Institute (InCor) HCFMUSP − University of São
Paulo Medical School, São Paulo, Brazil
Low plasma levels of HDL-cholesterol are typical of Acute Coronary Syndromes
(ACS) and predict risk of recurrent cardiovascular events. Is such high risk
related to HDL-mediated atheroprotection? Blood was withdrawn (i) during the
first 24 hours after diagnosis in the absence of statin therapy (Phase I) and
(ii) 8 days after initiation of therapy (Phase II). Patients with STEMI displayed
low HDL-C levels. For evaluation of HDL functionality, HDL subpopulations
were fractionated from plasmas of ACS patients (n = 11) and matched healthy
controls (n = 10); physicochemical properties, cholesterol efflux capacities
in macrophage-like human THP-1 cells, and capacity to protect LDL from
oxidative stress were assessed. As compared to their counterparts in controls,
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dense HDL3b and 3c subpopulations from ACS patients in Phase I exhibited
diminished antioxidative activity (up to −35%, p < 0.05, on a particle mass basis).
In addition, HDL3b from ACS patients displayed reduced cholesterol efflux
capacity (−22%, p < 0.05) relative to controls. Moreover, all HDL subpopulations
from ACS patients in Phase I displayed altered chemical composition, with
enrichment in core triglyceride and depletion in cholesteryl ester (up to +65%
and −30% respectively), suggesting altered CETP activity. Marked decrease
in the abundance of apoAI and apoAII in dense HDL3 potentially reflecting
binding of acute phase proteins was equally observed. Statin therapy enhanced
the defective antioxidative activity of HDL3 after 8 days of treatment (+18%,
p < 0.05). Clearly, the atheroprotective activities of small, dense HDL3 particles
are impaired in the early phase of ACS; such anomalies are attenuated by statin
treatment
201 ATORVASTATIN AND HORMONE REPLACEMENT THERAPY
AFFECT EXPRESSION OF GENES REGULATING THE REVERSE
CHOLESTEROL TRANSPORT IN MONONUCLEAR CELLS FROM
POSTMENOPAUSAL WOMEN
A. Cerda1 , F.D. Genvigir1 , C.B. Rohde1 , M. Issa1 , S.A. Cavalli1 ,
M.C. Bertolami2 , M.H. Hirata1 , R.D. Hirata1 . 1 School of Pharmaceutical
Sciences/University of São Paulo, 2 Dante Pazzanese Institute of Cardiology,
São Paulo, Brazil
Menopause status is associated with changes in lipid levels that result in
increased risk of development of atherosclerosis and cardiovascular events.
Hormone replacement therapy (HRT) and atorvastatin have been used to
improve lipid profile lowering rates of heart diseases in postmenopausal
women. Effects on serum lipids and expression profile of genes involved in
the reverse cholesterol transport (RCT) − APOA1, ABCA1, ABCG1, SCARB1
and LXRa − were evaluated in peripheral blood mononuclear cells (PBMC)
from 87 postmenopausal hypercholesterolemic women treated with atorvastatin
(AT, n = 17), estrogen or estrogen plus progestagen (HRT, n = 34) and estrogen
or estrogen plus progestagen associated with atorvastatin (HRT+AT, n = 36).
Gene expression was measured by TaqMan® quantitative PCR, using HPRT1
as reference gene. Total cholesterol (TC), LDL-c and apoB were reduced
after each treatment (p < 0.001) but no difference was observed in HDL-c
(p > 0.05). Triglycerides, VLDL-c and apoAI were reduced by atorvastatin,
whereas triglycerides and VLDL-c were increased after HRT. HRT women
had lower reduction on TC, LDL-c and apoB than AT and HRT+AT groups
(p < 0.05). ABCA1 expression was reduced after all treatments, whereas APOA1
was increased by atorvastatin and SCARB1 was reduced after HRT (p < 0.05).
Although expression of LXRa, an important transcription factor controlling
expression of genes involved in RCT, was not modified after treatments, it
was correlated with ABCA1, APOA1 and SCARB1 RNAm values before and
after treatments (p < 0.05). In conclusion, HRT and atorvastatin improve the
lipid profile; however these treatments have a differential impact on expression
profile of genes controlling the RCT in PBMC.
202 TANGIER DISEASE: A PLASMA PROTEOME APPROACH
M. Puntoni1 , C. Boccardi1 , F. Sbrana2 , S. Rocchiccioli1 , F. Bigazzi2 , L. Citti1 ,
T. Sampietro1 . 1 Institute of Clinical Physiology − CNR, 2 Fondazione G.
Monasterio CNR-Regione Toscana, Pisa, Pisa, Italy
Background: Tangier disease (TD) is a rare autosomal recessive disorder
characterized by a deficiency or absence of high-density lipoprotein caused
by mutations in the adenotriphosphate-binding cassette transporter-1 gene
(ABCA1). These lead to a defect in cellular cholesterol removal causing the
deposition of cholesterol esters throughout reticuloendothelial system.
Methods: We enrolled a homozygous TD patient and the heterozygous father.
Whole plasmatic extracts were processed and mass spectrometry analyses
of peptide fragments were performed on MALDI TOF/TOF equipment. Trypsin
digestion produces a constant set of peptide fragments distinctive of each
starting protein (finger print). Peptide ions are automatically processed by
MASCOT and GPS Explorer software, which provide us the identification
of starting proteins. Peaks, indicative of different peptides, also subjected
to mass/mass analysis by means of fragmentation in a collision chamber.
Such second mass analysis provided aminoacid sequence of peptide and the
identification of possible post-translational modifications.
Results: The very high sensitivity of the method allowed us to identify plasma
proteins less peresented (<1.2 pg/ml). Apolipoprotein A-I, haptoglobin, alpha-2
macroglobulin, fibrinogen beta chain and isoform 1 of alpha-1 antitrypsin (C.I.
95%) resulted to be downregulated, while serotransferrin and Ig kappa resulted
upregulated in the homozygous TD patient respect to the heterozygous. The
same pattern was also observed by routine assays.
Conclusions: The downregulation of the acute phase proteins observed in this
case was unexpected. Proteome analyses may help in identification of abnormal
metabolic pathways eventually activated in TD.
45
203 EFFECT OF EZETIMIBE ON COMPONENTS OF HDL SUBCLASS
IN PATIENTS WITH END-STAGE RENAL DISEASE
Y. Ito1 , T. Hirano2 , K. Nohtomi2 , N. Nakanishi2 , T. Watanabe3 , T. Hyodo4 ,
T. Taira4 . 1 Research and Development Department, Denka Seiken Co., Ltd.,
Niigata, 2 Department of Diabates, Metabolism and Endocrinoligy, 3 Department
of Biochemistry, Showa University School of Medicine, Tokyo, 4 Department of
Nephrology, Yokohama Daiichi Hospital, Kanagawa, Japan
Background and Aims: The aim of this study was to investigate the effects
of Ezetimibe monotherapy on HDL subclass in patients with end-stage renal
disease (ERSD).
Methods: 26 ERSD patients receiving hemodialysis (HD) were given ezetimibe
(10 mg/d) for 6−8 weeks. HDL3 was separated from serum by a single
precipitation method established by our group. The components of HDL3 were
measured after separation. HDL2 was estimated by subtracting HDL3 from total
HDL.
Results: Ezetimibe significantly reduced RLP-C, LDL-C, and apo B without
affecting triglyceride, HDL-C and LCAT activities. HDL2-C levels were lower
and HDL3-C was substantially lower in the HD patients than in the controls.
Ezetimibe increased HDL2-apoAI but decreased HDL3-apoAI without affecting
serum apoAI or AII. Ezetimibe decreased HDL-SAA by 43%, and this inhibitory
effect was exclusively attributable to a 72% reduction in HDL3-SAA in response
to the ezetimibe treatment. The reduction of HDL3-SAA was significantly
associated with increased HDL2-apo AI and reduced HDL3-apo AI.
Conclusions: Ezetimibe treatment decreased “inflammatory” (SAA-containing)
HDL3, and may thus have restored the anti-atherogenic function of HDL
particles in ESRD patients.
204 OLIVE OIL CONSUMPTION EFFECT ON CHOLESTEROL EFFLUX
AND INFLAMMATION MARKERS
O. Helal, H. Berrougui, A. Khalil. Medecine, CDRV Sherbrooke University,
Sherbrooke, QC, Canada
The ATP-binding cassette transporters ABCA1 and ABCG1 play a crucial
role in the cholesterol efflux (CE) from macrophages to apoA-1 and to HDL
respectively. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine
secretion by modulating cholesterol content in the plasma. Several studies have
shown that extra virgin olive oil (EVOO) consumption may have hypocholesterol
and anti-inflammatory action.
Aims: The aims of this study were to examine the effect of 12 weeks of EVOO
consumption on inflammation and CE and the possible link between these two
parameters.
Methods: Whole HDL was isolated from 26 healthy volunteers before and after
12 weeks EVOO consumption (25 ml/day). HDL-mediated CE was evaluated
using THP-1 and ABCA1-upregulated J774 cells. The human inflammatory
markers (ICAM and E-Selectin) were measured by ELISA.
Results: Our results show that 12 weeks of EVOO consumption induces a
significant reduction of ICAM (−8.8%, p < 0.001) and E-Selectin levels (−7.9%,
p < 0.040) and a significant increase in the capacity of HDL to mediate CE from
THP-1 and ABCA1-upregulated J774 cells (+13.6% and +23.3% respectively).
Moreover, our result show that at baseline (before EVOO consumption), the
E-selectin level was positively and significantly correlated to the capacity of HDL
to mediate CE from THP-1 and from J774 macrophages (r = 0.534, p < 0.033
and r = 0.653, p < 0.005 respectively) but this correlations disappeared after 12
weeks of EVOO consumption.
Conclusion: Our data confirm the relationship between HDL-mediated
cholesterol efflux and inflammation in a process partly dependent on cholesterol
efflux via ABCA1 and ABCG1.
205 EFFECT OF CHRONIC RENAL FAILURE ON SERUM AND
HDL CHOLESTEROL EFFLUX CAPACITY AND MACROPHAGE
ABCA1/ABCG1 EXPRESSION
S. Schreier1 , M. Hollaus1 , E. Pemberger1 , S. Fruhwürth1 , C. Röhrl1 ,
F. Liebscher1 , B. Englinger1 , J. Becker2 , A. Schmidt3 , H. Scharnagl4 ,
C. Aufricht2 , H. Stangl1 , W. Strobl1 . 1 Dept. Medical Chemistry, 2 Dept.
Pediatrics, 3 Dept. Internal Medicine III, Medical University Vienna, Vienna,
4
Dept. Laboratory Medicine, Medical University Graz, Graz, Austria
Background: Patients with chronic renal failure (CRF) rapidly develop extensive
atherosclerosis. In CRF uptake of carbamylated LDL (cLDL) by macrophages
contributes to foam cell formation. The effects of CRF on the transporters and
acceptors involved in cholesterol efflux, however, are unclear.
Aims: To study the effect of cLDL on macrophage ABCA1 and ABCG1
expression and to determine whether CRF alters the capacity of serum or HDL
to accept cellular cholesterol (cholesterol efflux capacity, CEC).
Methods: THP-1 cells were incubated with cLDL vs. native LDL and
ABCA1/ABCG1 mRNA and protein was measured. CEC was determined for
serum, HDL and apoB depleted serum from 11 adult and 12 pediatric patients
with CRF (DOQI 3−5) without dialysis and from matched controls, and for HDL
carbamylated in vitro (cHDL) using [3 H] cholesterol loaded THP-1 macrophages
treated with LXR agonist or primary human monocyte/macrophages.
46
Results: Incubation of THP-1 cells with cLDL increased cellular free cholesterol
and cholesteryl ester, ABCA1 and ABCG1 mRNA and protein (1.5 to 3 fold,
p < 0.05) and [3 H] cholesterol efflux to apoA-I twofold (p < 0.05). The CEC of
serum, HDL, and apoB depleted serum did not differ between patients and
controls. The CEC of cHDL, however, was reduced to 50±10% of control
(p < 0.05), whereas the specific cell association of [125 I]cHDL was increased.
Conclusion: CRF does not appear to alter the CEC of HDL or serum. The
increase of ABCA1/ABCG1 expression and cholesterol efflux to apoA-I in
macrophages exposed to cLDL may represent a compensatory mechanism
to reduce cholesterol accumulation.
206 SKIN AUTOFLUORESCENCE IS INVERSELY RELATED TO HDL
ANTI-OXIDATIVE CAPACITY IN TYPE 2 DIABETES MELLITUS
D.J. Mulder1 , J.F. De Boer2 , R. Graaff3 , R. De Vries4 , W. Annema2,5 ,
J.D. Lefrandt1 , A.J. Smit1 , U.J.F. Tietge2,5 , R.P.F. Dullaart4 . 1 Department of
Internal Medicine, 2 Department of Pediatrics, Center for Liver, Digestive and
Metabolic Diseases, 3 Department of Biomedical Engineering, 4 Department of
Endocrinology, University Medical Center Groningen, Groningen, 5 TI Food
and Nutrition, Wageningen, The Netherlands
Objectives: High density lipoprotein (HDL) particles protect lipoproteins from
oxidative modification. An impaired anti-oxidative functionality of HDL in type 2
diabetes mellitus (T2DM) might contribute to enhanced formation of oxidative
stress products, such as Advanced Glycation Endproducts (AGEs). We tested
whether in T2DM the HDL anti-oxidative capacity is related to the accumulation
of AGEs in the skin.
Methods: Skin autofluorescence (AF), a non-invasive marker of AGEs, and
HDL anti-oxidative capacity, i.e. the ability of HDL to protect against LDL
oxidation in vitro, were assessed in 67 non-smoking T2DM patients without
complications (median age: 60 (53−65), 60% males, 6.5 (5.2−8.5) years of
diabetes duration).
Results: HDL anti-oxidative capacity correlated inversely with plasma glucose,
HbA1c , and triglycerides (P < 0.05 to <0.001) as well as with skin AF in
univariate analysis (r = −0.306, P < 0.02). Multiple linear regression showed
that skin AF remained inversely related to HDL anti-oxidative capacity (partial
r = −0.324, P = 0.011) taking account of age, sex, plasma glucose, HbA1c, total
cholesterol/HDL cholesterol ratio and triglycerides.
Conclusions: Skin AF is inversely related to the anti-oxidative ability of
HDL. This suggests that a decreased anti-oxidative functionality of HDL may
contribute to the tissue accumulation of AGEs in tissues and thereby also to
long-term diabetic complications.
207 A HIGH-FAT CHOLESTEROL-CONTAINING DIET PROMOTES
REVERSE CHOLESTEROL TRANSPORT FROM MACROPHAGES
TO FECES IN VIVO THAT DEPENDS ON ABCG5/G8
J.C. Escolà-Gil1,2 , G. Llaverias1,2 , J. Julve1,2 , D. Santos2 , J. MéndezGonzález1 , F. Blanco-Vaca1,2 . 1 IIB Sant Pau, 2 CIBER de Diabetes y
Enfermedades Metabólicas Asociadas. CIBERDEM, Barcelona, Spain
Background: A high saturated fatty acid- and cholesterol-containing (HFHC)
diet is considered an important contributor to atherosclerosis. However, HFHC
diet increases plasma cholesterol and HDL cholesterol levels. In the present
study, we determined the effects of this atherogenic diet on reverse cholesterol
transport (RCT) from macrophages to feces in vivo.
Methods: [3 H]cholesterol-labeled mouse macrophages were injected intraperitoneally into mice fed a low-fat low-cholesterol (LFLC) diet, a HFHC diet or the
HFHC diet without cholesterol added (HFLC) for 8 weeks, and radioactivity was
determined in plasma, liver and feces.
Results: As expected, HFHC diet caused a significantly increase in plasma
cholesterol, HDLc and liver cholesterol. HFHC diet, but not LFLC or HFLC
diets, enhanced macrophage-derived [3 H]cholesterol flux to feces of C57BL/6
mice. These changes were sex-independent and not associated with obesity
or insulin resistance. Unlike LFLC or HFLC diets, HFHC diet increased liver
ABCG1 expression and increased [3 H]cholesterol efflux to plasma by 40%.
HFHC diet also upregulated liver ABCG5/G8, which was associated with a
3-fold increase in HDL-derived [3 H]tracer fecal excretion, while plasma HDL
fractional catabolic rate remained unchanged. Interestingly, HFHC diet had no
effect on fecal macrophage-derived [3 H]cholesterol excretion in ABCG5/G8deficient mice.
Conclusion: Despite its deleterious effect on atherosclerosis, HFHC diet
promotes macrophage-to-feces RCT. Our data provide direct in vivo evidence
of the crucial role of dietary cholesterol and liver ABCG5/G8 in HFHC dietmediated induction of macrophage-specific RCT.
208 THE ANTIATHEROGENIC PROPERTIES OF HDL AFTER BARIATRIC
SURGERY
O. Amin Hussein1,2 , I. Shams3,4 , K. Abu Jabal3 , I. Waksman5 , I. Dashkovsky5 ,
S. Szvalb6 , N. Volkova7 , B. Fuhrman7 , M. Aviram7 . 1 Internal Medicine a,
Ziv Medical Center, Safed, 2 Faculty of Medicine, Technion, Haifa, 3 Internal
Medicine A, 4 Laboratory for Atherosclerosis Research, 5 Surgery Department,
6
Pathology Department, Ziv Medical Center, Safed, 7 Lipid Research
Laboratory, Rappaport Faculty of Medicine, Technion-Israel Institute of
Technology, Haifa, Israel
Background: High-density lipoprotein cholesterol (HDL-C) may not always be
atheroprotective and in some conditions, it paradoxically enhances the process
of atherosclerosis.
The cardiovascular protective effects of HDL have been mainly attributed to its
role in reverse cholesterol transport and to many other protective properties
towards atherosclerosis as anti-oxidative effect.
Aim of the study: To investigate the functional properties of HDL in patients
after bariatric surgery.
Patients and Methods: Forty candidate patients for bariatric surgery were
enrolled. Blood was drawn before, 3 weeks (n = 19) or 6 months (n = 21)
after bariatric surgery. Serum PON arylesterase activity was measured by
using phenylacetate as substrate. Serum lipid peroxides (PD) were measured
spectrophotometrically at 340 l. PD was measured at baseline and after
oxidation with 2,2 -Azobis(2-methylpropionamidine) dihydrochloride (AAPH).
[3 H]-Cholesterol efflux was measured by incubating J744 macrophages with
serum. Radioactivity was measured by b counter in the cell lysate and the
medium.
Statistical methods: Student’s t-test for paired samples was used. p < 0.05 was
cosidered significant.
Results: Serum mediated cholesterol efflux did not change after 3 weeks, but
increased significantly by 14% after 6 months. Both basal and AAPH-oxidized
peroxides were significantly decreased 3 weeks and 6 months after surgery.
PON lactonase and arylesterase activities decreased and liver function tests
normalized already within 3 weeks.
Conclusion: Bariatric surgery reduced serum peroxides as early as 3 weeks
while the increase in serum-mediated cholesterol efflux was observed after 6
months.
209 GLYCOXIDATION IMPAIRS THE HDL-MEDIATED OXYSTEROLS
EFFLUX AND INDUCES STEROL ACCUMULATION IN
MACROPHAGES
R. Tallada Iborra1 , A. Machado-Lima1 , G. Castilho1 , V.S. Nunes1 ,
E.R. Nakandakare1 , D.S. Abdalla2 , M. Passarelli1 . 1 Lipids Laboratory (LIM-10),
Faculty of Medical Sciences, University of São Paulo. São Paulo, Brazil.,
2
Department of Clinical and Toxicology Analysis, Faculty of Pharmaceutical
Sciences, University of São Paulo, São Paulo, Brazil
Introduction: Advanced glycation reduces the expression of ABCA-1 and
ABCG-1, inducing lipid accumulation in macrophages (macs). We analyzed
the content of total sterols and oxysterol subfractions in macs submitted to
glycoxidation.
Methods: J774 macs were incubated with oxidized LDL (50 mg/mL of DMEM) for
43 h and then treated with 0.5 mM glycolaldehyde (GAD-macs) or DMEM alone
(C-macs) for 5h, in the absence or presence of the LXR-agonist, T0901317
(1mM). Cells were carefully washed and incubated with 50 mg/mL of HDL (5
h) in the absence or presence of T0901317. Oxysterols and total cholesterol
were determined by gas chromatography/mass spectrometry in cell and culture
media lipid extracts by utilizing 5a cholestane as internal standard.
Results: In incubations with HDL+T0901317 the total content of intracellular
sterols (cholesterol + oxysterols) and of cholesterol-5a,6a-epoxide and
cholesterol-5b,6b-epoxide was, respectively, 1.4, 2.0 and 2.2 times higher in
GAD-macs as compared to C-macs. In culture media, total sterols content was
similar between C and GAD-macs and increased similarly after addition of HDL
or HDL+T0901317. A 20% and 23% reduction was observed in the amount
of respectively 7-ketocholesterol and 7b-hydroxycholesterol in culture media
from GAD-macs incubated with HDL alone. The addition of HDL+T0901317
increased these oxysterols similarly to C-macs.
Conclusion: Glycoxidation reduces the exportation of 7-ketocholesterol and
7b-hydroxycholesterol to HDL and induces macrophage sterol accumulation.
This may contribute to atherogenesis in DM.
Funding: FAPESP (Brazil)
79th EAS Congress
210 KEY ROLE OF LOW HDL CHOLESTEROL FOR THE ASSOCIATION
OF METABOLIC SYNDROME WITH INFLAMMATION IN PATIENTS
WITH PERIPHERAL ARTERIAL DISEASE
P. Rein1,2,3 , J. Ortmann4 , C.H. Saely1,2,3 , C. Boehnel1,3 , S. Beer1,2,3 ,
A. Vonbank1,2,3 , I. Baumgartner4 , H. Drexel1,2,3,5 . 1 VIVIT Institute, 2 Internal
Medicine, Academic Teaching Hospital Feldkirch, Feldkirch, Austria, 3 Private
University of the Principality of Liechtenstein, Triesen, Liechtenstein,
4
Department of Angiology, Inselspital Bern, Bern, Switzerland, 5 Drexel
University College of Medicine, Philadelphia, PA, USA
Background: The association of the metabolic syndrome (MetS) and of the
individual MetS stigmata with inflammation in patients with peripheral arterial
disease (PAD) has not been investigated yet.
Methods: We enrolled 410 consecutive patients who underwent routine duplex
sonography for the evaluation of suspected or established PAD Fontain stages
I-III and in whom PAD was verified sonographically. According to National
Cholesterol Education Programme Adult Treatment Panel III criteria, the MetS
was defined in the presence of at least 3 out of the 5 quantitatively defined
criteria large waist circumference, low HDL cholesterol, high triglycerides, high
blood pressure, and elevated fasting glucose.
Results: In univariate analyses, CRP was higher in patients with the
MetS (n = 200) than in those who did not have the MetS (0.94±1.88 vs.
0.56±1.18 mg/dl; p = 0.001), and also was higher in patients who fulfilled
the large waist (0.93±1.93 vs.0.59±1.16 mg/dl; p = 0.009) and the low HDL
criteria (1.10±1.66 vs. 0.61±1.52 mg/dl;p < 0.001) than in those who did not.
Importantly however, after adjustment for gender, smoking, BMI and LDL
cholesterol by means of ANCOVA only the low HDL cholesterol criterion
(F=6.06;p = 0.014) remained significantly associated with CRP. The significant
and independent association of low HDL with CRP was confirmed after
additional adjustment for all other MetS traits (F=7.76; p = 0.006).
Conclusion: We conclude that among patients with PAD, low HDL cholesterol
drives the association between the MetS and subclinical inflammation. This
observation is well in line with the paramount role of low HDL cholesterol as a
marker of cardiovascular risk.
211 HDL LOSES ITS ANTI-INFLAMMATORY PROPERTY AND EVEN
INDUCES MORE INFLAMMATION IN S100B OR LPS-STIMULATED
MACROPHAGES PREVIOUSLY EXPOSED TO AGE-ALBUMIN
L. Shimabukuro Okuda, G. Castilho, D.D.F.M. Rocco, E.R. Nakandakare,
S. Catanozi, M. Passarelli. Lipid Laboratory − LIM10, Faculty of Medical
Science − São Paulo University, São Paulo, Brazil
We investigated if HDL is able to prevent the release of inflammatory markers
by macrophages treated with advanced glycated albumin (AGE-albumin) and
S100B calgranulin or lypopolissacharides (LPS).
Low endotoxin AGE-albumin was prepared by incubating albumin with 10mM
glycolaldehyde and control (C) albumin with PBS alone (4 days, 37ºC).
Cholesterol-enriched macrophages were treated with C or AGE-albumin
(2 mg/mL) in the absence or presence of HDL (100 mg/mL) for 72 hours.
Then, cells were incubated with S100B (20 mg/mL) or LPS (1 mg/mL), for 24
h. Cytokines were determined in culture media by ELISA.
Macrophages treated with AGE-albumin presented a higher secretion of TNF-a,
IL-6 and MCP-1 induced by S100B or LPS in comparison to cells treated
with C-albumin. The S100B-induced TNF-a, IL-6 and MCP-1 secretion was
respectively, 57%, 72% and 50% reduced by HDL in macrophages treated with
C-albumin, in comparison to the same incubations in the absence of HDL. In
incubations with C-albumin and HDL, the secretion of TNF-a, IL-6 and MCP-1
induced by LPS was, respectively, 54%, 58% and 42% reduced comparing to
incubations in the absence of HDL. In treatments with AGE-albumin and S100B,
HDL was unable to reduce TNF-a and increased IL-6 (54%) and MCP-1 (20%).
The LPS-induced TNF-a and MCP-1 secretion was unchanged and IL-6 was
enhanced (16%) by HDL in cells exposed to AGE-albumin.
HDL loses its anti-inflammatory property and even induces more inflammation in
S100B or LPS-stimulated macrophages previously exposed to AGE-albumin.
Funding: FAPESP (Brazil)
212 V156K-APOA-I IN LIPID-BOUND STATE IS MORE RESISTANT TO
MODIFICATION OF FRUCTOSYLATION IN STRUCTURAL AND
FUNCTIONAL PROPERTIES
J.-H. Yoon1 , K.-H. Cho2 . 1 School of Biotechnology, 2 Yeungnam University,
Non-enzymatic glycation of protein can cause severe modification of structural
and functional properties of serum apolipoproteins. Since plasma apoA-I is
also sensitive to the glycation by fructose, we compared the sensitivity of the
fructosylation using wild-type (WT) and a point mutant of apoA-I. V156K, a
point mutant of apoA-I, has been reported to have anti-inflammatory and antiatherosclerotic activities. At the same concentration of fructose (5 mM final)
for 48 hr, WT-rHDL showed increased production of advanced glycated end
(AGE) products with proteolytic fragmentation. However, V156K-rHDL was more
resistant to fructosylation and protein cleavage. By glycation, WT-rHDL severely
47
lost antioxidant activity and anti-atherosclerotic activity, including inhibition
activity against cholesteryl ester transfer protein (CETP) and LDL phagocytosis,
and cholesterol efflux. However, V156K-rHDL showed much less production of
AGE products and glycated V156K-rHDL retained stronger antioxidant ability
and inhibition for CETP activity and LDL phagocytosis. Cholesterol efflux was
significantly enhanced by V156K-rHDL in the native and glycated state. In
stimulation activity for insulin secretion from pancreatic b-cell, glycated WT
apoA-I treated cell showed 25−35% less insulin secretion than that of native
WT, while glycated and native V156K showed similar insulin secretion activity.
In conclusion, by the fructation, anti-atherosclerotic and insulin secretion activity
of WT-apoA-I was almost deprived, but V156K-rHDL did not lose the beneficial
activity.
213 HDL-ASSOCIATED PARAOXONASE 1 (PON1) HYDROLYZING
ACTIVITY AGAINST MACROPHAGE OXIDIZED LIPIDS PROTECTS
AGAINST MACROPHAGE FOAM CELL FORMATION AND
ATHEROGENESIS
M. Aviram. Technion Faculty of Medicine/Rambam Medical Center, Haifa,
Israel
PON1 possesses several hydrolyzing activities against oxidized lipids which
are related to its histidine diad active site. Studies in mice revealed the in
vivo anti-atherogenicity of HDL-associated PON1 as the enzyme lactonizing &
lactonase activities can convert atherogenic hydroperoxide phospholipids
into non atherogenic hydroxycarboxilic acid, and lysophospholipids. Indeed,
lysophosphatidylcholine (LPC) can mimic the anti-atherogenic properties of
PON1 on macrophages. PON1 was found to stimulates cholesterol efflux from
macrophages via the ABCA1 transporter and this effect required the enzyme
active site and involves LPC formation. The other macrophage transporter for
HDL, the SRB-I receptor, was found to be up regulated by PON1. Whereas
PON1-induced increased ABCA1 resulted in enhanced cholesterol efflux, the
PON1-increase in cellular SRB-I was associated with decreased macrophage
apoptosis by HDL. PON1 is inactivated by oxidative stress and in order to
improve HDL-PON1 status, we next studied the effect of pomegranate juice
punicalagin polyphenolic on PON1-HDL association, and observed some most
beneficial anti atherogenic effects.
Finally, we analyzed HDL-PON1 status in association with protection against
atherosclerosis development in humans. The intima media thickness (IMT) in
patients with proven carotid artery stenosis (CAS) that consumed pomegranate
juice for one year, not only did not progressed, but was in fact attenuated
by 35%. We thus conclude that PON1 is a potent HDL-associated antiatherogenic enzyme due to its ability to breakdown specific oxidized lipids in
the atherosclerotic lesion.
214 SERUM PARAOXONASE ESTERASE AND LACTONASE ACTIVITIES
CORRELATE WITH INTERMEDIATE SIZE HDL PARTICLES
A. Gugliucci1 , T. Menini2 , K. Kotani3 , R. Hermo2 , R. Caccavello2 . 1 Touro
University-California, 2 Research, Touro University, Vallejo, CA, USA, 3 Clinical
Pathology, Jichi Medical School, Tochigi, Japan
Background: PON1 in HDL protects LDL from oxidation and decreases
homocysteine-thiolactone damage via its lactonase activity. Different HDL
subclasses have been linked to different degrees of cardioprotection. Little is
known about the distribution of PON1 across HDL subclasses.
Hypothesis: We tested the hypothesis that there might be differential
distribution of PON1 activity across different HDL subclasses.
Methods: In this cross-sectional study we enrolled 30 healthy subjects (14
males, 16 females) with HDL-cholesterol ranging from 20–110 mg/dl. We
employed paraoxon (triesterase activity) 5 (thiobutyl)butyrolactone (TBBL)
as well as dihydrocoumarin for lactonase and phenylacetate for PON1
monoesterase activity. HDL subfractions were analyzed by the Lipoprint HDL
system (Quantimetrix, CA, USA). Nine classes of HDL sizes can be quantified
using this procedure.
Results: HDL subclasses were stratified as high size (HDLa-c, Rf 0–0.15, HDL
2b), intermediate size (HDLd-f, Rf 0.20–0.29, HDL 2a-HDL3a), and low size
(HDLg-1, Rf 0.38–0.53, HDL3b-c). All PON1 activities correlated significantly
and positively with intermediate size HDL concentrations: lactonase r = 0.72,
p 0.0005; triesterase r = 0.69, p 0.001; arylesterase r = 0.70, p 0.001. No
correlation was found with high or low sized HDL subclasses.
Conclusions: We show that PON1 lactonase and other activities strongly
correlate only with a discrete size distribution of HDL3 and small HDL2 . Our
data suggest that specific HDL particles are responsible for most PON1 activity
and pave the way for future studies on the isolated particles themselves.
TBBL substrate was a generous gift from Dan Tawfik, Weizmann Inst. of
Science, Rehovot 76100, Israel.
48
215 ROLE OF HOMOCYSTEINE THIOLACTONASE ACTIVITY AND
ADMA LEVELS IN HEMODIALYZED AND RENAL TRANSPLANTED
PATIENTS
G. Paragh1 , F. Sztanek1 , M. Harangi1 , L. Lőcsey2 , J.T. Padra1 , L. Asztalos2 ,
I. Seres1 . 1 First Department of Medicine, 2 Institute of Surgery, University of
Debrecen, Medical and Health Science Center, Debrecen, Hungary
Previous data indicate that HDL-associated human paraoxonase-1 (PON1)
enzyme also has homocysteine thiolactonase (HTLase) activity. HTLase may
prevent proteins from homocysteinylation; therefore it may be potentially
protective against the progression of atherosclerosis. Previous studies have
demonstrated decreased PON1 paraoxonase activity in hemodialysed (HD)
and renal transplant (TRX) patients; however HTLase activity has not been
investigated.
We aimed to determine the paraoxonase and HTLase activities, and to clarify
the relationship between HTLase activity and some cardiovascular risk factors,
such as homocysteine, cystatin C and asymmetric dimethylarginine (ADMA) in
HD and TRX patients and in healthy controls (C).
114 HD and 80 TRX patients, and 54 healthy controls were involved in the study.
Paraoxonase and HTLase activities were measured spectrophotometrically.
ADMA level was determined with sandwich ELISA.
Both HD and TRX patients had significantly lower HTLase activites compared to
the C group (p < 0.001). Significantly lower HTLase and paraoxonase activities
were found in HD patients compared to the TRX group (p < 0.05). There were
significant negative correlations between HTLase activity and ADMA level in the
whole study population (p < 0.001). Significant positive correlations were found
between paraoxonase and HTLase activities in the whole study population
(p < 0.001). Multiple regression analysis showed that only paraoxonase activity
and homocysteine level are independent predictors of HTLase activity.
HTLase activity may be a new predictor of cardiovascular risk in renal failure,
however, it may be modulated by other risk factors. Measurement of HTLase
activity can be recommended in future studies on HD and TRX patiens.
216 INTERRELATIONSHIP BETWEEN LIPID METABOLISM AND
MACULAR PIGMENT OPTICAL DENSITY
S. van der Made1 , E. Kelly2 , T. Berendschot2 , J. Plat1 , R.P. Mensink1 . 1 Human
Biology, 2 University Eye Clinic, Maastricht University Medical Center+,
Maastricht, The Netherlands
Background: Lutein accumulation in the macular region might help against the
deterioration of the retina as seen in age-related macular degeneration (AMD).
As lutein is predominantly transported by high-density lipoproteins (HDL), we
studied if effects of lutein supplementation on macular pigment optical density
(MPOD) − a measure of the lutein content in the eye − depends on baseline
HDL concentrations.
Methods: 80 men and women participated in a trial with a 12 weeks intervention
period in which one egg per day (N = 15), a buttermilk drink containing one egg
yolk (N = 14), a lutein-egg (N = 19) or a zeaxanthin-egg (N = 16) was consumed.
The control group (N = 16) did not change their habitual egg intake. Blood was
sampled to measure serum lipids, lipoproteins and carotenoids, and MPOD was
measured at the beginning of the study (week 1) and at weeks 6 and 12.
Results: At baseline, lutein and MPOD (r = 0.28; p= < 0.05 correlated positively.
Using mixed model analysis, HDL-C (P < 0.001) and LDL-C (P < 0.001) were
significantly related to serum lutein levels. As expected, also the type of
intervention determined lutein concentrations during the course of the study.
In another model, however, no evidence was found that baseline HDL-C levels
predicted changes in MPOD (P= 0.60).
Conclusion: After 12 weeks of supplementation, changes in MPOD after lutein
supplementation do not depend on baseline HDL cholesterol concentrations.
217 IMPACT OF CHOLESTERYL ESTER TRANSFER PROTEIN
INHIBITION ON NUCLEAR MAGNETIC RESONANCE DERIVED
LIPOPROTEIN PARTICLE PARAMETERS
N. Rashedi1 , D. Brennan1 , J.J. Kastelein2 , S.E. Nissen1 , S. Nicholls1 .
1
Cardiovascular Medicine, Cleveland Clinic, Cleveland, OH, USA, 2 Academic
Medical Center, Amsterdam, The Netherlands
Background: Cholesteryl ester transfer protein (CETP) facilitates the exchange
of esterified cholesterol and triglyceride between high-density lipoproteins
(HDL) and atherogenic lipoprotein particles. CETP inhibitors elevate levels of
HDL cholesterol and lower low-density lipoprotein (LDL) cholesterol in statintreated patients. The impact of CETP inhibition on lipoprotein subclasses has
not been investigated.
Methods: 910 patients with angiographic coronary artery disease were
treated with either placebo (n = 446) or the CETP inhibitor torcetrapib
(n = 464) in addition to background atorvastatin. Changes in nuclear magnetic
resonance derived measures of lipoprotein particle concentration and size were
characterized.
Results: Treatment with torcetrapib was associated with a beneficial impact on
HDL cholesterol (52.9%±0.7 vs. −0.6%±0.7, p < 0.001) and LDL cholesterol
(−14%±0.9 vs. 5%±0.9, p < 0.001) levels. Torcetrapib was associated with an
increase in mean HDL particle size (10%±.2 vs. 0.08±0.2, p < 0.001) and
concentration of large HDL particles (117.5%±2.9 vs. + −11%±2.9, p < 0.001),
but not the concentration of HDL particles (1%±1.1 vs. −5.8%±1.1, p < 0.001).
In contrast, decreases in the concentration of total (−35.3%±1.6 vs. −1%±1.7,
p < 0.001) and small (−55.5%±4.2 vs. −8.3%±4.2, p < 0.001) LDL particles was
observed in association with torcetrapib administration. Correlation between
changes in total LDL and HDL particles (r = 0.11 p = 0.04) and total LDL and
large HDL particles (r = −0.37 p < 0.001) was observed.
Conclusions: Inhibition of CETP is associated with an increase in the
concentration of large HDL particles and reduction in the concentration of
atherogenic small LDL particles. The cardiovascular impact of CETP inhibitors
without off-target toxicity remains to be determined.
218 SEX HORMONES MODULATE REVERSE CHOLESTEROL
TRANSPORT IN HUMAN MACROPHAGES
A.N. Smirnov1 , T.A. Shchelkunova1 , I.A. Morozov2 , P.M. Rubtsov2 ,
I.A. Sobenin3,4,5 , I.V. Adrianova5 , E.G. Rudymov1 , N.M. Mukhamedova6 ,
D.M. Martirosyan7 , A.N. Orekhov3,4 . 1 School of Biology, M.V. Lomonosov
Moscow State University, 2 V.A. Engelhardt Institute of Molecular Biology,
Russian Academy of Sciences, 3 Institute of General Pathology and
Pathophysiology, Russian Academy of Medical Sciences, 4 Institute for
Atherosclerosis Research, Russian Academy of Natural Sciences, 5 Intitute
of Experimental Cardiology, Russian Cardiology Research Center, Moscow,
Russia, 6 Baker IDI Heart and Diabetes Institute, Melbourne, SA, Australia,
7
Nutrition and Food Science Department, Texas Woman’s University,
Richardson, TX, USA
Gender differences in risks of cardio-vascular disorders are believed to arise
from effects of sex hormones on both lipid metabolism in the liver and on
several cell types in the vessel wall. The efficacy of a system of cholesterol
reverse transfer from the vessel wall performed mainly by macrophages is
important antiatherogenic factor that might serve as a target for sex hormones.
In this study, the effects of hormones on expression of mRNAs encoding for key
components of cholesterol reverse transfer and their transcriptional regulators
(neutral cholesterol ester hydrolase CEH, ATP-binding cassette transporters
ABCA1 and ABCG1, auxiliary factors SR-BI and ApoE, as well as lipid
sensors and transcriptional regulators of lipid metabolism LXRa/b, PPARa/g,
and SREBP1/2) in human macrophages using qPCR were measured. Estradiol
(E2), testosterone and 5alpha-dihydrotestosterone (DT) singly or in combination
with modified low density lipoproteins (mLDL) were enable to induce statistically
significant changes in the contents of ABCA1, ABCG1, SR-BI, PPARa, and
SREBP1 mRNAs. Notably, E2 potentiated while DT diminished stimulatory
effect of mLDL on ABCG1 mRNA only in female cells. Thus, sex hormones
had action upon about a half from studied mRNAs related to performance
or regulation of cholesterol reverse transfer in macrophages. We speculate
that these effects can contribute to gender differences in atherosclerosis
susceptibility.
Supported by Russian Ministry of Education and Science.
219 THE NASCENT HDL-LIKE PARTICLES CONTAINING APO A-I
OR APO A-II ARE INDEPENDENTLY GENERATED DURING
PHOSPHOLIPIDS MEDIATED HDL REMODELING
A. Wolska, M. Czyżewska, K. Wołoszyn, A. Szutowicz, M. Wróblewska.
Laboratory Medicine, Medical University of Gdansk, Gdansk, Poland
We have demonstrated that reaction between ultracentrifuged HDLs and
egg yolk lecithin liposomes caused the generation of new pre-b mobility
particles containing apolipoproteins A-I and/or A-II. This study investigated
the impact of the reaction time and temperature on the dynamics of the
transfer of apolipoproteins from HDLs to pre-b mobility fraction and nondisrupted liposomes, at liposomal phospholipids to HDL-phospholipids ratio 5:1.
We also studied how the transfer proceeds in case of HDLs non modified
by ultracentrifugation. We shown no significant effect of temperature on the
dynamics of apo A-II transfer from ultracentrifuged HDLs. The entire loss of
apo A-II by HDLs (17 and 13% of the initial HDL content at 10 and 37ºC,
respectively) occurred during the first 5 minutes of reaction. Pre-b mobility
fraction generated by the reaction between native HDLs and liposomes also
contained apo A-II, however at 10 and 37ºC only approximately 2.5% of initial
HDL apo A-II content transferred from HDLs to liposomes and pre-b fraction.
After 5 minutes of reaction loss of apo A-I by both ultracentrifuged and native
HDLs was similar at 37ºC and 10ºC (approximately 7% of initial HDL content).
Prolonged incubation caused a further loss of apo A-I only at 37ºC (up to 20%
of initial HDL content after 24h incubation). The dissociation of apolipoproteins
from HDLs is a key process for the generation of new pre-b mobility HDLlike particles. Our results indicated that apo A-I or apo A-II containing pre-b
mobility particles are generated independently during HDL remodeling mediated
by PL.
79th EAS Congress
220 DOES APO A-II CYCLE BETWEEN MATURE a-HDL AND NASCENT
PRE-b HDL A-II PARTICLES?
A. Wolska, M. Czyżewska, K. Wołoszyn, A. Szutowicz, M. Wróblewska.
Laboratory Medicine, Medical University of Gdansk, Gdansk, Poland
During the interaction between HDLs and lecithin liposomes a new pre-b mobility
fraction containing apo A-I and apo A-II is generated. We incubated HDLs and
liposomes at liposomal phospholipids (PL) to HDL PL ratio 5:1. We analysed
the relationship between the acquisition of PL either by ultracentrifuged or
native HDL in serum depleted of other lipoproteins and the transfer of apo
A-I and apo A-II from HDLs to pre-b mobility fraction and residual liposomes.
At 37ºC the transfer of liposomal PL to native HDLs was significantly higher
than to ultracentrifuged HDLs in each of the tested incubation times (from 5
minutes to 24h). On the contrary, native HDLs lost significantly less apo A-II
than ultracentrifuged HDLs. While the transfer of PL to HDLs was temperaturedependent, the transfer of apo A-II to pre-b fraction and liposomes did not
depend on the incubation temperature. Our results demonstrated that the
dissociation of apo A-II from HDLs occurred regardless of the enrichment of
HDL in PL. We assessed that the incubation mixture of native HDLs with
liposomes contained approximately 6 fold less of apo A-II containing pre-b
particles than mixture of liposomes with ultracentrifuged HDLs. We believe,
that it was not caused by the violation of HDLs stability by ultracentifugation,
because this procedure did not influence the amount of apo A-I that dissociated
from HDL. Presumably, the newly generated pre-b particles containing apo A-II
were rapidly incorporated back to a-HDL. Putative candidates for the mediators
of this process are LCAT and PLTP.
221 HDL FROM ELDERLY AND RECONSTITUTED HDL
CONTAINING GLYCATED APOLIPOPROTEINS A-I SHARE
PRO-ATHEROSCLEROTIC AND PRO-SENESCENT PROPERTIES
WITH INCREASED CHOLESTEROL INFLUX
K.-H. Park, K.-H. Cho. School of Biotechnology, Yeungnam University,
High-density lipoprotein (HDL) is a strong antioxidant, anti-inflammatory,
and anti-senescence molecule. However, in the current study HDL from
the elderly (E-HDL) exhibited increased glycation with apolipoprotein (apo)
A-I multimerization and decreased phospholipid content. Similarly, glycated
apoA-I (gA-I) by fructosylation has a covalently multimerized band without
a crosslinker and impaired phospholipid-binding ability. Treatment of human
dermal fibroblasts (HDFs) and macrophages with E-HDL and gA-I caused more
severe cellular senescence and foam cell formation, respectively; however,
treatment with HDL from a young group (Y-HDL) and native apoA-I (nA-I)
suppressed senescence and atherosclerosis. E-HDL3 and reconstituted HDL
(rHDL) containing gA-I showed enhanced cholesterol influx into macrophages
compared to Y-HDL3 and nA-I-rHDL. In conclusion, E-HDL and gA-I-rHDL share
similar physiologic properties in macrophages and HDFs. E-HDL and gA-I-rHDL
exacerbated cellular senescence and atherosclerosis with increased cellular
cholesterol influx.
222 PARAOXONASE AND HOMOCYSTEINE THIOLACTONASE
ACTIVITIES AND ASYMMETRIC DIMETHYL-ARGININE (ADMA)
LEVEL IN HEMODIALYZED PATIENTS
I. Seres1 , F. Sztanek1 , L. Lőcsey2 , M. Harangi1 , G. Paragh1 . 1 First Department
of Medicine, 2 Institute of Surgery, University of Debrecen, Medical and Health
Science Center, Debrecen, Hungary
Elevated homocysteine level and oxidative stress contribute to cardiovascular
complications in hemodialysis patients. HDL-associated human paraoxonase-1
(PON1) has antioxidant effect by inhibiting LDL oxidation. PON1 has lactonase
activity, by which hydrolyzes homocysteine-thiolactone. The renal elimination of
ADMA − an endogenous inhibitor of NO synthase − is impaired in hemodialyzed
patients contributing to endothelial dysfunction.
Our aim was to examine the connection between paraoxonase and homocystein
thiolactonase (HTLase) activities of PON1 and the correlation between ADMA
level and HTLase activity in hemodialyzed patients.
114 hemodialyzed patients were enrolled into our study. Patients were divided
into groups based on their BMI: malnourished (n = 25), normal weight (n = 50)
and overweight (n = 39) groups. ADMA levels were measured with competitive
ELISA. Paraoxonase, arylesterase and HTLase activities of PON1 were
measured spectrophotometrically.
Our results showed that paraoxonase and HTLase activities of PON1 in both
overweight and normal group were significantly lower than in the malnourished
group. The paraoxonase and HTLase activities of PON1 showed significant
positive correlation (r = 0.39, p < 0.01). The correlation between PON/HDL ratio
and HTLase activity was also significant (r = 0.336, p < 0.01). ADMA level was
significantly higher than the normal range. ADMA concentration did not differ
significantly among the three patient groups, but in the normal and malnourished
groups it correlated negatively with BMI. The HTLase activity of PON1 showed
significant inverse correlation with ADMA levels in the malnourished and normal
weight groups.
49
Decreased PON1 paraoxonase and HTLase activity, nutritional status and
elevated ADMA levels may contribute to premature atherosclerosis in
hemodialyzed patients.
223 TRANSFER OF LIPIDS TO HDL IN PATIENTS WITH TYPE 2 DIABETES
MELLITUS WITH AND WITHOUT CORONARY ARTERY DISEASE
M.C.O. Sprandel, W.A. Hueb, A. Casella-Filho, C.A. Segre, F.R. Freitas,
R.C. Maranhão. Heart Institute of the Medical School Hospital, University of
São Paulo, São Paulo, Brazil
Introduction: Systematic assessment of metabolic and functional aspects of
HDL is important for the understanding of HDL anti-atherogenic role. Lipid
transfer between HDL and the other lipoproteins, a process mediated by CETP
and PLTP, is a crucial step in HDL metabolism and determinant for HDL
functions in cholesterol esterification and reverse cholesterol transfer.
Objective: Investigate whether the susceptibility of patients with type 2 diabetes
mellitus (DM2) to develop coronary artery disease (CAD) is related with
alterations in lipid transfers to HDL.
Methods: 83 patients with DM2 and CAD (DM2-CAD) and 82 with DM2
(DM2 group) were studied. They were of both genders, aged 40−80 yrs.
Fasting plasma samples were incubated for 1h at 37o C with a donor artificial
nanoemulsion labeled with 14 C-cholesteryl-esters and 3 H-triglycerides or 3 Hfree-cholesterol and C14 -phospholipids. Radioactive lipids transferred from the
donor nanoemulsion to HDL were quantified in the supernatant after chemical
precipitation of non-HDL fractions and nanoemulsion.
Results: In DM2-CAD, LDL-cholesterol and triglycerides were higher
than in DM2 patients, but HDL-cholesterol was lower. DM2-CAD showed
diminished transfer to HDL of free cholesterol (DM2-CAD= 4.23±0.2426;
DM2= 5.66±0.1384, p < 0.0001) and of triglycerides (1.65±0.1361 vs
3.08±0.1170, p < 0.0001). In contrast, cholesteryl-ester transfer (3.67±0.1690
vs 2.25±0.1339, p < 0.0001) was increased in DM2-CAD whereas the phospholipid transfer was equal (15.37±0.6004 vs 15.18±0.2059, p = 0.7696).
Conclusion: Reduction of free-cholesterol transfer to HDL may hinder
cholesterol esterification and reverse cholesterol transport. Alterations in
triglyceride and cholesteryl-ester transfer may affect lipoprotein stability. Those
disturbances in HDL metabolism may have facilitated the atherogenesis process
in DM2-DAC.
224 ITINERARY OF HIGH DENSITY LIPOPROTEINS AND
APOLIPOPROTEIN A-I TRANSPORT THROUGH THE
ENDOTHELIUM
D. Perisa, L. Rohrer, A. von Eckardstein. Institute for Clinical Chemistry,
University Hospital Zürich, Zürich, Switzerland
Atherosclerosis is an ongoing progressive accumulation of lipids in the arterial
intima leading to plaque formation.
Epidemiological studies show a negative correlation of the high density
lipoprotein (HDL) blood concentration to atherosclerosis.
HDL has multiple anti-atherogenic functions: Some of these take place in the
vessel walls in the intima. To get access to the intima and the lipid-laden
macrophages within, HDL has to pass the endothelial barrier.
Previously, we showed that HDL is transcytosed through endothelial cells (ECs)
by interaction with ATP binding cassette transporter G1 (ABCG1) and scavenger
receptor B I (SR-BI).
To elucidate endocytosis and transcytosis of HDL in ECs, we investigated
the uptake, location and distribution of fluorescently labelled HDL. Moreover
we studied colocalisations of HDL with ABCG1 and SR-BI as well as with
established compartment markers in primary ECs.
Further, we are analysing the change in HDL distribution inside the ECs upon
stimulation with pharmacological substances known to interfere with cellular
steady state of vesicles and trafficking routes through the cells.
We are corroborating our findings in the endothelium from aortic tissue. Live
microscopy and electron microscopy experiments will help us to visualise and
understand the itinerary of HDL through aortic endothelial cells
225 MODIFIED APOLIPOPROTEIN (APO) A-I BY ARTIFICIAL
SWEETENER CAUSES SEVERE PREMATURE CELLULAR
SENESCENCE AND ATHEROSCLEROSIS
J. Seo, K.-H. Cho. School of Biotechnology, Yeungnam University, Gyeongsan,
Republic of Korea
Long-term consumption of artificial sweeteners (AS) has been the recent focus
of safety concerns. However, the potential risk of the AS in cardiovascular
disease and lipoprotein metabolism has not been investigated sufficiently. We
compared the influence of AS (aspartame, acesulfame K, and saccharin) and
fructose in terms of functional and structural correlations of apolipoprotein (apo)
A-I and high-density lipoproteins (HDL), which have atheroprotective effects.
Long-term treatment of apoA-I with the sweetener at physiological concentration
(3 mM for 168 hr) resulted in loss of antioxidant and phospholipid binding
activities with modification of secondary structure. The AS treated apoA-I
exhibited proteolytic cleavage to produce 26 kDa-fragment. They showed proatherogenic properties in acetylated LDL phagocytosis of macrophages. Each
50
sweetener alone or sweetener-treated apoA-I caused accelerated senescence
in human dermal fibroblasts. These results suggest that long-term consumption
of AS might accelerate atherosclerosis and senescence via impairment of
function and structure of apoA-I and HDL.
226 LOW HDL IN SOUTH ASIAN IMMIGRANTS: COULD
DYSFUNCTIONAL HDL BE THE MISSING LINK?
S. Dodani1 , L. Dong2 , S. Reddy3 . 1 Internal Medicine, University of Kansas,
Leawood, 2 University of Kansas Medical Center, Kansas City, KS, 3 University
of California at Los Angeles, Los Angeles, CA, USA
Coronary artery disease (CAD) is the leading cause of mortality and morbidity
in US, and South Asian immigrants (SAIs) have a higher risk for CAD compare
to other ethnic groups. Traditional risk factors do not completely explain
excess CAD risk, therefore search for new markers is needed. We assessed
dysfunctional (pro-inflammatory) high density lipoprotein (Dys-HDL) in SAIs and
assessed its association with sub-clinical CAD and metabolic syndrome (MS).
Methods: A prospective cross-sectional study on SAIs aged 35−65 years
was conducted. Sub-clinical CAD was measured using carotid intima media
thickness (IMT) as a surrogate marker of atherosclerosis. Dys-HDL was
determined by novel cell free assay and HDL inflammatory Index.
Results: Of the total sample 148 subjects, twenty-six percent (25.6%) had HDL
inflammatory index 1 suggesting Dys-HDL. Sub-clinical CAD using CCA-IMT
0.8 mm was seen in 31.46%% and MS in 29.7%. On the Fisher Exact test
analysis, both presence of MS and Dys-HDL was significantly correlated with
CCA-IMT, even after age and gender adjustment. The odds of having Dys-HDL
with CCA-IMT were 5 times (confidence interval 2.00, 13.27) than with those
without Dys-HDL.
Conclusions: The measurement of HDL level and function plays an important
role in CAD risk assessment beside HDL levels only. Further large studies are
required to explore association of Dys-HDL with CAD in order to understand
the causes of excess CAD in SAIs so that specific preventive and treatment
guidelines can be developed.
227 REVERSE CHOLESTEROL TRANSPORT KEY PROTEINS AND
ATHEROSCLEROSIS
E. Demina1 , V. Miroshnikova1 , A. Denisenko2 , A. Schvarzman1 . 1 Petersburg
Nuclear Physics Institute of RAS, Gatchina, 2 Saint-Petersburg Institute of
Experimental Medicine, Saint Petersburg, Russia
Reverse cholesterol transport (RCT) − excess of cholesterol by high density
lipoproteins (HDL) from macrophages back to the liver, plays an important
protective role in the development of atherosclerosis. ATP binding cassette
transporter G1 (ABCG1) and apoproteinA1 (ApoA1) are known to play important
role in RCT. Also ApoA1 is a major protein of HDL.
According to our hypothesis variations in expression levels of RCT key proteins
(ABCG1 and ApoA1) contribute to atherosclerosis development.
Using real-time quantitative polymerase chain reaction the mRNA levels of
RCT key proteins were measured in monocytes derived macrophages in
selected groups (mean age 45.9): 20 patients with angiographically proved
atherosclerosis and 10 healthy individuals.
Reduction of macrophage ABCG1 mRNA level is associated with reduction
in content of HDL cholesterol (R > 0.65, p < 0.02) and increasing of the rate
of arterial occlusion among patients (R < −0.63, p = 0.05). But there is no
significant difference between relative levels of ABCG1 mRNA between group
of patients and control group: 0.87±0.66 versus 1.26±0.55 (p = 0.076). These
results indicate that ABCG1 transporter regulates HDL metabolism and it’s
cholesterol content and protects against atherosclerosis.
We have investigated association of ApoA1 gene polymorphism with
atherosclerosis development. Allelic frequencies of polimorphic variants
(−75)G/A and 83C/T were determined in the group of patients (N = 177) and in
control group (N = 229). Frequency of allele 83T of ApoA1 gene in the group
of patients was lower than in the control group (0.025 versus 0.065, p = 0.005)
this allele is associated with lower risk of atherosclerosis development among
Saint-Petersburg population (RR((TT+CT) vs CC) = 0.39).
228 SERUM PARAOXONASE PHENOTYPE DISTRIBUTION IN EXUDATIVE
AGE RELATED MACULAR DEGENERATION AND ITS RELATIONSHIP
TO HOMOCYSTEINE AND OXIDIZED LOW DENSITY LIPOPROTEIN
A. Ghorbanihaghjo, A. Javadzadeh, E. Bahreini, N. Rashtchizadeh, H. Argani,
S. Alizadeh. Biotechnology Research Center, Tabriz University of Medical
Sciences, Tabriz, Iran
Purpose: Disequilibrium between oxidative stress and antioxidant levels has
been proposed as an important case of Exudative Age Related Macular
degeneration (E-ARMD). The aim of the present study was to investigate
homocysteine (Hcy) level and antioxidant paraoxonase 1 (PON1) activity within
its phenotypes together oxidized LDL (OX-LDL) levels in the patients with
E-ARMD.
Methods: Serum PON1 activity and plasma Hcy and OX-LDL levels were
analyzed in 45 E-ARMD patients and compared with 45 healthy controls.
Results: The distribution of PON1 phenotypes was significantly different
between the patients with E- ARMD and control subjects (P = 0.01). AAphenotype with low activity was significantly more frequent in E-ARMD
patients compared with healthy subjects (62.2% vs. 35.6%, respectively). Other
phenotype frequencies in the patients compared with controls were as ABphenotype (intermediate activity) 28.9% vs. 46.7% and BB-phenotype (high
activity) 8.9% vs. 17.8%, respectively. Except in BB phenotype patients with
AA and AB phenotypes had higher plasma Hcy levels in comparison to those
of controls. The mean OX-LDL levels, in all three phenotypes (P < 0.05), and
OX-LDL/HDL ratio, in AA and AB phenotypes (P = 0.001, P = 0.1, respectively)
were significantly higher in the patients than controls. In comparison between
three PON1phenotypes of the patients, no significant differences were found in
Hcy and OX-LDL levels.
Conclusion: Increased plasma Hcy as an independent risk factor in induction of
OX-LDL induces oxidative stress in macula area. PON1 activity is not a sufficient
factor in prevention of oxidative stress in occurrence of E-ARMD disease.
229 UNFOLDING OF EXCHANGEABLE APOLIPOPROTEINS
A. Dergunov. Biochemistry, National Research Centre for Preventive Medicine,
Moscow, Russia
Guanidine-hydrochloride (GuHCl)-induced denaturation of a-helices in human
plasma apoA-I, apoA-IV, apoE3 and three recombinant apoE isoforms in
solution and discoidal complexes of apoA-I, apoA-II, apoA-IV and apoE3 with
phosphatidylcholine was studied. The raw data, obtained by us and others,
were analyzed with the three equilibrium schemes. The protein conformational
stability (DG(H2 O)) and a slope of linear dependence of free energy of unfolding
on GuHCl concentration (m-value) were estimated. The data for all proteins,
except apoA-II, fit with the three-state model, thus evidencing two-domain
structure both in solution and in complexes. The predicted folding rate of
the four apoE in solution correlated with their conformational stability, but
the dependence disappeared at the inclusion of apoA-I and apoA-IV into
analysis. In addition, the m-values, adjusted for residue number in helices
(mrh ), differed between those for apoE and apoA-I/apoA-IV due to the different
structural motifs and/or folding rates. However, if analyzed together, the mrh values for six proteins correlated positively with the fractional change in
accessible surface area at unfolding for Phe, Lys and Asn, while negatively
for Arg, Ala and Gly residues. The difference between the adjusted DGrh (H2 O)
values for apolipoproteins in complexes and in solution decreased at the
increase of reduced temperature T red , calculated as T red = (T obs − T t )/T t . Twodomain structure of apolipoproteins in complexes was much more unified.
The induction of intrinsic disorder by arginine residues may be of primary
importance in metabolism and function of exchangeable apolipoproteins, while
their stability in nascent discoidal HDL is controlled by the physical state of
phosphatidylcholine.
230 CHANGES IN FUNCTION AND STRUCTURE OF HIGH-DENSITY
LIPOPROTEIN IN OBESE PATIENTS COMPARED WITH HEALTHY
CONTROLS
S. Hama, H. Soran, Y. Liu, V. Charlton-Menys, R. Zadeh, M. France, R. Yadav,
M. Jeziorska, B. Ammori, P. Durrington. University of Manchester, Manchester,
UK
Background: There is an inverse relationship between HDL and cardiovascular
disease. We hypothesised that HDL’s anti-oxidant function is altered in obese
patients, with or without type 2 diabetes.
Methods: 19 obese patients, 8 with type 2 diabetes, and 13 healthy controls
were recruited. HDL’s antioxidant ability was tested by measuring lipid peroxide
(LPO) formation after incubation of HDL with Cu2+ at baseline and at 3 hours.
ApoAI was determined in HDL and its sub-fractions. Serum PON1 activity was
measured by a semi-automated method.
Results: There was no significant difference in LPO concentration at baseline
between controls and obese patients (30 [23−50] vs. 39 [22−94] nmol/ml). LPO
formation was, however, significantly greater in obese patients after 3 hours (39
[4−93] vs. 67 [10–129] nmol/ml; p < 0.05). PON1 activity tended to be lower in
the obese group with (77 [24–271] nmol/ml/min) or without diabetes (142 [26–
216] nmol/ml/min) compared with controls (178 [38–457] nmol/ml/min). ApoAI in
HDL2 of the obese-diabetic group was significantly lower compared with healthy
controls (0.77 [0.69–0.85] vs. 1.01 [0.64–1.74] g/l; p < 0.05).
Conclusions: LPO formation in HDL from obese patients was higher than in
healthy HDL, suggesting the presence of pro-inflammatory HDL. Deterioration
of HDL’s ability to protect itself against oxidation could indicate deterioration of
its protective functions and this could be linked to re-distribution of apoAI in
HDL sub-fractions and lower PON1 activity.
79th EAS Congress
231 ASSESSMENT OF 2 METHODS FOR ISOLATION OF HIGH-DENSITY
LIPOPROTEIN (HDL)
S. Hama, H. Soran, V. Charlton-Menys, Y. Liu, R. Yadav, M. France,
B. Ammori, P. Durrington. University of Manchester, Manchester, UK
Background: HDL is heterogeneous. It can sub-divided into HDL2 and HDL3.
HDL2 is thought to offer more protection than HDL3 against atherosclerosis.
Two main techniques are commonly used to separate HDL subfractions,
ultracentrifugation and precipitation. Our literature search revealed that the
precipitation methods are reported to give higher results for HDL3 cholesterol
(HDL3-C) than ultracentrifuge methods, but there have been few direct
comparisons.
Methods: Blood was drawn from 12 healthy volunteers after overnight fasting
and serum total HDL-cholesterol (HDL-C) was determined by a 3rd generation
automated assay. We also used sequential ultracentrifugation to isolate total
HDL (density >1.063 g/ml), HDL2 (density 1.063–1.125 g/ml) and HDL3 (density
>1.125 g/ml) and assayed cholesterol in these fractions. In another 12 healthy
volunteers we used a Dextran/MgCl2 precipitation method to isolate total HDL
and HDL3 and HDL2-C was calculated by subtraction.
Results: Serum total HDL-C for the two groups was similar (1.76±0.70
vs. 1.69±0.30 mmol/l; mean±SD). Total HDL-C at density >1.063 g/ml
was 1.65±0.49 mmol/l (94%) compared with 1.48±0.32 mmol/l (88%) by
precipitation. HDL3-C by ultracentrifugation was 0.35±0.09 mmol/l (23% of total
HDL-C) and much higher by precipitation at 1.24±0.36 mmol/l (84% of total
HDL-C) (P < 0.0001).
Conclusions: Our study draws attention to widespread disagreement in the
literature about the distribution of cholesterol in HDL subfractions, which would
appear to be method specific. There is a need to resolve these differences and
to establish a universally agreed reference method.
232 HDL CHOLESTEROL/APOLIPOPROTEIN AI RATIO INVERSELY
LINKED WITH ACUTE CORONARY SYNDROME, INDEPENDENT
TO HDL CHOLESTEROL
S.Y. Lim1 , H.-S. Seo1 , E.J. Kim1 , J.O. Na1 , C.U. Choi1 , H.E. Lim1 , S.-W. Rha2 ,
C.G. Parl2 , D.J. Oh2 . 1 Korea University Guro-Hospital, 2 Cardiovascular Center,
Korea University Guro-Hospital, Seoul, Republic of Korea
The risk in patients with low LDL-C is explained by low HDL-C levels. However,
the role of HDL-Cin CVD is still not conclusive in some situations. Moreover, few
studies were performed to evaluate the HDL function independent to HDL-C on
acute coronary syndrome (ACS) as yet. The aim of this study is to compare
the level of HDL-C, ApoAI, and its ratio between patients with ACS and those
without ACS. We analyzed 143 patients in each group (ACS and control group)
with propensity matching. We measured level of HDL-C and ApoAI and, its ratio
(HDL-C/ApoAI). The serum level of HDL-C, LDL-C and ApoAI showed higher
values in control group than ACS group. HDL-C/ApoAI also showed difference
between both groups. Regression analysis showed that HDL-C, ApoAI, and
HDL-C/ApoAI were strongly associated with occurrence of ACS event even
after adjusting for possible affecting risk factors or variables. Furthermore, efflux
HDL-C/ApoAI ratio was a strong inverse predictor of coronary disease status. In
an analysis adjusted for age and sex, the quartile of lowest HDL-C/ApoAI ratio
was associated with a increased risk of ACS, as compared with the quartile
of highest HDL-C/ApoAI ratio. These findings suggest that HDL-C/ApoA1 ratio
is predictive of residual vascular risk of ACS in addition to HDL-C and ApoA1
among Korean patients who are in even normal concentrations of LDL-C. Not
only the level of HDL-C, but also the functional status of reverse cholesterol
transport by HDL estimated through the ratio of HDL to ApoAI decreased during
ACS.
51
differences were not statistically significant. In contrast, HDL2 cholesterol levels
were significantly lower in those who had albuminuria compared with those who
had normoalbuminuria (0.50 vs 0.57 mmol/L, p = 0.01).
Conclusion: In this study we provided the evidence that higher levels of HDL,
and particularly HDL2 cholesterol, may be protective against the development
of albuminuria in DM1. The relationship between lipids and albuminuria has
important implications for treatment because studies with patients with diabetes
indicated that statins therapy reduced albuminuria by 25%.
234 IMPAIRED HIGH-DENSITY LIPOPROTEIN ANTIOXIDANT CAPACITY
IN TYPE II DIABETES
R. Mingpakanee1 , N. Charoensa2 , Y. Phrawisat3 , W. Dahlan4 . 1 Department
of Transfusion Medicine, 2 Medical Technology Program, 3 Medical Technology
Program, Faculty of Allied Health Sciences, 4 Halal Science Center,
Chulalongkorn University, Bangkok, Thailand
The high incidence of atherosclerosis in type II diabetic (DM) patients is
associated with dyslipidemia; including hypertriglyceridemia, increase in lowdensity lipoprotein (LDL) cholesterol and decrease in high-density lipoprotein
(HDL) cholesterol). LDL oxidation is a crucial step in atherosclerosis, process
that can be inhibited by HDL through its associated enzymes; paraoxonase
(PON) and platelet-activating factor acetylhydrolase (PAF-AH). The aim of this
study was to investigate the effect of HDL on copper-induced LDL oxidation.
HDL was isolated from type II DM patients (n = 20) and non DM volunteers
(n = 20), age between 40 to 60 years. Antioxidant capacity of HDL on LDL
oxidation (measured as %Inhibition of HDL), serum paraoxonase activity and
LDL oxidation (measured as lipid peroxide), were evaluated. In comparison to
control group, lipoprotein profiles, of Type II DM groups contained less HDLcholesterol. The inhibitory effect of HDL on LDL oxidation and PON activity
were lower in type II DM group. Furthermore LDL isolated from type II DM was
prone to oxidized. Our results demonstrate that HDL from type II DM patients
exhibit impairment in its antioxidant ability which increases the risk of developing
atherosclerosis.
235 CARDIOVASCULAR PARAMETERS IN HIV POSITIVE PATIENTS
WITH CHRONIC PERIODONTITIS
R.V. Orellana1 , A. Monteiro2 , M.A. Gidlund3 , A. Mantesso4 , E. Ramos
Samches, H. Fonseca. 1 Imunology of Atherosclerosis, 2 Imunology of
Atherosclerosis ICB 4, 3 Imunofisiopatologia da Aterosclerose, 4 Pathology,
University of São Paulo, São Paulo, Brazil
Introduction: Recently, some studies show the oral health, especially
periodontal disease risk factors related to atherosclerosis, few studies have
linked periodontal disease in HIV positive patients. Thus, the purpose of this
study is to investigate the association between chronic periodontitis and risk
markers for atherosclerosis in HIV positive patients.
Methods: We selected 20 patients with chronic periodontitis and 20 patients
without periodontal disease groups both HIV positive. Were analyzed: total
cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL),
triglycerides, interleukin1and 10, total and differential count of blood cells.
Results: The levels of total cholesterol, triglycerides, HDL-C and LDL-C
were similar in both groups (p = 0.5338, p = 0.7660, p = 0.2444 p = 0.9461).
Interleukin-1 and -10, leukocytes, lymphocytes, neutrophils were not significant
in patients with chronic periodontitis (p = 0.7557, p = 0.1479, p = 0.3717,
p = 0.6359 p = 0.1805).
Conclusion: Our results suggest no association between periodontitis and risk
factors for atherosclerosis in HIV positive patients.
233 RELATIONSHIP BETWEEN ALBUMINURIA AND SERUM LIPIDS
IN TYPE 1 DIABETES MELLITUS
T. Bulum, I. Prkačin, L. Duvnjak. University Hospital Merkur, Vuk Vrhovac Clinic
for Diabetes, Endocrinology and Metabolic Diseases, School of Medicine,
University of Zagreb, Zagreb, Croatia
236 SPECIFIC AGONIST OF THE CONSTITUTIVE ANDROSTANE
RECEPTOR IMPROVES REVERSE CHOLESTEROL TRANSPORT
AND CHOLESTEROL HOMEOSTASIS IN HYPERLIPIDEMIC MICE
D. Masson, A.L. Sberna, T. Gautier, J. Grober, A. Athias, M. Assem,
L. Lagrost. INSERM UMR866, Institut Fédératif de Recherche Santé-STIC,
Université de Bourgogne, Dijon, France
Introduction: In a number of studies, an increase in LDL and decrease in
HDL cholesterol levels has been found to be a risk factor for nephropathy in
individuals with type 1 diabetes (DM1). In this study we compared the level of
urinary albumin excretion with serum lipids in DM1.
Patients and Methods: 304 patients (37.9±11.3 years, 166M/138F,
BMI > 24.2±3.1 kg/m2 , HbA1c 7.22±1.6%, systolic and diastolic blood
pressure 126±15, 79±8 mmHg, serum creatinine 92.5±15.0 mmol/L, creatinine
clearance 1.81±0.48 ml/sec, urinary albumin excretion 15.3 mg/24h (0.9–
748.2)), have been evaluated in this study.
Results: From 304 patients, 246 (80.9%) had normoalbuminuria (<30 mg/24h),
and 58 (19.1%) albuminuria (>30 mg/24h). Only two patients had albumin
excretion over 300 mg/24h. Patients with albuminuria had higher level of
total (5.07 vs 5.02 mmol/L, p = 0.666), LDL (2.8 vs 2.8 mmol/L, p = 0.778),
VLDL cholesterol (0.57 vs 0.48 mmol/L, p = 0.151), triglycerides (1.27 vs
1.08 mmol/L, p = 0.158) and lower levels of HDL (1.68 vs 1.73 mmol/L,
p = 0.293) and HDL3 cholesterol (1.15 vs 1.16 mmol/L, p = 0.812), these
Background and Aims: The constitutive androstane receptor (CAR) is known
to be mainly involved in xenobiotic metabolism. The aim of our study was to
assess whether pharmacological CAR activation could also affect cholesterol
homeostasis in hyperlipidemic mouse models.
Methods: Wild type, Car−/−, ApoE−/− and low-density lipoprotein receptor
(Ldlr) −/− mice fed a western-type diet were treated with the CAR agonist
TCPOBOP.
Results: CAR activation was associated with a decrease in fecal cholesterol
output but with a marked increase (up to three fold, P < 0.01) in the elimination
of fecal bile acids. It was related to the induction of genes involved in synthesis,
conjugation and excretion of bile acids as well as the repression of the ileal
apical sodium-dependent bile acid transporter. In the same time, cholesterol
accumulation was reduced and marker (C4) of bile acid synthesis was increased
in the liver of TCPOBOP-treated animal. In all cases, TCPOBOP had no effect
in Car−/− mice. Kinetic studies performed with high-density lipoproteins (HDL)
labelled with (3)H-cholesteryl esters indicated that TCPOBOP-treated mice
52
excreted more HDL cholesterol-derived bile acids in their feces. Finally, longterm CAR activation was associated with decreases in cholesterol content of
the whole body and atherosclerosis lesions in aortic arches.
Conclusions: CAR is new player in cholesterol metabolism and CAR activation
has a beneficial impact on cholesterol homeostasis and atherosclerosis in
hyperlipidemic mice.
237 A RAPID EVALUATION MODEL FOR INFLAMMATORY DEATH USING
ZEBRAFISH LARVAE BY OXIDIZED LOW-DENSITY LIPOPROTEIN
AND DYSFUNCTIONAL HIGH-DENSITY LIPOPROTEINS
J. Park, K.-H. Park, K.-H. Cho. School of Biotechnology, Yeungnam University,
Oxidation and inflammation is a leading cause of almost all chronic metabolic
disorders, and plays major role in cardiovascular disease, cancer, and chronic
age-dependent disease. High-density lipoprotein (HDL) and apolipoprotein A-I
is a strong antioxidant and anti-inflammatory molecule in plasma. Fructose
induced non-enzymatic glycation of apoA-I can lead to the production of
dysfunctional apoA-I and HDL. In order to compare physiological effect,
reconstituted HDL containing native apoA-I (nA-I) or glycated apoA-I (gA-I) was
injected into zebrafish embryo under presence of inflammatory molecule. Coinjection of reconstituted HDL containing gA-I (gA-I-rHDL) and lipopolysaccaride
(LPS) resulted in acute death of embryos, while rHDL containing nA-I (nA-IrHDL) and LPS resulted in significantly enhanced the survivability. Co-injection
of oxLDL and nA-I-rHDL elevated the embryo survival, while oxLDL and
gA-I-rHDL exacerbated the survival with increase of inflammatory response.
Furthermore, co-injection with oxLDL and HDL2 (5 ng of protein) or HDL3 (15
ng of protein) from the young group (22±2 years old) showed significantly
increased embryo survivability compared with the same co-injection of HDL
from elderly (71±4 years old). In conclusion, our assay system provides a
rapid and economical method to screen an anti-oxidant and anti-inflammatory
agent using zebrafish embryos.
238 APOLIPOPROTEIN M BINDS OXIDIZED PHOSPHOLIPIDS AND
INCREASES THE ANTIOXIDANT EFFECT OF HDL
S.E. Nielsen1 , J. Ahnström2 , C. Christoffersen1 , A.N. Hoofnagle3 ,
P. Plomgaard1 , J.W. Heinecke4 , H. Björkbacka5 , B. Dahlbäck2 , L.B. Nielsen1 .
1
Department of Clinical Biochemistry KB3011, Rigshospitalet, Department
of Biomedical Science, University of Copenhagen, Copenhagen, Denmark,
2
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund
University, University Hospital Malmö, Malmö, Sweden, 3 Department of
Laboratory Medicine, 4 Department of Medicine, University of Washington,
Seattle, WA, USA, 5 Department of Clinical Sciences, Lund University,
University Hospital Malmö, Malmö, Sweden
Objective: Oxidation of LDL plays a key role in the development of
atherosclerosis. HDL may in part protect against atherosclerosis by inhibiting
LDL oxidation. Overexpression of HDL-associated apolipoprotein M (apoM)
protects mice against atherosclerosis through a not yet clarified mechanism.
Being a lipocalin, apoM contains a binding pocket for small lipophilic molecules.
Here, we report that apoM likely serves as an antioxidant in HDL by binding
oxidized phospholipids, thus enhancing the antioxidant potential of HDL.
Methods and Results: HDL was isolated from wild type mice, apoMdeficient mice, and two lines of apoM-Tg mice with ~2-fold and ~10-fold
increased plasma apoM, respectively. Increasing amounts of apoM in HDL were
associated with a concentration-dependent increase in the resistance of HDL
to oxidation with Cu2+ or 2,2 -azobis 2-methyl-propanimidamidedihydrochloride
and to an increased ability of HDL to protect human LDL against oxidation.
Oxidized phospholipids, but not native phospholipids, quenched the intrinsic
fluorescence of recombinant human apoM, suggesting selective binding of
oxidized phospholipid in the lipocalin-binding pocket of apoM.
Conclusions: The results suggest that apoM can bind oxidized phospholipids
and that it increases the antioxidant effect of HDL. This new mechanism may
explain at least part of the antiatherogenic potential of apoM.
239 ANTI-ATHEROGENIC EFFECTS OF PERSIMMON (DIOSPYROS
KAKI) PEEL IN LDLR−/− -DEFICIENT MICE: PLAQUE REDUCTION,
INHIBITION OF OXLDL/b2GPI AUTOANTIGEN AND THROMBOXANE
PLATELET ACTIVATION
E. Matsuura1 , N. Quan2 , K. Kobayashi1 , Y. Matsunami1 , M. Ide1 ,
M. Makarova3 , L. Shen4 , S. Ohno5 , Y. Zheng1 , H. Kobayashi6 , L.R. Lopez7 .
1
Cell Chemistry, Okayama University Graduate School of Medicine, Okayama,
Japan, 2 Cardiovascular Center, First Hospital, Jilin University, Changchun,
China, 3 Pathology, I.M. Sechenov Moscow Medical Academy, Moscow,
Russia, 4 Bacteriology, Okayama University Graduate School of Medicine,
Okayama, 5 Okayama Agriculture Development Institute, Akaiwa, 6 Regional
Cooperative Research Organization, Okayama Prefectural University, Soja,
Japan, 7 Corgenix, Inc, Broomfield, CO, USA
Introduction: The fruit persimmon (Diospyros Kaki) contains several bioactive
compounds and persimmon peel (PP) is particularly rich in carotenoids and
polyphenols with antioxidant, tyrosinase activities and hypolipidemic effects. It
has been postulated that PP may have dietary anti-atherogenic properties. We
studied the effect of PP on the development of atherosclerosis in low-density
lipoprotein receptor (LDLR−/− )-deficient mice by assessing lipid metabolism,
aortic plaque size, platelet activation and immune response to atherogenic
autoantigens associated with oxidative stress.
Methods: Male LDLR−/− -deficient mice were fed with normal chow, plain
high fat diet (controls) or supplemented with 10% dried PP for 12 weeks.
Sections of aortic atherosclerotic plaques were stained with Sudan IV
and plaque size estimated by Scion Image analysis. Plasma oxidized
LDL/b2-glycoprotein I (oxLDL/b2GPI) complexes (atherogenic autoantigen), IgM
anti-oxLDL antibodies and adiponectin were measured by ELISA. Urinary
11 dehydro-thromboxane B2 (11dhTxB2) (platelet activation marker) and
8-hydroxy-2-deoxyguanosine (8OHdG) (oxidative stress marker) were also
measured by ELISA.
Results: At 12 weeks, dietary PP significantly reduced plasma cholesterol
and triglycerides. The size of aortic atherosclerotic plaques in PP fed mice
decreased by 70% compared to control mice (p < 0.005). Mice with PP
supplementation had significantly lower levels of oxLDL/b2GPI complexes
(p < 0.05). PP also significantly reduced urinary levels of 11dhTxB2 (p < 0.05) to
the same level of mice receiving aspirin. Urinary 8OHdG, plasma adiponectin
and IgM anti-oxLDL were not affected.
Conclusions: These results demonstrate that persimmon peel (PP) may have
anti-atherogenic properties mediated by normalization of lipid metabolism,
inhibition of platelet activation and reduction of oxLDL/b2GPI atherogenic
complexes.
240 VITAMIN K ROLE IN ARTERIAL STIFFNESS DURING METABOLIC
SYNDROME IN RAT: DIRECT OR INDIRECT EFFECT VIA LIPID
METABOLISM
L. Emonnot1 , N. Côté2 , P. Mathieu2 , É. Levy1 , P. Moreau1 . 1 Université de
Montréal, Montréal, 2 Laval Hospital, Quebec, QC, Canada
The vitamin K-dependent enzymatic complex activates vascular anticalcification
proteins. To test whether quantitative alterations in this enzymatic complex may
explain cardiovascular stiffness occurring in metabolic syndrome (MS). To this
end, 3 groups of 4-week-old Wistar rats were employed:
i. control rats (Con) on regular rodent chow;
ii. experimental rats (LS) on a diet enriched in lipid and sucrose to produce
MS; and
iii. LS rats with subcutaneous administration of vitamin K (LSK) (2.5 mg every
2 days).
After 2 months, blood samples were taken. All groups displayed similar body
weight but the MS group was mostly characterized by hyperinsulinemia,
hypertriglyceridemia and decreased HDL-cholesterol levels. Moreover, the MS
group exhibited high pulse wave velocity (PWV) and arterial blood pressure.
Treatment with vitamin K normalized plasma levels of triglycerides and insulin
while reducing blood pressure and PWV. Interestingly, the MS group did
not show vascular calcification along with raised plasma levels of calcium
metabolism markers. Fibronectin expression was examined to determine
the contribution of fibrosis to PWV elevation, and our findings undeniably
documented its marked increase in MS rats with a concomitant significant
decrease with the administration of vitamin K. The induction of arterial fibrosis
in aortas was essentially due to free fatty acid mobilization as noted in our in
vivo experiments. Our studies indicate that vitamin K plays a functional role in
various alterations elicited by an experimental model of metabolic syndrome.
Ongoing experiments will elucidate whether vitamin K represents a potential
therapeutic tool for this cardiometabolic disorder.
241 OXIDIZED LDL TRIGGER ENDOPLASMIC RETICULUM STRESS
AND AUTOPHAGY: PREVENTION BY HDL
C. Vindis1 , C. Muller1 , R. Salvayre2 , A. Nègre-Salvayre1 . 1 INSERM U858,
2
Faculty of Medecine, Toulouse, France
Introduction: Oxidized LDL (oxLDL) exhibit atherogenic properties including
foam cell formation, inflammatory response, cell proliferation at low concentration, and apoptosis at higher concentration. In contrast, the anti-atherogenic
high-density lipoproteins (HDL) inhibit LDL oxidation, and counteract adverse
biological effects, such as cytotoxicity and inflammation triggered by cytokines
and oxLDL.
Objectives: To more deeply investigate the mechanisms mediating the
protective effects of HDL, we examined whether ER stress and autophagy
were implicated in oxLDL-induced apoptosis and whether HDL prevented these
stress processes.
Methods and Results: In human endothelial cells, HDL prevent the
oxLDL-induced activation of ER stress sensors and subsequent activation of
the pro-apoptotic mediators JNK and CHOP. OxLDL also trigger the activation
of autophagy, as assessed by LC3 processing and Beclin-1 expression. The
autophagic process is independent of the pro-apoptotic arms of ER stress,
but Beclin-1 contributes to PS exposure and phagocytosis of oxLDL exposed
79th EAS Congress
cells. Induction of autophagy and PS exposure by oxLDL are prevented by
HDL. Finally, the cytosolic Ca2+ deregulation triggered by oxLDL is a common
signalling pathway that mediates ER stress-induced cell death and autophagy,
all these events being blocked by HDL.
Conclusions: OxLDL-induced ER stress is associated to autophagy activation
which is not involved in oxLDL-mediated cell death machinery but contribute to
phagocytosis of oxLDL exposed cells. HDL by blocking the Ca2+ deregulation,
and consequently both ER stress and autophagy, may reduce the level of
ER stress and autophagy markers that are detected in early and advanced
atherosclerotic lesions.
242 PKC D IS INVOLVED IN OXIDIZED LDLS-INDUCED APOPTOSIS
OF VASCULAR SMOOTH MUSCLE CELLS
C. Ingueneau1 , P. Larroque1 , M.E. Reyland2 , R. Salvayre1 , A. Nègre-Salvayre1 ,
C. Vindis1 . 1 INSERM U1048, University of Toulouse III, Toulouse, France,
2
Anschutz Medical Center, University of Colorado, Denver, CO, USA
Introduction: Oxidized LDL (oxLDL) exhibit many atherogenic properties by
inducing foam cell formation, inflammatory response and apoptosis. Recently,
it has been demonstrated that apoptosis of vascular SMCs (VSMCs) alone is
sufficient to induce features of plaque vulnerability in apoE−/− mice. Among the
protein kinase C (PKC) isoforms, the PKCd isoform is described as an important
mediator of apoptosis in VSMCs.
Objectives: The aim of this work was to study the role of PKCd in oxidized
LDL-mediated apoptosis of vascular smooth muscle cells.
Methods and Results: We report that, in human VSMCs, oxLDLs trigger
PKCd activation as shown by its phosphorylation and nuclear translocation.
Mouse embryonic fibroblasts invalidated for PKCd (MEF PKCd −/− ) showed a
strong resistance to oxLDL thus supporting the role of PKCd in oxLDL-induced
apoptosis. By contrast to hSMC, the protective effect observed in MEF PKCd
−/−
was not dependent on the inhibition of the intrinsic calcium-dependent
mitochondrial pathway induced by oxLDL. Protective effect of caspase-8
inhibitor and increased mRNA expression of Fas receptor showed that, in this
cellular model, PKCd is involved in the extrinsic apoptotic pathway activated by
oxLDL.The detection of phophorylated PKCd in human carotid plaques strongly
highlights the critical role of PKCd in regulating apoptosis of vascular cells.
Conclusions: Our results show that PKCd play a major role in oxLDL-induced
apoptosis by acting in two different signalling pathways that participate in plaque
formation and instability.
243 PHOSPHATIDYLCHOLINE HYDROPEROXIDE-INDUCED RAC
ACTIVATION: ITS INVOLVEMENT IN THP-1 CELL ADHESION TO
ICAM-1 VIA ACTIN POLYMERIZATION
A. Asai1 , K. Nakagawa2 , F. Okajima1 , Y. Nakajima1 , M. Nagao1 , T. Miyazawa2 ,
S. Oikawa1 . 1 Endocrinology and Metabolism, Nippon Medical School, Tokyo,
2
Food and Biodynamic Chemistry Laboratory, Tohoku University Graduate
School of Agricultural Science, Sendai, Japan
Background and Aims: The accumulation of phosphatidylcholine hydroperoxide (PCOOH), a primary oxidation product of phosphatidylcholine, in
blood plasma and tissues has been reported to be associated with various
pathological conditions including atherosclerosis. Although PCOOH is a
common precursor of a variety of atherogenic secondary oxidation products of
phosphatidylcholine, the biological functions of PCOOH per se have rarely been
investigated. Recently, we demonstrated that PCOOH induces THP-1 monocytic
cell adhesion to ICAM-1 via F-actin-rich membrane protrusion formation. Here,
we investigated the underlying mechanism of the induction of cell adhesion.
Methods: ICAM-1-immobilized microplates were used for cell adhesion assay.
Membrane protrusion morphology was observed fluoroscopically after F-actin
was stained with Alexa Fluor 488 phalloidin. Rho family GTPase activations
were evaluated by pull-down assay.
Results: PCOOH-induced THP-1 cell adhesion and membrane protrusion
formation were suppressed by the pretreatment of fluvastatin (isoprenoid
depletion). The inhibitory effect of fluvastatin was abrogated by mevalonate
or geranylgeranyl pyrophosphate, but not by cholesterol. The cell adhesion
and protrusion formation was also suppressed by a geranylgeranyltransferase
inhibitor GTI-286. Pull-down assays demonstrated that Rac1 and Rac2 were
activated in PCOOH-treated cells. Correspondingly, Clostridium difficile toxin
B (pan-Rho family GTPase inhibitor) and NSC23776 (synthetic Rac-specific
inhibitor) also suppressed cell adhesion. Furthermore, RNA interference of
the Rac isoforms led to decreased cell adhesion and membrane protrusion
formation in PCOOH-treated cells.
Conclusion: The present findings demonstrate that Rac GTPases play an
important role in PCOOH-induced THP-1 cell adhesion to ICAM-1 via actin
polymerization.
53
244 APOE*3LEIDEN.CETP TRANSGENIC MICE AS MODEL FOR THE
METABOLIC SYNDROME
A.M. van den Hoek1,2 , J.W.A. van der Hoorn1,2 , M.C.E. Maas1 , C.M. van
den Hoogen1 , A. van Nieuwkoop1 , E.O. Offerman1 , E.J. Pieterman1 ,
L.M. Havekes1,2 , H.M.G. Princen1 . 1 TNO-Metabolic Health Research, 2 Leiden
University Medical Center, Leiden, The Netherlands
Background: The metabolic syndrome is characterized by the co-occurrence
of several risk factors i.e. increased body weight (bw) and insulin resistance (IR)
and at the same time adverse changes in plasma lipids as observed in diabetic
dyslipidemia, with increased triglycerides and apoB-containing lipoproteins and
decreased HDL.The aim was to investigate whether the APOE*3Leiden.CETP
(E3L.CETP) mouse is a translational model for the metabolic syndrome.
Methods: Male E3L.CETP mice were put on a high fat diet and fructose
in drinking water for 12−16 weeks to induce diet-induced obesity and IR.
Thereafter, mice were treated with either rosiglitazone (3 and 11 mg/kg/d),
liraglutide (0.2 mg/kg/d), HSD-1 inhibitor (0.1 mg/kg/d), resveratrol (75 mg/kg/d),
(fenofibrate 12 mg/kg/d), atorvastatin (10 mg/kg/d) or niacin (720 mg/kg/d) for 4
weeks. The effects on bw, plasma lipids and IR (via plasma glucose/insulin
and/or hyperinsulinemic euglycemic clamps) were assessed.
Results: Dietary treatment resulted in a human-like lipoprotein profile with
a TC/HDL-C ratio of 3−4. Anti-diabetic compounds rosiglitazone, liraglutide
and HSD-1 inhibitor significantly decreased glucose and insulin levels or IR.
Liraglutide and HSD-1 inhibitor also decreased bw. Established lipid-lowering
compounds atorvastatin, fenofibrate and niacin, and resveratrol improved the
dyslipidemia.
Conclusion: The data indicate that the E3L.CETP mouse is a good
translational animal model combining all important features that underlie
the metabolic syndrome and mimic the human response to clinically used
treatments. We conclude that the E3L.CETP mouse is a promising model to
investigate the effects of new drugs, alone or in combination, that affect IR and
diabetic dyslipidemia.
245 SUBCLINICAL HYPOTHYROIDISM IS RELATED TO A SPECIFIC
LIPID PROFILE INDEPENDENTLY OF TSH LEVELS
A. Hernández-Mijares1 , A. Jover2 , C. Bañuls2 , M. Rocha2 , E. Solá1 , L. Bellod2 .
1
Endocrinology Unit, Hospital Universitario Dr. Peset, 2 Hospital Doctor Peset
Research Foundation, Valencia, Spain
Background and Objectives: Subclinical hypothyroidism (SCH) is a frequent
condition defined biochemically as a normal serum free thyroxine concentration
in the presence of an elevated serum thyrotropin (TSH) concentration. Overt
hypothyroidism is associated with an increased risk of cardiovascular disease
(CVD), but whether SCH does remains controversial.
The aim of this study was to assess classic and emerging risk factors in
patients with SCH clustered in two groups according to TSH levels compared
to euthyroid subjects.
Patients and Methods: Ninety-five middle-aged women with a recent diagnosis
of SCH were grouped according to TSH levels: 5−9.9 mIU/ml (69 patients) and
10 mIU/ml (26 patients). Lipoprotein subclasses, homocysteine and insulinresistance were measured, and conventional cardiovascular risk markers were
recorded. Results were compared with those obtained in 65 euthyroid controls
matched by age and BMI.
Results: Patients with SCH showed significantly higher homocysteine and
triglycerides levels, and higher total/HDL-cholesterol and atherogenic index of
plasma [Log(Triglycerides/HDL-Cholesterol)], and significantly lower levels of
HDL-cholesterol than controls. Both groups of SCH patients had similar lipid
concentrations, but those with TSH levels 10 mIU/ml showed smaller LDLcholesterol particles. No differences were found in insulin-resistance or in blood
pressure in all groups.
Conclusion: Our findings suggest that SCH in middle-aged women is
associated with an atherogenic lipid profile, not only when TSH levels are
10 mIU/ml, but also with milder increases.
246 AMLODIPINE RESTORES ENDOTHELIAL FUNCTION ALTERED
BY IRREVERSIBLY GLYCATED LOW DENSITY LIPOPROTEINS
L. Toma, C.S. Stancu, G.M. Sanda, A.V. Sima. Lipoproteins and
Atherosclerosis, Institute of Cellular Biology and Pathology ‘N. Simionescu’,
Bucharest, Romania
Aim: To evaluate the potential of amlodipine to restore endothelial dysfunction
induced by irreversibly glycated LDL (AGE-LDL).
Methods:
i. evaluation of the oxidative and inflammatory status of human endothelial
cells (EA.hy926) incubated with human AGE-LDL (100ug protein/ml) in the
absence/presence of 10uM amlodipine;
ii. determination of the mechanism of action of amlodipine, p38 MAPK and
NF-kB signaling pathways.
AGE-LDL was prepared by incubation of native LDL with 0.2M glucose at 37ºC,
under sterile conditions, with antioxidants, one month. The gene expression
of p22phox , MCP-1 and VCAM-1 was determined by Real Time PCR and
54
the protein expression of iNOS, MCP-1, phospho-p38 MAPK and phosphop65 subunit of NF-kB by Western Blot. The cellular NADPH activity, the
3-nitrotyrosine in the culture medium, and the adhesion of freshly isolated
human monocytes to the cells were measured by chemiluminescence, Dot Blot,
and spectrofluorimetric techniques.
Results showed that amlodipine determines in cells incubated with AGE-LDL
a statistically significant decrease of:
i. the oxidative stress, as demonstrated for p22phox (40%) and iNOS (70%)
expression, NADPH oxidase activity (60%) and 3-nitrotyrosine level (30%);
ii. the inflammatory stress, as demonstrated for MCP-1 (50%) and VCAM-1
(40%) expression and for monocytes adhesion (10%);
iii. the phosphorylation of p38 MAPK and p65 subunit of NF-kB.
Conclusion: Amlodipine ameliorates endothelial dysfunction induced by
glycated LDL through mechanisms involving p38 MAPK and NF-kB. The
results suggest that amlodipine may successfully be used as a complementary
treatment in diabetes.
Funding: Supported by the Ministry for Education, Research, Youth and Sport,
Romania, grant 1227/2009−11.
247 ANALYSIS OF EICOSANOIDS IN HUMAN LDL BEFORE AND AFTER
CU-INDUCED OXIDATION
J. Dorow1 , L. Kortz1 , S. Becker1 , J. Thiery2 , S. Hauschildt3 , U. Ceglarek1,2 .
1
LIFE − Leipzig Research Center for Civilization Diseases, University
Leipzig, 2 Institute of Laboratory Medicine, Clinical Chemistry and Molecular
Diagnostics, University Hospital Leipzig, 3 Institute for Bioscience, Pharmacy
and Psychology, University Leipzig, Leipzig, Germany
Lipoprotein oxidation is a key stage in the development of atherosclerosis.
Oxidized low-density lipoprotein (oxLDL) has many atherogenic properties.
Eicosanoids are lipid mediators, oxidized from arachidonic acid by enzymatic or
non-enzymatic peroxidation. The aim of our study was to develop a standardized
preanalytical protocol for the analysis of eicosanoids in native LDL and Cuinduced oxLDL from healthy volunteers.
Plasma-LDL was isolated by density-gradient ultracentrifugation and subsequently oxidized with CuCl2 . After solid phase extraction eicosanoids
were quantified by fast liquid chromatography combined with tandem mass
spectrometry (5500 QTRAP Applied Biosystems).
A storage of LDL for 7 days at 4ºC resulted in a significant increase
of 11 eicosanoids. Particularly hydroxyeicosatetraenoic acids (e.g 11-HETE)
increased up to 400%. After Cu-induced oxidation HETEs, prostaglandins,
hydroperoxyeicosatetraenoic acids (HpETE), oxotetraenoic acids (oxETE), and
isoprostanes were detected in oxLDL. Applying a standardized analytical
protocol for LDL-storage and oxidation we were able to generate the eicosanoid
panel with a mean variability of 11% (6−15%). Comparing LDL and oxLDL from
different subjects we found a high variability in eicosanoid concentrations (15–
147%). In LDL from subjects with elevated LDL/HDL cholesterol ratio (>3.9,
n = 4) significantly higher eicosanoid concentrations were found compared to
healthy subjects (n = 4) (e.g. PGE2 : 653 pg/mg protein versus 170 pg/mg
protein).
In conclusion we developed a standardized protocol for the reproducible
analysis of eicosanoids in LDL and oxidized LDL. A high variability of eicosanoid
concentrations in LDL and oxLDL from different subjects was observed in
association with an elevated LDL/HDL ratio.
248 A GENETIC VARIANT WITHIN CAVEOLIN-3 PROTECTS AGAINST
STATIN-INDUCED MYOPATHY
E. Giannakidou-Jordan1 , C. Gelsinger1 , U. Kassner1 , H. Knoblauch2 ,
S. Spuler2 , A. Busjahn3 , E. Steinhagen-Thiessen1 . 1 Lipid Ambulatory
Clinic, Charite Berlin, Campus Virchow Klinikum, 2 Muscle Research Clinic,
Experimental and Clinical Research Center, Charite Berlin, Campus Berlin
Buch, 3 HealthTwist, Berlin, Germany
Myopathy is a frequent and potentially life-threatening complication of statin
therapy. We hypothesized that there is genetic susceptibility to statin-induced
myopathy could be associated with skeletal muscle-related genes. We enrolled
a cohort of 400 patients, 250 with muscular symptoms and 150 matched
asymptomatic individuals, after due approval. We collected all demographic
and laboratory data, including genomic DNA. Muscular symptoms associated
with statin intake were defined as muscle pain and/or muscle cramps and/or
elevated CK.
Laboratory and clinical data were available from all patients, including CK, lipids
and liver enzymes. We genotyped single-nucleotide polymorphisms (SNPs) in
Caveolin-3, a gene associated with “painful myopathy”. We found a significant
effect (p < 0.009) for CAV3 rs1974763 in men. The T allele was protective
against statin-induced myopathy, while the C allele appeared as a risk factor.
The overall frequency of the alleles was 86% and 13% for C and T respectively.
The frequency of the T-allele was 21% in male controls compared to 10% in
male cases. In females the T-allele had a frequency of 8% in controls and
13% in cases (n.s.). Caveolae are flask-like plasma membrane invaginations
involved in a variety of signal transduction pathways, for example GLUT4 and
Src kinase signalling. In addition, there is emerging evidence of the tight link
between caveolae and cholesterol metabolism and the impact of statins on
caveolae structure and function. To our knowledge, this is the first association
study linking genetic variants within a skeletal muscle-related gene, namely
Caveolin-3 with statin-induced myopathy.
249 DIFFERENTIAL EFFECT OF ASPIRIN ON OXIDATIVE STRESS,
THROMBOXANE, NITRIC OXIDE METABOLITES AND P-SELECTIN
IN TYPE 2 DIABETES MELLITUS
L.R. Lopez1 , I. Muncy1 , E. Matsuura2 , J. Batuca3 , A. Oregon-Miranda4 ,
I. Garcia De La Torre4 , P.R. Ames5 . 1 Corgenix, Inc, Broomfield, CO,
USA, 2 Cell Chemistry, Okayama University Graduate School of Medicine,
Okayama, Japan, 3 Pharmacology, New University of Lisbon, Lisbon, Portugal,
4
Immunology & Rheumatology, Hospital General de Occidente, Zapopan,
Mexico, 5 Haematology & Rheumatology, Airedale General Hospital, Steeton,
UK
Introduction: Aspirin (ASA) prevents CVD by irreversibly inhibiting platelet
cyclo-oxygenase, leading to decreased thromboxane-mediated platelet activation/aggregation. We evaluated additional effects of ASA treatment (100 mg/day
x 7days) on oxidative stress, nitric oxide metabolites and P-Selectin in diabetes
mellitus (DM).
Methods: Serum and urine were obtained before ASA ingestion (baseline)
and after 7 days (post-ASA) from 73 DM (mean age 54.3 years, mean disease
duration 9.6 years) and 86 age/sex matched healthy controls. Urinary 11dhTxB2
and 8-isoPGF2a, and serum P-Selectin were measured by ELISA. Serum nitrite
(NO−2 ) and nitrate (NO−3 ) by modified Griess reaction.
Results: Compared to baseline controls, baseline DM had higher mean levels
of 11dhTxB2 (3,665±2,465 vs 2,450±1,572 pg/mg creatinine, p = 0.002),
8-isoPGF2a (1,457±543 vs 1,009±412 pg/mg creatinine, p < 0.0001), NO−2
(11.8±7.3 vs 4.8±5.3 mM, p < 0.0001), NO−3 (50.4±39.3 vs 20.9±16.7 mM,
p < 0.0001) and P-Selectin (120.8±56.7 vs 93.0±26.1 ng/mL, p = 0.02). Post
ASA inhibition of 11dhTxB2 was 71.5% (3,665 to 996) in DM and 75.1% (2,450
to 624) in controls, but DM had twice as many ASA non-11dhTxB2 responders
(>1,500) than controls (14.8% and 8.4%). Post-ASA 11dhTxB2 levels in DM
(996±845) were significantly higher than controls (624±509, p < 0.0001). ASA
had no effect on 8-isoPGF2a, NO−2 , NO−3 or P-Selectin in DM or controls.
Conclusion: ASA had no significant effect on oxidative stress, nitric oxide
metabolites or P-Selectin in either DM or controls. The degree of 11dhTxB2
inhibition was similar in DM and controls though ASA non-responders were
more frequent in DM despite greater baseline 11dhTxB2 concentration. The
latter aspect warrants further investigation.
250 LDL FROM PATIENTS WITH METABOLIC SYNDROME OR TYPE
2 DIABETES SHOW INCREASED LIPID PEROXIDATION AND
ACTIVATE PLATELETS
C. Calzada1 , R. Colas1 , M. Guichardant1 , A. Sassolas1 , C. Cugnet-Anceau2 ,
M. Moret2 , P. Moulin1 , M. Lagarde1 . 1 Université de Lyon, INSA-Lyon, CarMeN,
INSERM U1060, IMBL, Villeurbanne, 2 Hospices Civils de Lyon, Lyon,
France
Aims: The objectives were to determine different complementary indices of
lipid peroxidation in LDL from patients with metabolic syndrome (MetS) or type
2 diabetes and to investigate ex vivo the effects of LDL obtained from these
patients on control platelets.
Methods: LDL were isolated by ultracentrifugation from plasma of patients
and healthy control subjects. The profile of lipids, fatty acids and fatty acid
oxidation products was determined. The effects of LDL on platelet arachidonic
acid signaling cascade and platelet aggregation were investigated.
Results: Compared to LDL from healthy subjects, LDL from MetS
and type 2 diabetic patients were smaller (25.1±0.2 vs 24.4±0.2 vs
24.4±0.1 nm), had lower cholesteryl esters, lower ethanolamine plasmalogens
and higher triglycerides levels. The concentrations of linoleic acid, the
main polyunsaturated fatty acid, were decreased in phosphatidylcholine and
cholesteryl esters, whereas increased concentrations of hydroxylated fatty acids
issued from linoleic acid (hydroxy-octadecadienoic acids, HODE) (+50%), and
malondialdehyde (x2) were found in LDL from MetS and diabetic patients.
Addition of LDL from patients with MetS or diabetes to control platelets
resulted respectively in a 3-fold and 3.5-fold increase of collagen-induced
platelet aggregation. Both LDL from MetS and diabetic patients stimulated
phosphorylation of platelet p38 MAPK and cytosolic phospholipase A2 , and
increased by 2-fold thromboxane B2 production. LDL from healthy subjects had
no activating effects on platelets.
Conclusion: The presence of a MetS, with or without diabetes, is associated
with lipid modifications and increased lipid peroxidation in LDL which trigger
platelet activation.
79th EAS Congress
251 DO MTTP MUTATIONS OVERCOME HETEROZYGOUS FAMILIAL
HYPERCHOLESTEROLEMIA? SEGREGATION ANALYSIS IN A
FAMILY WITH BOTH LDLR AND MTTP MUTATIONS
M. Di Filippo1,2 , N. Peretti2,3 , P. Moulin2,4,5 , C. Jacobs1 , S. Chebel1 ,
A. Lachaux3,5 , A. Sassolas1,2 . 1 Dept of Biochemistry, Hospices Civils de Lyon,
2
INSERM U1060 CarMeN, 3 Dept of Pediatric Gastroenterology, 4 Dept of
Endocrinology, Hospices Civils de Lyon, 5 University Lyon 1, Lyon, France
Objectives: To investigate genetic and clinical abnormalities and treatment
efficacy in a Turkish consanguineous family with combined inheritance of both
familial hypercholesterolemia and abetalipoproteinemia.
Methods: Lipid profiles and systematic sequencing of MTTP, LDLR, ApoE and
ApoB genes were performed for both parents and the three children.
Results: Abetalipoproteinemia (homozygous c.59del17 MTTP) was first diagnosed in child 1 (1 year old, LDLC < 0.02 g/l); father (24 years) LDLC was 1.39
g/l and mother (19 years) was 2.09 g/l. Five years later, the third child (4 years)
showed a severe hypercholesterolemia (LDLC 4.64 g/l, IMTc 0.52 mm), leading
to the study of the LDLR gene: he was homozygous for p.V523M (12−25%
residual LDLR activity) and was not carrier of the MTTP mutation. Both parents
were found subsequently heterozygous for both mutations, child 2 was unaffected. Simultaneously, the father showed an increase in LDLC (2.21 g/l) and a
recent corneal arc (IMTc 0.85 mm). Following statin treatment LDLC decreased
to: 1.48 g/l for the father (atorvastatine 10 mg), 1.32 g/l for the mother (fluvastatine 80 mg) and 1.45 g/l for the child (atorvastatine 10 mg, ezetimibe 30 mg);
interestingly, the child normalized IMTc (0.39 mm) after 2 years treatment.
Conclusion: Weak residual LDLR activity could explain the excellent treatment
responsiveness of the homozygous LDLR mutation carrier. Normal LDLC
concentration in the father with delayed hypercholesterolemia suggests that
partial inhibition of MTP may strongly alter the phenotype of heterozygous
hypercholesterolemia.
252 PLAQUE OXYSTEROLS INDUCE IMBALANCED UP-REGULATION
OF MMP-9 IN MACROPHAGIC CELLS THROUGH REDOX-SENSITIVE
SIGNALING PATHWAYS
S. Gargiulo, P. Gamba, B. Sottero, G. Poli, G. Leonarduzzi. Clinical and
Biological Sciences, University of Turin, Orbassano (Torino), Italy
55
although the incidence was small (statin: 0.35%, statin+ezetimibe: 0.56%;
p = 0.017). A significantly greater number of subjects reported 1 AE, drugrelated, hepatitis-related and gastrointestinal-related AEs, and CK elevations
10×ULN (all p 0.008) in the first-line vs. second-line therapy studies in both
treatment groups. AEs were generally similar between treatments in subgroups,
and similar rates of AEs were reported between age and race subgroups;
however, generally higher AE rates were reported for women.
Conclusions: Statin+ezetimibe and statin monotherapy demonstrated generally similar tolerability profiles. Ongoing statin treatment at study entry likely
screened out participants for previous statin-related AEs and tolerability issues.
Concerns regarding AEs should not limit lipid-altering therapy for subjects 65,
though tolerability may be more of an issue for women.
254 PITAVASTATIN HAS PLEIOTROPIC ANTI-OXIDATIVE AND ANTIINFLAMMATORY BENEFITS IN PATIENTS WITH DYSLIPIDAEMIA:
RESULTS FROM A PHASE III STUDY PROGRAMME
N. Hounslow. Kowa Research Europe, Wokingham, UK
Aims: To determine the pleiotropic effects of pitavastatin by evaluating its antioxidative actions (reduction in pro-atherogenic oxidized low-density lipoprotein
[Ox-LDL]) and anti-inflammatory effects (reductions in high sensitivity C-reactive
protein [hs-CRP]) in patients at high risk of coronary heart disease (CHD).
Methods: Evaluation of changes in Ox-LDL and hs-CRP (measured in a central
research laboratory) in patients recruited to a 52-week open-label extension
study of pitavastatin (NK-104–307), to two blinded active-controlled 44-week
extension studies (NK-104–309 and NK-104–310) or to a 60-week open-label
extension study (65 years of age; NK104–308). The daily dose of pitavastatin
was 4 mg or 2 mg (NK-104–308). Patients had mixed (combined) dyslipidaemia
or primary hypercholesterolaemia and a range of CHD risk factors, including
type 2 diabetes and hypertension, and entered the extension studies from 12week blinded core studies.
Results: Pitavastatin 2−4 mg consistently decreased levels of Ox-LDL and hsCRP during long-term treatment (Table).
Table: Mean change (%) in Ox-LDL and hs-CRP
Ox-LDL (U/L)
The rupture of a vulnerable atherosclerotic plaque is a major cause of thrombus
formation leading to acute coronary syndrome. Connective tissue integry
depends on a balance between degradation and repair of the extracellular
matrix (ECM). The degradation of ECM by MMPs contribute to fibrous cap
thinning and plaque rupture. Macrophages are considered to be the major
source of these enzyme in vulnerable regions of the atherosclerotic plaques.
The MMPs are inhibited by forming 1:1 complexes with TIMPs. MMP-9 has a
prominent role in plaque formation, destabilization and rupture. The oxidized
LDL contain numerous lipid oxidation products and among them, oxysterols
are able to modulate several biochemical effects in the pathogenesis of
atherosclerosis.
To clarify the role of these compounds, we have studied the effect of an oxysterol
mixture, whose composition is similar to that found in the human carotid
plaques, on the MMP-9 expression by promonocytic U937 cells. Our results
indicate that the oxysterols induce a significantly increase of mRNA, protein and
activity of MMP-9, whereas they don’t affect the expression of the endogenous
inhibitors TIMP-1 and TIMP-2. The cell treatment with the pharmacological inhibitors and siRNA demonstrate that this effect is mediated by NADPH oxidasedependent ROS over-production and activation of the MAP kinases ERK and
JNK which, in turn, may act on the transcription factors AP-1 and NF-kB.
Our data show that oxysterols can contribute to the plaque instability through
the upregulation of MMP-9, key enzyme in the degradation of fibrous cap of the
advanced atherosclerotic plaques.
253 SAFETY PROFILE OF STATINS ALONE OR COMBINED WITH
EZETIMIBE: A POOLED ANALYSIS OF OVER 21,000 PATIENTS
P.P. Toth1 , W. Weintraub2 , D. Morrone2 , M.E. Hanson3 , R.S. Lowe3 , J. Lin3 ,
A. Shah3 , A.M. Tershakovec3 . 1 Preventive Cardiology, Sterling Rock Falls
Clinic, Sterling, IL, 2 Christiana Care Center for Heart & Vascular Health,
Christiana Care, Newark, DE, 3 Merck, Whitehouse Station, NJ, USA
Objective: Assess the safety and tolerability profiles of various statins +
ezetimibe vs statin monotherapy using a pooled analysis of data.
Methods: Data were combined from 27 double-blind, placebo-controlled or
active-comparator studies that randomized adult hypercholesterolemic patients
to statin or statin+ezetimibe for 6−24 weeks. In the full cohort, percent
patients with adverse events (AEs) within treatment groups (statin: N = 10,517;
statin+ezetimibe N = 11,714) was assessed by a logistic regression model with
terms for first-/second-line therapy (statin-naı̈ve/ongoing statin at study entry),
trial within first-/second-line therapy, and treatment. The same model was fitted
for age (<65, 65 years), gender, and race (white, black, other) subgroups with
additional terms for subgroup and subgroup-by-treatment interaction.
Results: In the full cohort, the only significant difference between treatments
was in consecutive AST or ALT elevations 3× upper limit of normal (ULN),
Study
Core
study
baseline*
hs-CRP (mg/L)
Change
at end of
core study
Change
at end of
extension
study
Core study
baseline
Change
at end of
core study
Change
at end of
extension
study
NK-104–307
(4 mg)
81.8
(n = 641)
−30.8%
−36.8%
3.19
(n = 1338)
−13.5%
−25.4%
NK-104–309
(4 mg)
79.8
(n = 120)
−33.4%
−37.6%
3.53
(n = 120)
−19.0%
−11.9%
NK104–310
(4 mg)
72.0
(n = 141)
−30.0%
−32.4%
3.38
(n = 141)
N/A
−9.5%
NK-104–308
(2 mg)
79.1
(n = 511)
−26.0%
−38.4%
3.35
(n = 509)
−0.6%
−14.9%
*Following a dietary and washout period of 6−8 weeks.
Mean percentage changes are shown for individual patients from core study baseline.
N/A, not available.
Conclusions: Pitavastatin exerts beneficial anti-oxidative and anti-inflammatory
effects in addition to its direct effects on cholesterol levels.
255 CORRELATION BETWEEN LP(A) AND PARAOXONASE IN
PSORIASIS
G. Ferretti1 , T. Bacchetti1 , A. Campanati2 , O. Simonetti2 , A. Offidani2 .
1
Dipartimento di Biochimica, Biologia e Genetica, 2 Dipartimento di Medicina
Clinica e Biotecnologie Applicate, Università Politecnica delle Marche, Ancona,
Italy
Modifications of lipoprotein levels (LDL, HDL and VLDL) and composition play
a role in inflammation and atherosclerosis. Recently, it has been demonstrated
that also lipoprotein (a) [Lp(a)] is involved cardiovascular disease.
Aim of the study was to investigate the relationship between Lp(a) levels,
markers of oxidative stress and activity of paraoxonase (PON1), an antioxidant
and antiinflammatory enzyme associated with HDL. The study was carried
out in healthy subjects (n = 10) and patients affected by psoriasis (n = 23), an
inflammatory disease associated with an increased risk of atherosclerosis.
The results showed higher levels of Lp(a) in serum of psoriasis patients compared to controls (29.6±18.3 mg/dL vs 12.6±2.4 mg/dL; p < 0.001). Higher levels of lipid hydroperoxides (5.05±1.72 mmol/L vs 2.05±0.77 mmol/L, p < 0.001)
and C-reactive protein (CRP) (0.69±0.56 mg/dL vs 0.32±0.15 mg/dL, p < 0.05)
were observed in patients with respect to controls, confirming that psoriasis
is associated with inflammation and oxidative stress. The mean value of
serum PON1 activity, was lower in psoriasis patients (207.2±101.6 U/mL)
with respect to controls (298.6±106.3 U/mL), but the differences were not
statistically significant. Positive correlations were established between Lp(a)
and lipid hydroperoxides (r = 0.71, n = 23, p < 0.001) or CRP levels (r = 0.56,
n = 23;p < 0.01) in psoriasis subjects; in the same patients, a negative
56
correlation was observed between Lp(a) levels and PON1 activity (r = −0.76,
n = 23, p < 0.001). PON1 exerts a protective effect against lipid peroxidation of
lipoproteins and membranes, therefore our results suggest that patients with
higher Lp(a) levels are more exposed to oxidative damage.
256 EFFECT OF EZETIMIBE ON MARKERS OF OXIDATIVE STRESS
IN PATIENTS WITH HYPERCHOLESTEROLEMIA
M.S. Kostapanos1 , A.T. Spyrou1 , C.C. Tellis2 , I.F. Gazi1 , A.D. Tselepis2 ,
E.N. Liberopoulos1 , M.S. Elisaf1 . 1 Department of Internal Medicine, University
of Ioannina School of Medicine, 2 Laboratory of Biochemistry, Department of
Chemistry, University of Ioannina, Ioánnina, Greece
Ezetimibe effectively reduces low-density lipoprotein cholesterol (LDL-C). In this
study, we tested the hypothesis that ezetimibe monotherapy may also decrease
markers of oxidative stress in subjects with hypercholesterolemia.
Methods: Subjects with hypercholesterolemia and no evidence of cardiovascular disease were randomized to open-label ezetimibe monotherapy 10 mg/day
(EZT group) or therapeutic lifestyle changes (TLC group). At baseline and 12
weeks post-treatment serum lipoprotein and apolipoprotein levels as well as
oxidative stress parameters, including oxidized LDL (ox-LDL), 8-isoprostanes
(8-epiPGF2a) and reactive oxygen metabolites (d-ROMs) levels, were blindly
determined.
Results: A total of 60 patients were included; 30 in each group. Despite a
significant decrease in ox-LDL levels (by 20.8%, p < 0.001 vs baseline; p < 0.001
vs TLC group) in the EZT group no change in the ratio ox-LDL to LDL-C
was noticed following ezetimibe treatment. A trend towards a decrease in
8-epiPGF2a (by 15.2%, p = 0.09 vs baseline; p = 0.10 vs TLC group) and
d-ROMs levels (by 10.0%, p = 0.11 vs baseline; p = 0.09 vs TLC group)
in the EZT group was proved unrelated to the degree of LDL-C lowering
associated with ezetimibe administration. A significant decrease in 8-epiPGF2a
and d-ROMs levels (by 20.4% and 18.2%, respectively, p < 0.01 vs baseline for
both), was noted only among patients who exhibited ‘high oxidative stress’ at
baseline. No change in any of oxidative stress parameters was noted in the
TLC group.
Conclusion: Ezetimibe may decrease markers of oxidative stress in
hypercholesterolemic subjects. This benefit may be more profound among
hyperlipidemic patients who exhibit ‘high oxidative stress’ at baseline.
257 ASSOCIATION OF HYPERTENSION WITH SMALL, DENSE
LOW-DENSITY-LIPOPROTEIN IN PATIENTS WITHOUT METABOLIC
SYNDROME
Y.K. Kim, H.S. Seo, J.O. Na, C.U. Choi, H.E. Lim, E.J. Kim, S.-W. Rha,
C.G. Park, D.J. Oh. Korea University Guro-Hospital, Seoul, Republic of
Korea
A higher proportion of small, dense low-density-lipoprotein (sdLDL) is known to
be associated with a high prevalence of cardiovascular disease in association
with metabolic syndrome (MS). Hypertension (HTN) is one of the known risk
factors for MS. However, whether HTN is associated with sdLDL in patients
without MS is not yet clear.
Methods: The lipid profiles, including LDL subfractions, of 383 consecutive
subjects were evaluated. Seventy-seven of these patients had MS and 306
subjects did not. The patients without MS consisted of 198 hypertensive patients
(non-MS/HTN group) and 108 normotensive subjects (non-MS/non-HTN group).
Peak and mean particle diameter of LDL were measured by gradient gel
electrophoresis; lipid profiles and blood sugar and apolipoprotein levels were
also measured.
Results: Plasma total cholesterol, LDL-cholesterol, high-density-lipoprotein
(HDL)-cholesterol, triglyceride, HDL-C/Apo A1, LDL-C/ApoB, and apolipoprotein (A1, B, CII and E) levels did not differ between the non-MS/non-HTN and
non-MS/HTN groups. When analyzing LDL subfraction, the absolute amount
of patterns A and B was not different between the non-MS/non-HTN and
non-MS/HTN groups. When comparing the non-MS/non-HTN and the nonMS/HTN groups, the proportion of sdLDL was higher in the non-MS/HTN group
(37.7% versus 39.9%, P = 0.046). The body mass index, homeostasis model
assessment index, and C-reactive protein levels were not different between the
non-MS/non-HTN and non-MS/HTN groups.
Conclusions: The proportion of sdLDL to total LDL was significantly higher in
hypertensive subjects, even those without MS, than in normotensive subjects.
This result suggests that HTN can contribute to atherosclerosis and endothelial
dysfunction associated with mechanical vascular injury by altering LDL size.
258 LIPID PROFILE IN PATIENTS WITH INFLAMMATORY
POLYARTHRITIS PRIOR TO THERAPY
A.Z. Alhusain1 , S.M. Verstappen1 , H. Mirjafari1 , D. Symmons1 , V. CharltonMenys2 , P. Durrington2 , D. Bunn3 , I.N. Bruce1 . 1 Arthritis Research UK
Epidemiology Unit, 2 Cardiovascular Research Group, University of Manchester,
Manchester, 3 Norfalk Arthritis Research Group, University of East Anglia,
Norwich, UK
Rheumatoid arthritis (RA) is a chronic inflammatory condition with established
disease-associated dislipidaemia. Previous studies noted that lipid profiles of
patients with active RA are mainly characterised by reduced serum levels of high
density lipoprotein (HDL-C) while contrasting results of serum total cholesterol
TC and low density lipoprotein (LDL-C) were found. The objectives of this study
were i) to determine lipid profiles in a group of patients with early inflammatory
polyarthritis (IP) not exposed to treatment and ii) to examine the association
between inflammation and clinical markers of disease activity with serum lipid
profiles.
Patients with early IP (2 swollen joints for 4 weeks) from The Norfolk Arthritis
Register (NOAR), were included in this study. 339 (37%) of them were not
on any treatment at time of recruitment. Baseline clinical assessments and
examinations were conducted and blood samples were collected to measure
autoimmune and lipid profile.
Baseline results are; age = 50(19.7), TC 4.79 [3.85, 5.58] mmol/l, HDL 0.98
[0.75, 1.3] mmol/l, LDL 2.96 [2.14, 3.75] mmol/l, TG 1.56 [1.15, 2.05] mmol/l,
apoA-1 1.82 [1.47, 2.18] U/l, and apoB 0.83 [0.62, 1.04] U/l. Regression analysis
revealed a negative association between CRP and TG (b coefficient [95% CI])
= (−0.07 [−0.01, −0.003]) which remained significant even after adjustment for
age and gender (−0.09 [−0.02, −0.002]). A similar association with DAS28 score
was observed (−0.1[−0.17, −0.03]) after adjustment (−0.1 [−0.2, −0.01]).
In patients with early arthritis, inflammation mainly affects TG; alterations in TG
may contribute to atherogenesis in the active phase of RA.
Table 1
Variable
Mean (SD)/Median (IQR)
Age
TC
TG
HDL
LDL
CRP
DAS-28
50.7 (19.7)
4.79 (3.85, 5.58)
1.56 (1.15, 2.05)
0.98 (0.75, 1.3)
2.9 (2.1, 3.7)
14.6 (20)
3.5 (1.2)
259 PROTEIN DISULFIDE ISOMERASE INHIBITION BY
4-HYDROXYNONENAL-ADDUCTS CONTRIBUTES TO OXIDIZED
LOW DENSITY LIPOPROTEIN-INDUCED APOPTOSIS
C. Muller, J. Bandemer, C. Vindis, R. Salvayre, A. Negre-Salvayre. INSERM
U1048, University of Toulouse III, Toulouse, France
The proteine disulfide isomerase (PDI), is an endoplasmic reticulum (ER)associated chaperon protein, that plays a role in neosynthesized protein
maturation, and protects against apoptosis induced by various stress-inducing
agents. Its role in atherosclerosis is not known so far. We aimed to investigate
the role of PDI in apoptosis elicited by oxidized LDLs (oxLDLs) in human
vascular cells (HMEC-1) and in macrophagic U937 cells.
Toxic pro-apoptotic oxLDL concentrations inhibit PDI activity, together with an
increased expression of proapoptotic ER stress markers (CHOP and spliced
XBP1mRNAs). PDI inhibition by bacitracine or by transfection of a vector coding
for a mutant enzymatically inactive form, increased the expression of CHOP and
promoted apoptosis at non-toxic oxLDL concentrations, in both HMEC-1 and
U937, suggesting a protective function for this chaperon against oxLDL-induced
cell death. The inhibitory effect on PDI oxLDLs may result from an accumulation
of 4-hydroxynonenal (4-HNE)-adducts, which alter its enzyme activity. The
antioxidant N-acetyl cysteine (NAC) neutralized 4-HNE adduct formation and
restored the enzyme activity of PDI. 4-HNE-modified PDI was detected in
macrophages in human endarterectomy lesions, suggesting a possible loss
of function for this chaperon, in instable atherosclerotic plaques.
79th EAS Congress
260 INDUCTION OF CYTOSKELETON REORGANISATION BY OX-LDL
MEDIATES ID1-DEPENDENT ANGIOGENESIS
J. Qiu1 , G. Wang1 , J. Hu1 , Y. Zheng1 , Q. Peng1 , X. Luo2 , C. Tang1 . 1 Key
Laboratory of Biorheological Science and Technology (Chongqing University),
Ministry of Education; Chongqing Engineering Laboratory in Vascular Implants;
Bioengineering College of Chongqing University, Chongqing, China, 2 Burn
Research Institute, Southwest Hospital, Third Military Medical University,
Chongqing, China
Objective: The inhibitor of differentiation protein 1 (Id1), a helix-loop-helix
transcription factor, stimulates endothelial cell (EC) migration, proliferation, and
angiogenesis, and inhibits EC senescence. The aim of this study was to
investigate the role of the Id1 protein in mediating angiogenesis of EC exposed
to oxidised low-density lipoprotein (ox-LDL) and the related mechanism during
this process.
Methods and Results: Western blot analysis indicated that Id1, p53,
hypoxia-inducible factor-1 (HIF-1), and beta1-integrin were upregulated by
ox-LDL in a dose-dependent manner in human umbilical vein endothelial
cells (HUVECs). Treatment with low concentration of ox-LDL increased
EC angiogenesis, vascular endothelia growth factor expression, and actin
stress fibre formation. High concentration of ox-LDL decreased angiogenesis
and cytoskeleton organisation in HUVECs. Furthermore, Id1-overexpression
increased angiogenesis and restored actin stress formation and the disrupted
angiogenesis induced by high ox-LDL concentration. Silencing of Id1 expression
abolished EC tube formation and cytoskeleton formation induced by low ox-LDL
concentrations.
Conclusions: These findings suggested that Id1, at least partially, mediates
EC angiogenesis induced by ox-LDL and that a critical correlation between Id1
protein and oxidative stress induced-angiogenesis exsits.
261 THE CORRELATION BETWEEN APOLIPOPROTEINS B/A1
RATIO AND ARTERIAL STIFFNESS IN THE PATIENTS WITH
HYPERTENSION
L. Agoston-Coldea, S. Lupu, T. Mocan. “Iuliu Hatieganu” University of Medicine
and Pharmacy, Cluj-Napoca, Romania
Objectives: The aim of this study was to determine whether apolipoproteins
(apo) B and A-1 predict atherosclerosis in patients with arterial hypertension
and preserved left ventricular ejection fraction.
Methods: The study group included 60 consecutive hypertensive patients
and a control group of 44 healthy volunteers with an average age of
48.2(1.3) vs. 46.4(1.5) years. The dosage of the lipids fractions was done
by enzymatic method and those of the apoA-I and apoB was measured
by immunoturbidimetric method from Roche. Subclinical atherosclerosis was
also determined by measuring the intima–media thickness and carotid arterial
stiffness from beta index, pulse wave velocity (PWV) using B-mode and M-mode
by ultrasonography.
Results: In hypertensive patients, the apoB value and apoB/apoA1 ratio was
significantly greater compared to the control group. PWV increased with age
and elevated arterial tension. The arterial stiffness was correlated with level
apoB (r = 0.62, p = 0.05) and with apoB/apoA1 ratio (r = 0.75, p = 0.01). These
relations remained significant (p = 0.05) in models adjusted for non-lipid risk
factors. The areas under the receiver-operating characteristic (ROC) curves
(AUC) were similar for apoB (AUC=0.71; 95% CI 0.62–0.80) and apoB/apoA1
ratio (AUC=0.77; 95% CI 0.69–0.75) in detecting patients with increased PWV.
No significant correlation was obtained between the intima–media thickness
and the value of apolipoproteins.
Conclusions: The elevation of apoB and apoB/apoA1 ratio is associated
with increased arterial stiffness and reflects a lipoprotein profile predisposing
to the development of subclinical atherosclerosis in patients with arterial
hypertension.
262 LDL-APHERESIS FOR PREVENTION OF RECURRENT
HYPERTRIGLYCERIDEMIA-INDUCED ACUTE PANCREATITIS, A
CASE REPORT
A. Saad, P. Moriarty. University of Kansas Medical Center, Kansas City, KS,
USA
Background: LDL-apheresis therapy has been used with standard treatment to
manage hypertriglyceridemia-induced acute pancreatitis (HTGP). The utility of
LDL-apheresis includes reduction of plasma lipids, inflammatory markers and
blood viscosity. LDL-apheresis therapy for the chronic management of (HTGP)
is not well studied.
Methods: A 52 year old female with a history of Familial Combined
Hyperlipidemia, premature coronary artery disease (CAD), diabetes, and
twenty-one episodes of (HTGP) between 1994 and 2003 was presented to the
atherosclerosis and LDL-apheresis center at Kansas University. After failure with
standard medical management, she was approved for bi-weekly LDL-apheresis
(liposorber MA 01, Kaneka Inc.) therapy.
Results: Over the next four years LDL-apheresis lowered lipid levels (table 1)
and prevented both recurrent attacks of CAD and acute pancreatitis. In addition,
57
blood viscosity analysis (Rheolog, Inc.), at low shear rate (1(1/s)), was reduced
by 20% (pre 28.92 cP/post 23.00 cP). Treatment was discontinued after 4 years
when sustained pre-treatment LDL-C levels reached <100 mg/dL. Following 6
weeks without LDL-apheresis an episode of acute pancreatitis reoccurred and
the patient was restarted on regular LDL-apheresis therapy.
Lipids panel
(mg/dl)
Pre-LDL apheresis Pre-LDL apheresis Before last acute
initially
after four years
pancreatitis
LDL-C
HDL
TG
Total cholesterol
454
ND
17,600
1,212
81
31
677
204
473
Conclusion: LDL-apheresis therapy may be considered for patients with
chronic episodes of HTGP. The lowering of blood viscosity in addition to
triglycerides may benefit patients with HTGP.
263 INCREASED LEVELS OF LOW-DENSITY LIPOPROTEIN
S-HOMOCYSTEINYLATION IN CHRONIC KIDNEY DISEASE (CKD)
A. Zinellu1 , G. Loriga2 , A.E. Satta2 , L. Deiana1 , C. Carru1 . 1 Dept. of
Biomedical Sciences, 2 Dept. of Internal Medicine, University of Sassari,
Sassari, Italy
It has been recently reported as plasma low-density lipoprotein (LDL) are
susceptible to S-homocysteinylation (through a disulfide bond) and that this
modification may enhance LDL atherogenicity. We have investigated the levels
of homocysteine (Hcy) linked to LDL in CKD patients in which lipid abnormalities
highly contribute to the excess of morbidity and mortality.
30 patients with proteinuric chronic nephropathy and 60 healthy volunteers
has been selected. Capillary electrophoresis has been employed to measure
LDL-bound thiol Hcy, cysteine (Cys), cysteinylglycine (Cys-Gly), glutathione
(GSH), and glutamylcysteine (Glu-Cys). Lipid profile and total plasma thiols
has been also measured.
CKD has significantly low levels of HDL and increased levels of plasma
triglycerides and LDL. Moreover elevated levels of total plasma Hcy, Cys, has
been found in patients than in controls. LDL-S-Hcy was significantly increased
in nephropathic subjects (median 23.9 vs 12.0 nmol/mmol apoB, p < 0.001) like
also LDL-S-Cys (median 386 vs 301 nmol/mmol apoB, p < 0.01). By multiple
linear regression, we found that in healthy people, total Hcy was the most
important determinant of LDL-bound Hcy (t = 4.78, p < 0.0001) whilst Cys-Gly
was negatively associated with apoB-Hcy concentrations (t = −2.28, p < 0.03).
In CKD the most important determinant of homocysteinylation was creatinine
(t = −2.25, p < 0.03) while total plasma Hcy is weakly associated with apoB-Hcy
(t = 3.69, p < 0.001).
Considering our previous results on the toxicity of this lipoprotein modification,
the increased levels in Hcy-LDL observed in CKD suggest that, among
the other risk factors for cardiovascular disease in these individuals, LDL
homocysteinylation should also be considered.
264 CHANGES OF PLASMA ADIPONECTIN LEVEL AND LIPID PROFILE
IN SEVERELY OBESE PATIENTS BEFORE AND AFTER BARIATRIC
SURGERY
M.J. Hosseinzadeh-Attar1 , A. Golpaie1 , M. Hosseini2 , H. Darakhshanian1 ,
M. Talebpour3 . 1 Nutrition and Biochemistry, 2 Biostatistics, 3 Surgery, Tehran
University of Medical Sciences, Tehran, Iran
Introduction: Obesity is associated with a high risk of CAD morbidity and
mortality. Evidence suggests that adiponectin is an important adipokine in
vascular homoeostasis and also an independent risk factor for endothelial
dysfunction, HTN, CAD and MI. On the other hand, elevation of circulating
adiponectin can alleviate various vascular dysfunctions. The aim of this study
was to investigate the changes of plasma adiponectin and lipid profile, BP and
anthropometric indices in morbidly obese subjects before and one month after
bariatric surgery.
Methods: 37 morbid obese patients were studied prior to and 1 month after
bariatric surgey aged 37±9.1 yr. BMI, waist circumference, adiponectin, HDL,
LDL, TG and total cholesterol were measured. Data were compared between
groups by using dependent t-test. P values <0.05 were considered significant.
Results: Patients lost 12.9±4.3% of the initial body weight, serum adiponectin
levels increased from 36±11.7 to 41.1±11 mg/ml (P < 0.008). BMI reduced from
44.8 to 39±4.7kg/m2 and waist circumference decreased from 123.1±14.2
to 111.3±12.7 cm (P < 0.0001). LDL and TG reduced significantly from
111.9±28.8 to 104.1±24.7 and 179±118.3 to 134.5±48 respectively. The
changes of HDL were not significant.
Conclusion: Weight reduction 1 month after bariatric surgery was associated
with a significant increase in the circulating adiponectin levels and reduction of
LDL and TG in morbidly obese subjects but HDL changes were not significant
58
probably due to short time period. These findings suggest that increased
adiponectin may have a protective effect on cardiovascular disease.
265 INVESTIGATION OF THE PATHOGENESIS OF ATHEROSCLEROSIS:
ASSOCIATION BETWEEN OXIDIZED LOW DENSITY LIPOPROTEINS
AND ATHEROSCLEROSIS RISK FACTORS
Z.A. Kučinskienė1,2 , L. Bagdonaite1,2 . 1 Faculty of Medicine, Vilnius University,
2
Center of Laboratory Diagnostics, Vilnius University Hospital Santariskiu
Clinics, Vilnius, Lithuania
Objective: Studies demonstrated that patients with coronary heart disease
(CHD) have elevated plasma levels of oxidized low density lipoproteins (oxLDL), but not so many have investigated clinically silent individuals with high
CHD risk influenced by genetics. The aim of study was to investigate oxLDL levels in plasma samples of individuals with CHD and their clinically
healthy siblings and to assess the relationship between ox-LDL and biochemical
markers − atherosclerosis risk factors of metabolic and inflammatory origin.
Methods: This study consisted of group of 56 patients with CHD and group of
56 their siblings with clinically silent ultrasound assessed atherosclerosis. Both
of groups included 45±7-year-old men. Ox-LDL were measured by competitive
ELISA (Mercodia, Sweden). Other biochemical markers were detected by
enzymatic methods or by nephelometric assay.
Results: Statistically significant (p < 0.05) correlation was estimated in both
investigated groups between circulating ox-LDL and serum total cholesterol
(r = 0.37), LDL cholesterol (r = 0.42), triglycerides (r = 0.3) and apoB (r = 0.4).
Ox-LDL levels had tendency (not statistically significant) to be higher
among healthy siblings, than among individuals with CHD (94.3±44.8
versus 80.4±47.5, P = 0.14). There was no correlation between ox-LDL and
inflammatory markers (CRP, IL-6 and fibrinogen).
Conclusions: Elevated ox-LDL levels among siblings certify that ox-LDL
are associated with subclinical atherosclerosis. Significant association was
estimated between circulating ox-LDL and atherosclerosis risk factors of
metabolic origin in both groups of CHD patients and their siblings. This relation
support the concept, that ox-LDL may play major role in atherosclerosis
development and may be assessed as the marker of atherosclerosis
progression.
266 IRREVERSIBLY GLYCATED LDL INCREASE INTRACELLULAR
FREE CALCIUM CONCENTRATION IN HUMAN VASCULAR SMOOTH
MUSCLE CELLS
G.M. Sanda, C.S. Stancu, A.V. Sima. Lipoproteins and Atherosclerosis,
Institute of Cellular Biology and Pathology ‘N. Simionescu’, Bucharest,
Romania
Aim: To assess the direct effect of irreversibly glycated LDL (AGE-LDL) on
intracellular free calcium concentration ([Ca2+ ]i ) of human vascular smooth
muscle cells (hSMC).
Methods: Native LDL (nLDL) isolated from human plasma was incubated with
0.2M glucose, under sterile conditions, with antioxidant protection (4 weeks,
37ºC) for AGE-LDL or with 10mM CuSO4 (24h, 37ºC) for oxidized LDL (oxLDL)
preparation. [Ca2+ ]i was determinated by incubating hSMC with the calcium
fluorescent indicator Fura 2-AM. To investigate the role of the receptors for
nLDL and modified LDL, specific antibodies against these receptors were used.
In addition, the effect of a voltage-gated calcium channels blocker, amlodipine,
was assessed.
Results: Incubation of AGE-LDL or oxLDL with hSMC (2min) induced a 2
and 1.5-fold respectively, increase in [Ca2+ ]i , as compared with nLDL. Using
antibodies against the LDL-R-related protein (LRP1), scavenger receptor CD36
and the receptor for advanced glycation endproducts (RAGE) significantly
reduced [Ca2+ ]i in the cell suspensions exposed to AGE-LDL (by 93%, 47% and
77%, respectively) or oxLDL (by 92%, 35% and 77%, respectively). Amlodipine
induced a significant decrease of [Ca2+ ]i in both hSMC exposed to AGE-LDL
(by 85% and 78%, respectively) or nLDL. In contrast, its effect in cells incubated
with oxLDL was smaller (only 43%).
Conclusions: AGE-LDL exposure of hSMC determines an increased
mobilization of calcium ions, a process that involves specific modified LDL
receptors, LRP1, RAGE and CD36. Amlodipine reduces [Ca2+ ]i in hSMC
exposed to AGE-LDL.
Funding: Supported by the Ministry for Education, Research, Youth and Sport,
Romania, grant 1227/2009−11.
267 CLONING AND EXPRESSION OF A FLUOBODY RECOGNIZING
MINIMALLY MODIFIED LOW-DENSITY LIPOPROTEIN
T.E.S. Faulin1 , V.R. Hering2 , M.J. Politi2 , D.S.P. Abdalla1 . 1 Dept. of Clinical and
Toxicological Analyses, 2 Dept. of Biochemistry, University of São Paulo, São
Paulo, Brazil
Introduction and Objective: There are several evidences that minimally
modified LDL (mmLDL) contributes to the pathogenesis of atherosclerosis. The
aim of this study was to obtain a fluobody consisting of green fluorescent protein
(GFP) and anti-mmLDL single chain variable fragment (scFv) that, with gold
nanoparticles, can be used to detect mmLDL.
Material and Methods: The DNA sequence that encodes anti-mmLDL scFv,
previously obtained from a hybridoma secreting anti-mmLDL monoclonal
antibody, was inserted into the bacterial vector pET-28a(+) with the DNA
sequence of green fluorescent protein (GFP). Escherichia coli BL21(DE3) strain
was transformed with the vector to express the fluobody which was purified by
affinity chromatography on nickel column. Gold nanoparticles were synthesized
and added in increased concentrations into a solution of anti-mmLDL fluobody.
This solution was excited at 400 nm and the emission spectrum of fluobody was
measured before and after the addition of nanoparticles. All tests of interaction
between nanoparticles and fluobody were performed with a fluorimeter using a
quartz cuvette.
Results and Conclusion: The target plasmid was constructed successfully;
scFv and GFP were within the correct reading frame. The expressed fluobody
showed fluorescence under ultraviolet light, affinity to mmLDL but not to
unmodified LDL. The synthesized gold nanoparticles quenched the fluobody
fluorescence showing an extensive spectral overlap between them. The antimmLDL fluobody and gold nanoparticles are promising tools to be used to
detect mmLDL in studies of atherosclerosis.
Financial support: FAPESP (grants to D.S.P.A. and M.J.P.; scholarships to
T.E.S.F. and V.R.H.)
268 THE EFFECT OF PITAVASTATIN AND PRAVASTATIN ON THE
SERUM PHOSPHATIDYLCHOLINE HYDROPEROXIDE (PCOOH)
CONCENTRATION
S. Oikawa1 , F. Okajima2 , A. Asai2 , Y. Ishigaki3 , Y. Oka3 , S. Kono4 , J. Sasaki5 ,
H. Sugihara2 , K. Nakagawa6 , T. Miyazawa6 . 1 Department of Medicine,
2
Nippon Medical School, Bunkyoku, 3 Tohoku University School of Medicine,
Sendai, 4 Kyusyu University, Faculty of Medical Sciences, 5 International
University of Health and Welfare, Fukuoka, 6 Tohoku University, Graduate
School of Agriculture, Sendai, Japan
Objective: We have previously reported that aging, hyperlipidemia, and
diabetes mellitus increased serum level of phosphatidylcholine hydroperoxide
(PCOOH) known as oxidative stress maker. We evaluated the effect of strong
and mild statin on serum PCOOH levels in hypercholesterolemia.
Method: Hypercholesterolemic patients with type 2 diabetes mellitus (n = 123)
were enrolled and assigned randomly to the pitavastatin (1−2 mg/day) or to the
pravastatin group (10 mg/day). Serum lipids and atherosclerosis related factors
(PCOOH, hsCRP, Urinary 8OHdG, Adiponectin, and LPL mass) were measured
after three-month treatment. Hemolytic serum samples (n = 24) were excluded,
and the total number of PCOOH data was 99.
Results: PCOOH significantly decreased by 22.2% (509±116nM to
386±77nM, p < 0.001) after treatment with statins for 3 month. Total
Cholesterol (TC, 245±31 mg/dl to 191±30 mg/dl, 21.5%, p < 0.001), lowdensity lipoprotein cholesterol (LDL-C, 161±26 mg/dl to 112±25 mg/dl, 29.6%,
p < 0.001), triglyceride (TG, 159±89 mg/dl to 133±105 mg/dl, 10.5%, p < 0.01),
and non-HDL-C (192±29 mg/dl to 138±30 mg/dl, 27.8%, p < 0.001) were
significantly reduced. There was no significant difference in the other laboratory
data.
Conclusion: This study makes clear that statins are effective in lowering serum
lipid hydroperoxide as well as serum lipids. It suggests such an effect of statins
will be one of the mechanisms preventing atherosclerotic diseases.
269 THE ROLE OF CARDORIUM PLUS ON SERUM LIPIDS,
INFLAMMATION AND OXIDATIVE STRESS IN HYPERLIPIDEMIC
BROILER CHICKS
M. Devarakonda, R. Enjamoori. Department of Pharmaceutical Chemistry,
Shri Vishnu College of Pharmacy, Bhimavaram, India
The present study was aimed at investigating the efficacy of Cardorium Plus
on oxidative stress, inflammation and hypperlipidemia in high fat diet induced
hyperlipidemic chick model. Cardorium plus is a poly herbal formulation, per oral
administration of 80 mg significantly (p < 0.01) reduced the elevated triglycerides
(TG), total cholesterol (TC), low-density lipoprotein (LDL), atherogenic index
(AI), c-reactive protein, showed significant (p < 0.01) increase in high-density
lipoprotein (HDL) and observed significant (p < 0.01) reduction in thiobarbituric
acid reacting substances (TBARS), concomitant rise in erythrocyte reduced
glutathione (eGSH), erythrocyte catalase (eCAT), and erythrocyte superoxide
dismutase (eSOD) were evident when compared to fat diet fed group. Results
were compared with Lovastatin 4.65 mg/kg/day, Ascorbic acid 20 mg/kg/day and
piroxicam 0.3 mg/kg/day treated groups.
The interesting part of the study revealed that the beneficial effects of Cardorium
Plus on reducing hyperlipidemia, inflammation and oxidative stress compared
to other drug treated groups where other drug treated groups shown some
lacuna in preventing either of the facts which contributing to cardiovascular
diseases. So, Cardorium Plus had shown superiority over the other three treated
groups. This study could be a best suggestion that in complex diseases like
cardiovascular diseases where multiple factors play role in disease progression
79th EAS Congress
then traditional way of treating could a better option. Traditional medicine would
place infront numerous advantages over synthetic drugs.
270 MULTIPLEX MASSARRAY SPECTROMETRY ASSAY FOR
DETECTING 36 FH MUTATIONS/POLYMORPHISMS IN GREEK AND
CYPRIOT PATIENTS
E. Bashiardes1 , S. Xenophontos1 , G. Miltiadous2 , M. Elisaf3 , M. Cariolou1 .
1
Cardiovascular Genetics and Laboratory of Forensic Genetics, The Cyprus
Institute of Neurology and Genetics, 2 Internal Medicine, Hippocrateon Private
Hospital, Nicosia, Cyprus, 3 Internal Medicine, University of Ioannina, Ioannina,
Greece
Autosomal dominant FH is caused by mutations in the LDLR, APOB and PCSK9
genes. So far, ten mutations have been reported for the LDLR gene, in Northwestern Greek patients and three for Cypriot patients. No mutations have as
yet been detected in the APOB and PCSK9 genes in Cypriot or Greek patients.
Statins are widely prescribed in FH patients. A common variant, rs4149056,
has been reported to be strongly associated with an increased risk of statininduced myopathy. In this study, we developed a MassARRAY spectrometry
assay in order to identify 36 mutations/polymorphisms (29 in the LDLR, 5 in
the PCSK9, 1 in APOB and 1 in the SLCOIB1 genes) in the Greek and Cypriot
population.
Material and Methods: 90 DNA samples from FH patients with a previously
characterized mutation were selected and a further 50 new samples which had
not been screened for any mutations.
Results: Mutations were identified in all 90 FH samples with known mutations
using the iPLEX assay. For the remaining 50 samples a mutation was detected
in four samples and verified by sequencing. No false-positive results were
obtained using this assay. Overall, genotyping call rate was 93% for all 36
assays for the 140 samples.
Conclusions: The newly designed multiplex FH assay designed, covers 52%
and 94% of the known Cypriot and North-western Greek point mutations,
respectively. This iPLEX assay in its present form is suitable for large-scale
screening for FH mutations in patients and for population screening in Greeks.
271 OXIDIZED PHOSPHOLIPIDS (OXPLS) INDUCE PRODUCTION OF
STEM CELL FACTOR (SCF) AND ACTIVATION OF ITS RECEPTOR
C-KIT IN ENDOTHELIAL CELLS
T. Afonyushkin, O.V. Oskolkova, V.N. Bochkov. Vascular Biology, Medical
University Vienna, Vienna, Austria
OxPLs that accumulate in atherosclerotic vessels are increasingly recognized
as pathogenic factors in cardiovascular disease. We found that OxPLs
stimulated in cultured venous and arterial endothelial cells (ECs) expression
of mRNA encoding for SCF. SCF is a pleiotropic cytokine inducing a variety of
biological effects, and in particular those related to endothelial homeostasis, e.g.
stimulation of ECs migration and proliferation, protection of ECs from apoptosis,
and recruitment of circulating progenitor cells. OxPLs upregulated two mRNA
isoforms encoding for soluble and membrane-bound SCF. Treatment of ECs with
OxPLs upregulated levels of soluble SCF in cell culture medium. Furthermore,
conditioned medium from OxPLs-treated ECs stimulated phosphorylation of
SCF receptor, c-KIT. In addition to OxPLs, SCF was induced by electrophilic
agents such as OxLDL and iso-prostaglandin A2. Electrophilic stress response
played mechanistic role in SCF upregulation since siRNA against NRF2
suppressed induction of SCF by OxPLs. We found that OxPLs treatment
promoted survival of ECs under conditions of serum starvation; this protective
effect was blocked by c-KIT inhibitor imatinib. Our data allow hypothesizing that
OxPL-induced SCF may promote ECs survival in the lesion under conditions
of atherogenic cellular stress and induce endothelial repair via mobilization of
circulating progenitor cells. These responses may compensate for toxic action
of OxPLs and other deleterious agents present in the plaque.
272 METABOLIC SYNDROME IN SOUTH ASIAN IMMIGRANTS: MORE
THAN LOW HDL REQUIRING AGGRESSIVE MANAGEMENT
S. Dodani1 , M. Butler2 , J. Vacek2 , K. Gupta3 . 1 University of Kansas, Leawood,
2
University of Kansas, Kansas City, 3 univerity of Kansas, Kansas city, KS,
USA
Background: Aggressive clinical and public health interventions have resulted
in significant reduction in coronary artery disease (CAD) worldwide. However,
South Asian Immigrants (SAIs) exhibit the higher prevalence of CAD and its
risk factors as compared with other ethnic populations. The main objective of
the current study is to assess the prevalence of metabolic syndrome (MS), its
association with high density Lipoprotein (HDL) function, Apo lipoprotein A-I
(Apo A-I) polymorphisms, and sub-clinical CAD using common carotid intimamedia thickness (CCA-IMT) as a surrogate marker.
Methods: Community-based cross-sectional study on SAIs aged 35−65
years was conducted. Sub-clinical CAD was measured using CCA-IMT.
Dysfunctional/pro-inflammatory (Dys-HDL) was determined using novel cell free
assay and HDL inflammatory index.
59
Results: According to the International Diabetes Federation definition, MS
prevalence was 29.7% in SAIs without CAD. 26% had HDL inflammatory
index 1 suggesting Dys-HDL. Six novel APOA-1 gene polymorphisms were
discovered and on logistic regression, three single nucleotide peptides-SNPs
(G2, G3, and G5) were found to be significantly associated with MS (p = 0.397,
p = 0.386, p = 0.054). On multi-variate analysis, MS was significantly associated
with BMI > 23 (P = 0.005), Apo-A-I levels (pp = 0.01), and Lp [a] (p < 0.0001).
Conclusion: SAIs are known to be at a disproportionately high risk for CAD
that may be attributed to a high burden for MS. There is need to explore and
understand non-traditional risk factors with special focus to Dys-HDL, knowing
that SAIs have low HDL levels. Large prospective studies are needed to further
strengthen current study results.
273 LONG CHAIN N-3 POLYUNSATURATED FATTY ACIDS (LCN3PUFA),
EICOSAPENTAENOIC ACID (EPA) AND DOCOSAHEXAENOIC ACID
(DHA), MODULATE HUMAN THP-1 MACROPHAGE CHOLESTEROL
LEVELS
S. McCartan1,2 , A. McGinty1 , B. Green2 , C. Mercer1 , J. McEneny1 ,
L.A. Powell1 . 1 Nutrition and Metabolism, Centre for Public Health, 2 IAFLU,
School of Biological Sciences, Queen’s University Belfast, Belfast, UK
The effects of EPA and DHA on cholesterol levels in THP-1 monocyte-derived
macrophages were investigated. THP-1 monocytes were minimally (MD; 30
nM phorbol myristate acetate [PMA]), 24 hrs), or fully differentiated (FD; 100
nM; 48 hrs with 24 hrs recovery) and pretreated with EPA or DHA (50 mM; 24
hrs) prior to incubation with native or oxidised low density lipoprotein (n/oxLDL:
50 mg/mL; 24 hrs). Total (TC) and free cholesterol (FC) levels were analysed
using an Amplex red assay.
Table 1
Cholesterol (ng/mg protein)
Basal
Total
MD
FD
Free
MD
FD
nLDL
+EPA
+DHA
14.61
35.98
6.27
37.6
7.5
33.67
14.79
36.13
5.6
36.1
6.62
31.88
OxLDL
+EPA
+DHA
16.8
44.14
15.5
34.81
16.5
42.12
15.9
38.84
9.1
33.15
9.5
34.74
+EPA
+DHA
29.5
66.05
28.0
71.8
25.1
76.1
24.3
56.39
21.8
61.07
20.38
64.15
MD macrophages had significantly lower basal levels of FC and TC than FD
macrophages (p < 0.05). This significant difference between MD and FD was
maintained across all treatments with LC n-3 PUFA ± LDL.
NLDL treatment had no significant effects. OxLDL treatment increased
cholesterol levels in both MD (TC: p = 0.08, FC: p = 0.07) and FD macrophages
(TC: p < 0.01, FC: p < 0.05).
In MD macrophages EPA and DHA significantly reduced basal TC (p < 0.05
for both), and EPA reduced basal FC levels (EPA: p = 0.07). FC levels were
decreased in MD nLDL-treated macrophages, (EPA: p < 0.05, DHA: p = 0.05)
but LC n-3 PUFA had no effect on oxLDL treated macrophages.
In conclusion, absolute cholesterol levels are lower in MD macrophages.
Moreover, LC n-3 PUFA reduce basal and nLDL-dependant cholesterol in MD
but not FD macrophages.
274 APOLIPOPROTEIN B OF LOW-DENSITY LIPOPROTEIN
REDUCES NITRIC OXIDE-MEDIATED ENDOTHELIUM-DEPENDENT
RELAXATION IN RAT MESENTERIC ARTERIES
C.-B. Xu1 , W. Zhang2 , Y. Zhang1 , L. Edvinsson1 . 1 Lund University, Lund,
Sweden, 2 Xiamen University, Xiamen, China
Objective: To examine if low-density lipoprotein (LDL) and apolipoprotein B
(ApoB) alter endothelium-dependent vasodilatation of rat mesenteric arteries.
Methods and Results: Mesenteric arterial segments from male Sprague
Dawley rats were cultured in serum-free Dulbecco’s modified Eagle’s
medium in the presence of different concentrations of LDL or ApoB.
Endothelium-dependent relaxation induced by acetylcholine was studied in
a sensitive myograph. The vasodilatation derived from nitric oxide (NO),
endothelium-dependent hyperpolarizing factor (EDHF) and prostaglandin I2
(PGI2 ) pathways was characterized by using specific inhibitors. Real-time
PCR and immunohistochemistry were employed to assess mRNA and protein
expression for NO synthase (eNOS and iNOS) and cycloxygenase (COX),
respectively. The results showed that, co-incubation with either LDL or ApoB
for 24 hrs concentration-dependently attenuated the endothelium-dependent
vasodilatation. However, shorter incubations for 6 hrs with LDL had no effect.
LDL-induced reduction of vasodilatation was mainly due to impairment of NO
pathway, in parallel with suppressed mRNA and protein expression of eNOS and
60
iNOS, but not that of COX. Lipid peroxidation remarkably increased at 24 hrs
of organ culture, demonstrated by using thiobarbituric acid reactive substances
assay.
Conclusion: LDL reduces NO-mediated endothelium-dependent vasodilatation
in rat mesenteric arteries, which associates with LDL peroxidation and the
development of cardiovascular disease.
275 HEALTH EFFECTS OF OXIDIZED COD LIVER OIL IN HEALTHY
SUBJECTS: A RANDOMIZED CONTROLLED TRIAL
I. Ottestad1,2 , G. Vogt3 , M. Myhrstad1 , K. Retterstøl4 , A. Nilsson3 ,
J.-E. Haugen3 , G. Ravn-Haren5 , K.W. Brønner6 , L.F. Andersen2 , K. Holven2 ,
S. Ulven1 . 1 Akershus University College, Lillestrøm, 2 Department of Nutrition,
Institute for Basic Medical Sciences, University of Oslo, Oslo, 3 Nofima Mat AS,
Ås, 4 Lipid Clinic, Medical Department, Rikshospitalet-Oslo University Hospital,
Oslo, Norway, 5 Division of Toxicology and Risk Assessment, DTU Food,
Denmark, 6 TINE SA R&D Center, Oslo, Norway
Background: Oxidation of long chain fatty acids leads to formation of
secondary oxidation products such as 4-hydroxy-2-nonenal (4-HNE) and
4-hydroxy-2-hexenal (4-HHE) which may have negative health effects by
influencing processes involved in atherosclerosis such as oxidative stress and
inflammation.
Objective: The aim of the present study was to investigate health effects of
cod liver oil with different quality in a randomized controlled study.
Design: A seven weeks double-blinded randomized controlled study with
healthy subjects (n = 56) was conducted. After a four weeks wash-out period
the subjects were assigned into one of three groups receiving identical cod
liver oil capsules containing either 9 g/d (1.5 g/d EPA/DHA) with high or low
quality (measured by peroxide value [PV] and anisidine value [AV]) or 9 g/d of
sunflower oil.
Fasting blood samples were collected at week 0 and 7, and the level of
secondary oxidation products (plasma 4-HNE and 4-HHE) and oxidative stress
markers (plasma a-tocopherol and urine 8-iso-PGF2a ) as well as hsCRP were
analyzed.
Results: Preliminary results show no significantly differences in the circulating
levels of 4-HNE, 4-HHE, plasma a-tocopherol/plasma total lipids ratio, urine
8-iso-PGF2a pg/mg creatinine or hsCRP after 7 weeks of intervention with cod
liver oil of different quality.
Conclusion: This study shows that a daily intake of highly oxidized cod liver
oil for 7 weeks does not significantly change the level of circulating oxidation
products or affect oxidative stress markers.
276 PLASMA MALONDIALDEHYDE MEASUREMENT BY
FIELD-AMPLIFIED SAMPLE STACKING (FASI) CAPILLARY
ELECTROPHORESIS UV DETECTION
A. Zinellu, B. Scanu, E. Pisanu, M. Sanna, V. De Murtas, M.A. Pinna,
D. Cambedda, L. Deiana, C. Carru. Biomedical Sciences, University of
Sassari, Sassari, Italy
Oxygen-derived free radical damage resulting in lipid peroxidation is known
to play a key role in the aetiology of many diseases, including diabetes and
atherosclerosis. Due to the difficulty of measuring directly the free radicals
production, the lipid peroxidation measurement has become a commonly used
technique to evaluate oxidative stress. The determination of malondialdehyde
(MDA) is the most widely used method for the lipid peroxidation monitoring.
Analysis was performed in a MDQ capillary electrophoresis system using an
uncoated fused-silica capillary, 75 mm I.D. and 40 cm length injecting 2 mm
of water plug followed by electrokinetic sample injection of 10kV for 100s.
Electrophoresis was carried out in a 200 mmol/L Tris buffer at pH 5.00, 15ºC
and 12 kV at reverse polarity. Plasma samples were treated with one volume
of acetonitrile for protein elimination and supernatant were directly injected
on capillary without complex cleanup and/or sample derivatization procedures.
Using electrokinetic injection the detection limit in real sample was 3 nmol/L,
thus improving of about 100-fold the LOD of previous described methods
based on capillary electrophoresis. Precision tests indicate a good repeatability
of our method both for migration times (CV=1.11%) and areas (CV=2.05%).
Moreover, a good reproducibility of intra- and inter-assay tests was obtained
(CV=2.55% and CV= 5.14% respectively). The elevate sensitivity, the short time
needed for the sample pre-treatment, the relatively short analysis time (single
electrophoretic run time less than 10 min) and the simplicity make this method
suitable for large-scale applications in the study of lipid peroxidation.
277 OX-LDL INHIBITS THE MIGRATION OF VASCULAR SMOOTH
MUSCLE CELLS VIA UP-REGULATION OF P53 EXPRESSION
J. Hu, G. Wang, J. Qiu, Y. Zheng, Y. Teng, H. Chen, C. Tang, T. Yin. Key
Laboratory of Biorheological Science and Technology (Chongqing University),
Ministry of Education; Chongqing Engineering Laboratory in Vascular Implants,
Bioengineering College of Chongqing University, Chongqing, China
Introduction: The previous studies showed that there are less cell contents
in atherosclerotic plaque. This feature of vascular remodeling may have
relationship with the deposition of oxidized low density lipoprotein (ox-LDL)
in the vessel wall. A large number of recent studies showed that p53 is an
important factor involved in regulating cell migration. However, whether ox-LDL
affects the migration of vascular smooth muscle cells (VSMCs) by regulating
the expression and function of p53 is unclear.
Methods: In this study, we studied the effects of ox-LDL on VSMCs migration
through wound repair assay. And then, cell apoptosis and p53 expression
were analyzed by TdT-mediated dUTP-Xnick end labeling (TUNEL) assay and
Western blot respectively.
Results: We have shown that ox-LDL significantly impair cell migration. In
detail, cell migration was markedly retarded (0.56±0.06, healing area/wound
area; p < 0.05; n = 3) under high concentration (100 mg/ml) of ox-LDL compared
to control (PBS) (0.93±0.05, healing area/wound area; n = 3). Western blot
analysis showed that p53 expression of VSMCs increased with the increase
of the concentration of ox-LDL, respectively 0, 25, 50, 100 mg/ml (n = 3). But
TUNEL assay revealed that there was no cell apoptosis under the stimulation
of experimental concentration of ox-LDL (n = 3).
Conclusions: Our results indicated that high concentration of ox-LDL inhibits
the migration of VSMCs. This inhibitory process may not achieve by ox-LDL
induced VSMCs apoptosis, but via ox-LDL mediated up-regulation of p53
expression.
278 THE EFFECT OF OXIDATIVE STRESS UPON MEMBRANE AND
CYTOPLASMIC LIPIDS PEROXIDATION IN ERYTHROCYTES FROM
PATIENTS WITH CARDIOVASCULAR AND CEREBROVASCULAR
PATHOLOGIES
C.R.S. Revnic1 , C. Ginghina2 , F.G. Revnic3 , G. Prada4 , S. Prada5 , C. Pena6 ,
S. Botea7 . 1 Cardiology, UMF ‘Carol Davila’, 2 Cardiology, UMF Carol Davila,
Bucharest, 3 Biology of Aging, NIGG’ANA ASLAN’, 4 Gerontology and
Geriatrics, UMF Carol Davila, 5 Pharmacy, 6 Imunology, NIGG’ANA ASLAN’,
7
Molecular Pathology, Victor Babes Institute, Bucharest, Romania
Introduction: Under oxidative stress conditions, red blood cells can accumulate
membrane lipid peroxides and species derived from phospholipids during
pathological process such as stroke.
Objective: The aim of our study was to evaluate the effects of oxidative stress
generated by stroke upon red blood cells‘ membrane and plasma and upon
antioxidant enzymes in hyperthensive patinets associated with stroke.
Material and Method: This study has been done on 60 patients:30 male and
30 female aged between 30−70 years old with AHT associated with stroke
from C.C.Iliescu Cardiovascular InstituteRed blood cells of patients have been
processed by centrifugation at 3000 g and 4C. Lipid peroxidation in plasma
and red blood cell membrane as well as thiol groups and GGT, GST and
catalase antioxidant enzymes have been evaluated using stanard biochemical
techniques.
Results and Discussion: Our data have pointed out that there is less lipid
peroxidation in female patients with stroke (p < 0.05) where membrane fluidity is
decreased due to activation of plasma and erytrocyte superoxid dismutase and
catalase. The diminished catalase activity can be accounted for as a mechanism
of glutathion peroxidase regulation. In male with stroke the low Catalase
activity has been associated with an increased level of lipid peroxidation
(p < 0.05) both in membrane and plasma, while concentration of cytoplasm thiols
was low (p < 0.01). Concentration of free thiols in membrane is significantly
increased (p < 0.01) in stroke patients despite the age or sex.
Conclusion: Following stroke, there is a modification of antioxidant and
detoxifiant potential of erythrocyte by deacrising its capacity to uptake GSH
from plasma but also elimination of GSH conjugates.
279 YOUNGER PATIENTS WHO UNDERWENT MYOCARDIAL
INFARCTION, HAD INCREASED LEVELS OF
OXIDATION-SUSCEPTIBLE LIPIDS
P. Burchardt, B. Zuchowski, H. Wysocki. Division of Cardiology-Intensive
Therapy, Poznan University of Medical Sciences, Poznan, Poland
Dyslipidemia is a known risk factor for coronary heart disease (CHD). Onset of
this disease before the age of 45 suggests familial occurrence of CHD. Thus
the project assumed comparing patients who were undergoing hypolipidemic
treatment in two age groups (above and under 45 years old) in terms of routinely
used in clinical practice lipid parameters. Levels of apoB100, apoA1, LDL, TG,
HDL and TC along with indicators based on these parameters were assessed
in 140 consecutive patients.
Among patients who underwent myocardial infarction, in patients under
45 years old (N = 9) compared to the ones over 45 (N = 45) statistically significant higher levels of TC/apoA1 (1.64±39.5vs1.28±32.0,
p = 0.015), Lp(a) [g/l] (0.511±0.62vs. 0.17±0.195, p = 0.03), Lp(a)/apoA1
(0.34±0.42vs0.12±0.13 p = 0.03), apoB100[g/l] (1.227±0.43vs0.95±0.29,
p = 0.03) and apoB100/apoA1 (0.846±0.27vs0.65±0.2, p = 0.009) as well as
lower ratio of HDL/LDL (0.339±0.192vs. 0.486±0.22, p = 0.04) were observed.
The groups did not differ statistically in terms of use or dosage of hypolipidemic
drugs. In younger group, a relative increase in particularly susceptible to
79th EAS Congress
oxidation apoB100, Lp(a) levels was observed along with relative decrease
in antioxidative HDL and apoA1. These properties may be considered as the
explanation for the early onset of the coronary heart disease in people under
45 years old. A large study group is required in order to confirm the results of
the analysis.
280 EFFECT OF N-3 FATTY ACIDS AND STATINS ON OXIDATIVE
STRESS IN STATIN-TREATED HYPERCHOLESTORELEMIC AND
NORMOCHOLESTEROLEMIC WOMEN
M. Carrapeiro1 , M. Rogero2 , M. Bertolami3 , P. Botelho1 , N. Castro1 , I. Castro1 .
1
Department of Food and Experimental Nutrition, Faculty of Pharmaceutical
Sciences, University of São Paulo, 2 Department of Nutrition, Faculty of Public
Health, University of São Paulo, São Paulo, 3 Dante Pazzanese Institute of
Cardiology, São Paulo, Brazil
Introduction: Although the combination of statins with n-3 fatty acids seems to
be beneficial under the lipid profile aspect, there is little information about the
interaction of these two compounds on oxidative stress.
Objective: Evaluate the interaction between statins and n-3 fatty acids on
oxidative stress in women, using a 22 factorial design.
Methods: Forty-three women participated in this crossover design. They were
separated into two groups in which 20 were under statin treatment for more
than 6 months, and 23 were normolipidemic. Within each group, half of the
patients received capsules containing 2.4 g/day of a mixture of EPA and DHA
for 6 weeks, while the other half received a mixture of soya and corn oil. After
a period of 90 days of washout, the groups were switched, and received the
supplementation for 6 weeks more.
Results: Statins reduced serum LDL and increased SOD expression. N-3 fatty
acids increased the plasma malondialdehyde and SOD activity but reduced
catalase expression (p < 0.05). The interaction involving statins and n-3 fatty
acids was nearly significant to the serum triacylglycerol reduction (p = 0.054).
Conclusion: Combining statins and n-3 fatty acids is an excellent strategy to
improve the lipid profiles of women. However, n-3 fatty acids increased the
oxidative stress and the pleiotropic effect of statins seemed to not be enough
to counterbalance this result. Our data also suggested that the mechanism
by which n-3 fatty acids interfere in oxidative stress can be associated with
antioxidant enzymes expression and activity.
281 THE EFFECT (OF IDENTICAL TO THE TYPE AND DOSE) OF STATINS
TREATMENT IN PATIENTS WITH CAD DEPENDS ON SMOKING
P. Burchardt, B. Zuchowski, A. Palasz, H. Wysocki. Division of CardiologyIntensive Therapy, Poznan University of Medical Sciences, Poznan,
Poland
Objectives: There is few data defining the impact of smoking on lipid
metabolism disturbances among patients treated with statins.
The purpose was to compare lipid parameters (apoA1, apoB100, Lp(a), TG,
HDL, TC) among cigarette smokers (N = 30) and non-addicted patients (N = 91)
who (a) have used lipid-lowering therapy
Methods: The study was conducted among 140 consecutive patients
undergoing routine coronary angiography. ApoB100, apoA1, LDL, triglicerydes
(TG), HDL and total cholesterol (TC) levels were assessed with the use of
typical laboratory techniques. Coronary atherosclerosis was evaluated on the
basis of single stenosis significance as well as quantitatively by applying the
Gensini score.
Results: Cigarette smokers were characterized by significantly lower levels
of HDL (45.1±14.6±17.07 vs 52.15, p = 0.02), HDL/LDL (0.52±0.19±0.24
vs 0.35, p = 0.0002) and higher LDL levels (142.8±43.8 vs 117.3±50.04,
p = 0.002), ratio of LDL/apoA1 (97.16±29.43 vs 74.6±29.73, p = 0.0001),
triglycerides (196.4±155.1 vs 146.15±104.1, p = 0.00006), apoB100 (1.16
±0.29 vs 0.96±0.33, p = 0.0002), apoB/apoA ratio (0.81±0.24 vs 0.63±0.24,
p = 0.00004). Groups did not differ with respect to the application of the
dose or frequency of statins use. Among those with LDL levels below
the targeted 100 mg/dl, significantly higher proportion of total cholesterol to
apoA1 (1.32±0.25 vs 0.97±0.16, p = 0.001and higher levels of TG (204±115
vs 118±49, p = 0.0090) were observed in cigarette smokers. Smokers had
significantly more frequent history of myocardial infarction. Besides, the quantity
and quality of atherosclerotic lesions were similar in both studied groups.
Conclusions: The results indicate that lipid-lowering therapy in a group of
people smoking cigarettes gives worse results than the same dose therapy
in a group of nonsmokers. The study requires confirmation in a larger group of
subjects.
61
282 EZETIMIBE IMPROVES NAFLD AGGRAVATION INDUCED BY
DIETARY CHOLESTEROL OXIDATION PRODUCTS IN NON-HUMAN
PRIMATES
M. Deushi1 , K. Nakano2 , K. Osada3 , T. Kaizuka1 , K. Egashira2 , M. Yoshida1 .
1
Department of Life Science and Bioethics, Tokyo Medical and Dental
University, Tokyo, 2 Department of Cardiovascular Medicine, Kyusyu University,
Faculty of Medical Sciences, Fukuoka, 3 Department of Agricultural Chemistry,
School of Agriculture, Meiji University, Kanagawa, Japan
Background: Oxidative stress plays a dominant role in atherosclerosis and
nonalcoholic fatty liver disease (NAFLD). However, no prior studies examined
the effects of oxidized cholesterol products on NAFLD. The aims of this study
are to examine whether oxidized cholesterol products accelerate hepatic lipid
accumulation and inflammation in non-human primates. We also examined a
potential effect of NPC1L1 inhibitor, ezetimibe.
Methods and Results: Macaca fascicularis (male, 5 years-old) were fed
either a regular high-fat diet and cholesterol (control-HFD)/oxysterols (oxHFD; 0.015% of cholesterol was oxidized) were orally administrated for 24
weeks. Compared with control-HFD, ox-HFD did not affect plasma lipid levels,
but did affect hepatic lipid content [TC, 40.9 mg/g (ox-HFD) vs 3.2 (HFD);
TG, 28.0 (ox-HFD) vs 5.7 (HFD)]. Ox-HFD aggravated lipid accumulation as
well as recruitment of inflammatory cells when compared to control-HFD. We
then examined the effects of ezetimibe (Ez, 0.2 mg/kg/day for 12 weeks). In
addition to significant improvement of dyslipidemia, Ez improved biochemical
and pathological aspects of NAFLD.
Conclusion: Oxidized cholesterol products aggravated NAFLD in non-human
primates. Treatment of ezetimibe may be a novel therapeutic approach to
NAFLD via improving dyslipidemia and inflammation.
283 OXYSTEROL-BINDING PROTEIN-RELATED PROTEIN (ORP8) −
A NEW PRO-ATHEROGENIC FACTOR?
O. Beaslas1 , E. van Kampen2 , T. Vihervaara1 , G. Liebisch3 , R.B. Hildebrand2 ,
G. Schmitz3 , M. Van Eck2 , V.M. Olkkonen1 . 1 Minerva Foundation Institute
for Medical Research, Helsinki, Finland, 2 Leiden/Amsterdam Center for
Drug Research, Leiden, The Netherlands, 3 University Hospital Regensburg,
Regensburg, Germany
Oxysterol binding protein related protein 8 (ORP8) is expressed at highest levels
in macrophages. In macrophages of human coronary atherosclerotic advanced
lesions, the ORP8 mRNA is up-regulated as compared with healthy coronary
artery wall. However, the role of macrophage ORP8 in atherosclerotic lesion
development is unknown.
We created ORP8 knock-out (KO) mice. LDL receptor KO mice were
transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type
(WT) mice. Subsequently, they were challenged with a high cholesterol and high
fat Western-type diet (WTD) to induce atherosclerosis. Peritoneal macrophages
of the KO animals and RAW 264.7 cells silenced for ORP8 were used for gene
expression and lipidomic analyses.
After both 6 and 9 weeks WTD challenge, recipients of ORP8 KO BM displayed
a 20% reduction of lesion size as compared to WT animals. The composition
of the lesions was, however, similar. The KO BM recipient animals displayed
a significant increase of plasma triglycerides. Lipidome analyses of the KO
macrophages revealed gender-specific alterations in the abundancies of a
number of sphingolipid species. Similar changes were observed in Raw264.7
cells with ORP8 silenced. Gene expression analyses suggested reduced
expression of TNF-amRNA in the cells lacking ORP8.
The findings corroborate our hypothesis that macrophage ORP8 has the
capacity to act as a proatherogenic factor. Its mechanism of action may
involve modification of macrophage sphingolipid metabolism and inflammatory
signaling. ORP8 might thus be a potential target for development of new
preventive or therapeutic approaches for cardiovascular diseases.
284 TARGETED GENE DELETION IN THE TGF-BETA SIGNALING
PATHWAY AFFECTS ATHEROSCLEROSIS AND TH17 CELL
DIFFERENTIATION IN HYPERCHOLESTEROLEMIC MICE
A. Gisterå1 , A.K. Robertson1 , J. Andersson2 , D.F.J. Ketelhuth1 ,
A.M. Lundberg1 , M.O. Li3 , R.A. Flavell4,5 , G.K. Hansson1 . 1 Center for
Molecular Medicine and Dept of Medicine, 2 Karolinska Institutet at Karolinska
University Hospital − Solna, Stockholm, Sweden, 3 Immunology Program,
Memorial Sloan-Kettering Cancer Center, New York, NY, 4 Department of
Immunobiology, 5 Howard Hughes Medical Institute, Yale University School of
Medicine, New Haven, CT, USA
Atherosclerosis is an inflammatory process promoted by hypercholesterolemia.
Cellular immunity is highly involved in the disease development. TGF-beta is a
critical regulatory cytokine that has dual actions on T-cells; it can both inhibit
and promote inflammation in a context dependent manner. To study regulation
of TGF-beta, we developed mice with a T-cell specific deletion of Smad7, a
potent inhibitor of TGF-beta signaling that acts downstream of the TGF-beta
receptor. The mouse was generated using a cre-lox system, with the promoter
region of the Smad7 gene being floxed and the cre-enzyme expressed under the
62
CD4 promoter. Bone marrow from these mice was transplanted into low density
lipoprotein receptor knockout mice, which were fed a high fat diet for 8 weeks.
Surprisingly, smad7-deficient mice had significantly larger lesions in the aortic
root compared with matched controls, in spite of enhanced TGF-beta signaling.
Lesions from smad7-deficient mice contained a significant smooth muscle cap,
suggesting that they may be less vulnerable. In the para-aortic lymph nodes,
increased mRNA for the Th17 specific transcription factor ROR-gamma-T was
detected, arguing for a shift in the T-cell population towards the Th17-subset.
This is likely to be driven by unimpeded TGF-beta signaling in T-cells and
systemic inflammation induced by the high fat diet. Taken together, these
data show that deletion of smad7 in T-cells results in enhanced development
of atherosclerotic lesions and support the notion that modulation of cellular
immunity may be a useful disease-modifying target.
285 NANO- AND MICRO-STRUCTURE OF CALCIFICATION IN HUMAN
AORTIC VALVE, AORTA AND CORONARY WALL
S. Bertazzo1 , K.L. Cloyd1 , E. Gentleman1 , I. El-Hamamsy2 , A.H. Chester2 ,
M.H. Yacoub2 , M.M. Stevens1 . 1 Department of Materials, Department of
Bioengineering and Institute of Biomedical Engineering, Imperial College
London, 2 Harefield Heart Science Centre, Imperial College London, London,
UK
Calcification in the cardiovascular system has a tremendous impact upon
public health. For instance, calcification of the aortic valve affects around 29%
of the population over the age of 65. Here we investigated the nano- and
micro-structure and molecular composition of the calcified material found in
homographic and native aortic valves, aorta and coronary walls of 24 patients.
This investigation combines cutting-edge materials characterization techniques,
such as scanning electron microscopy, focused ion dual-beam, transmission
electron microscopy and bio-Raman micro-spectroscopy (Gentleman et al.
2009).
Our results suggest that cardiovascular calcification, independent of type
(homographic or native) or location (cardiac valves, aorta or coronary wall)
starts with the formation of calcified spherical structures from 100nm to 2 mm
in diameter, as presented in Fig. 1.
Fig. 1. Chromatic electron microscopy of calcified sphere present in diseased
cardiac valve samples.
In patients at a more advanced stage of the disease, the number of spheres
increases and the calcification process starts to affect the fibers that form valves
and the artery tissue. At the most advanced stage of calcification, the material
has some similarities in structure and chemical composition to bone. Bio-Raman
spectroscopy mapping of diseased tissues also showed that calcium phosphate
is confined to specific regions, confirming the micrographic studies.
Taken together, these results shed some light on the possible mechanism that
may control pathological calcification in the different regions of the circulatory
system and also the possibility to identify the cells that are responsible for the
mineralization.
Reference(s)
Gentleman, E. et al. Nat Mater 8, 763–770 (2009).
286 OSTEOPONTIN IS IMPORTANT FOR DEVELOPMENT OF UREMIC
ATHEROSCLEROSIS
T.X. Pedersen1,2 , N. Junker1 , M. Madsen1 , S. Bro3 , A. Hultgaardh-Nilsson4 ,
L.B. Nielsen1,2 . 1 Dept. of Biomedical Sciences, University of Copenhagen,
Copenhagen N, 2 Dept. of Clinical Biochemistry, 3 Dept. of Nephrology,
Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 4 Dept. of
Experimental Medical Science, University of Lund, Lund, Sweden
Objectives: Uremia is a strong risk factor for cardiovascular disease. The
multifunctional cytokine-like protein osteopontin (OPN) is highly expressed in
aortas of uremic apoE (E) KO mice. OPN may, at the same time, enhance
atherogenesis and attenuate vascular calcifications in uremia. To test this
idea, we explored the effect(s) of OPN-deficiency on aortic atherosclerosis and
calcifications in hyperlipidemic and uremic mice.
Methods and Results: Moderate uremia was induced by 5/6 nephrectomy in
apolipoprotein E (E) KO and OPN and E double KO mice (E/OPN KO). In E KO
mice, uremia increased aortic atherosclerosis (from 28±2% [n= 15], to 37±3%
[n= 20], p < 0.05). In contrast, aortic atherosclerosis was not increased by
uremia in E/OPN KO mice. Uremia increased the aortic OPN mRNA expression
(~2−5 fold, p < 0.05) and plasma OPN (from 270±14 ng/ml to 400±14 ng/ml,
p < 0.0001) in E KO mice. Remarkably, there was a close positive correlation
between plasma OPN and aortic atherosclerosis within the group of uremic E
KO mice (r = 0.69, p = 0.001). In primary macrophage-derived foam cells, OPN
deficiency reduced MCP-1 and IL-6 mRNA expression. In uremic E KO mice,
OPN deficiency abolished a uremia-mediated increase of aortic MCP-1 mRNA
and plasma MCP-1. Opposite to the effect on atherosclerosis, OPN-deficiency
led to increased calcifications within atherosclerotic plaques of uremic mice.
Conclusion: The data suggest that OPN increases macrophage inflammation
and promotes development of atherosclerosis in the setting of uremia.
287 SERINE PROTEASE INHIBITOR A3 INFLUENCES AORTIC
ANEURYSM FORMATION BUT NOT ATHEROSCLEROSIS
D. Wågsäter, D. Johansson, X. Zhou, P. Eriksson. Karolinska Institute,
Stockholm, Sweden
Background: Infiltration of neutrophils and mast cells plays an important role
in aneurysm formation and atherosclerosis. SerpinA3 is an inhibitor of several
proteases such as cathepsin G and chymase derived from mast cells and
neutrophils.
Objective: In this study, we investigated the putative role of serpinA3 in
aneurysm formation and atherosclerosis.
Methods and Results: Decreased mRNA and protein expression of serpinA3
was found in biopsies of human thoracic (TAA) and abdominal aortic aneurysm
(AAA) compared to non dilated aortas. Plasma levels of serpinA3 were
similar in patients with AAA or atherosclerosis as compared with age-matched
controls. CaCl2 -induced aneurysm formation was inhibited in transgenic mice
overexpressing the murine ortholog serpinA3n compared to wild-type C57Bl/6
mice. Histology staining and Western blot analysis showed that the transgenic
mice had lower expression of mast cell tryptase and less degraded elastin as
compared to wild type mice with aneurysm. SerpinA3n overexpression had no
effect on atherosclerosis development in ApoE−/− deficient mice.
Conclusion: Decreased expression of serpinA3 is associated with aneurysm
formation in humans, and CaCl2 -induced aneurysm formation is inhibited in
mice overexpressing serpinA3n. These results further support the importance
of certain proteases derived from neutrophils and mast cells in aneurysm
formation.
288 GLYCATED HEMOGLOBIN FOR THE DIAGNOSIS OF TYPE
2 DIABETES IN PERSONS UNDERGOING CORONARY
ANGIOGRAPHY: THE LURIC STUDY
G. Silbernagel1 , M.E. Kleber2 , T.B. Grammer3 , B.R. Winkelmann4 ,
B.O. Boehm5 , W. März3,6,7 . 1 Division of Endocrinology, Diabetology,
Nephrology, Vascular Disease, and Clinical Chemistry, Department of Internal
Medicine, Eberhard-Karls-University Tübingen, Tübingen, 2 LURIC Study
Nonprofit LLC, Freiburg im Breisgau, 3 Mannheim Institute of Public Health,
Social and Preventive Medicine, Mannheim Medical Faculty, University
of Heidelberg, Mannheim, 4 Cardiology Group Frankfurt-Sachsenhausen,
Frankfurt/Main, 5 Division of Endocrinology and Diabetes, Ulm University
Medical Center, Ulm University, Ulm, 6 Synlab Center of Laboratory Diagnostics
Heidelberg, Heidelberg, Germany, 7 Clinical Institute of Medical and Chemical
Laboratory Diagnostics, Medical University of Graz, Graz, Austria
Objective: Glycated hemoglobin 6.5% has been included in the 2010
American Diabetes Association (ADA) criteria for the diagnosis of diabetes. The
present analysis aimed to investigate the risk of all-cause and cardiovascular
death in persons at intermediate to high cardiovascular risk who were
additionally diagnosed as having type 2 diabetes according to the new
criterion.
Methods: We studied 2002 individuals who were referred for angiography and
did not have a history of diabetes. Oral glucose tolerance tests were performed
in 1772 subjects with fasting glucose <126 mg/dl. The mean (±standard
deviation) duration of the follow-up for all-cause and cardiovascular mortality
was 7.66±2.0 years.
Results: A total of 468 subjects had newly diagnosed type 2 diabetes according
to the ADA 2009 definition. Another 150 participants were diagnosed as having
type 2 diabetes using the ADA 2010 criteria. 346 deaths occurred during the
follow-up. Among these, 202 were due to cardiovascular diseases. Subjects
with type 2 diabetes according to the ADA 2009 criteria and those with
isolated elevation of glycated hemoglobin 6.5% had increased all-cause and
cardiovascular mortality compared to subjects without diabetes according to the
79th EAS Congress
ADA 2010 definition (all p < 0.03). All-cause and cardiovascular mortality were
not significantly different between subjects with type 2 diabetes according to
the ADA 2009 criteria and those with isolated elevation of glycated hemoglobin
(p = 0.360 and p = 0.894, respectively).
Conclusions: These results support the use of the ADA 2010 criteria for
diagnosing diabetes, at least in persons at intermediate to high cardiovascular
risk.
289 SPONTANEOUS PLAQUE DISRUPTIONS, DISSECTION, EROSION
AND INTRAPLAQUE HEMORRHAGE IN MURINE VEIN GRAFTS
CAN BE ATTENUATED BY TIMP-1 OVEREXPRESSION
M.R. de Vries1,2 , H.W.M. Niessen3 , C.W. Löwik4 , J.W. Jukema2,5 ,
P.H.A. Quax1,2 . 1 Vascular Surgery, 2 Einthoven Laboratory of Experimental
Vascular Medicine, Leiden University Medical Center, Leiden, 3 Pathology
and Cardiac Surgery, ICaR-VU, VU University Medical Center, Amsterdam,
4
Endocrinology, 5 Cardiology, Leiden University Medical Center, Leiden, The
Netherlands
Plaque rupture and erosion are the most common underlying causes of
cardiovascular death. Matrixmetalloproteinases (MMP) play a key role in these
processes. We hypothesize that overexpression of the endogenous inhibitor
of MMPs TIMP-1 results in decreased plaque disruptions in vein grafts in
hypercholesterolemic ApoE3Leiden mice.
Vein graft lesions in ApoE3Leiden mice consist of foamcells, smooth muscle
cells (SMC) and extracellular matrix with necrotic cores, calcifications and
inflammatory cells. Lesion neovessels consist of endothelial cells and basement
membranes, supporting mural cells were infrequently seen. 79% of 47 vein
grafts studied showed spontaneous disruption, 1/3 of these disruptions were
dissections, 1/3 were leaky microvessels with extravasated erythrocytes and
1/3 were erosions or intramural thrombosis. Also vein grafts with combined
leaky vessels and dissections or erosions were seen. Systemic overexpression
of TIMP-1 (and Luciferase as control) was obtained by plasmid electroporation.
Near InfraRed Fluorescense imaging was used to detect in vivo (inhibition of)
MMP activity and thus TIMP-1 plasmid functionality. TIMP-1 overexpression
resulted in a decrease of 73% in MMP activity in the vein graft-region after 28d
and a 40% reduction in vein graft thickening after 28d. The lesions showed
significant less disruptions (90%) than the control Luciferase group resulting in
a more stable phenotype with significant more SMC, collagen and significant
less macrophages and fibrinogen.
Here we show that murine ApoE3L vein grafts are a relevant model to study
plaque stability and subsequent disruptions. Furthermore we demonstrate that
MMP inhibition reduced plaque disruptions resulting in a more stable plaque
phenotype.
290 ATHEROPROTECTIVE EFFECTS OF ADIPONECTIN
OVER-EXPRESSION IN A MODEL OF ANGII-ACCELERATED
ATHEROSCLEROSIS
C.M.W. van Stijn, J. Kim, D. Becerra, R.K. Tangirala. Medicine/Endocrinology,
Diabetes & Hypertension, David Geffen School of Medicine at UCLA, Los
Angeles, CA, USA
Adiponectin, an adipocytokine derived from adipose tissue, exerts antiinflammatory and anti-atherogenic effects on vascular cells. Low plasma
adiponectin levels are associated with the metabolic syndrome, diabetes and
cardiovascular disease. It is unknown whether elevation of plasma adiponectin
levels provides protection against angiotensin II (AngII)-mediated vascular
inflammation and accelerated atherosclerosis. In this study, first, we investigated
adiponectin regulation of inflammatory gene expression in human and mouse
macrophages. Gene expression analyses of macrophages treated with TNF-a
or TNF-a + adiponectin revealed that adiponectin repressed several genes
inflammatory/atherogenic genes (ACE, MCP-1, IL-1b, IL-8 and PDGF-b).
We determined whether adenoviral adiponectin expression inhibits AngIIaccelerated vascular inflammation and atherosclerosis. Low-density lipoprotein
receptor-deficient (LDLR−/− ) mice fed high-fat and infused with Ang-II were
injected 5 X 108 particles of adenovirus expressing GFP (Ad-GFP, n = 16) or
mouse adiponectin (Ad-APN, n = 16). After eight weeks, plasma adiponectin
levels were 12-fold higher in Ad-APN than Ad-GFP (306.4 mg/dL vs 25.3 mg/dL)
accompanied by significant elevation of plasma HDL levels in Ad-APN mice
(23.6% increase). Quantification of atherosclerosis by en face method, revealed
a significant inhibition of atherosclerosis in Ad-APN (45% reduction, p < 0.01 vs.
Ad-GFP). Furthermore, adiponectin expression substantially inhibited mRNA
levels of inflammatory and atherogenic genes (ICAM-1, osteopontin, MCP-1
and CCR2) in the vessel wall. Interestingly, adiponectin also increased mRNA
expression of the reverse cholesterol transport genes, ABCA1 and ABCG1, in
the aorta. These data strongly support that concept that increasing plasma
adiponectin levels may be an effective therapeutic strategy to inhibit AngIImediated vascular inflammation and acceleration of atherosclerosis.
63
291 CORONARY ARTERY CALCIFICATION AND DIABETES MELLITUS:
SEX-SPECIFIC IMPACT ON INCIDENCE OF CARDIOVASCULAR
DISEASE − RESULTS OF THE HEINZ NIXDORF RECALL-STUDY
S. Moebus1 , S. Möhlenkamp2 , N. Dragano1 , U. Slomiany1 , S. Pechlivanis1 ,
R. Erbel2 , K. Mann3 , K.-H. Jöckel1 , Heinz Nixdorf Recall Study Group.
1
University Hospital, University of Duisburg-Essen, Institute for Medical
Informatics, Biometry and Epidemiology, 2 University Hospital, University of
Duisburg-Essen, West German Heart Center Essen, 3 University Hospital,
University Duisburg-Essen, Department of Endocrinology and Division of
Laboratory Research, Essen, Germany
Aims: To examine the impact of coronary artery calcification (CAC), a specific
marker of subclinical atherosclerosis, on cardiovascular disease (CVD) with
regard to diabetes status (DM).
Methods: We included 4,265 participants (aged 45−75 years) without overt
CVD at baseline of the population-based Heinz Nixdorf Recall Study. Baseline
examination included blood analyses and electron-beam tomography. DM is
based on self-report and/or intake of diabetes medication. We calculated hazard
ratios (HR) and 95%-confidence intervals (95%-CI) for events (myocardial
infarction/stroke) with cox proportional hazards models adjusting for age, CAC
(dichotomized by 75th age- and sex-specific percentile), waist circumference,
smoking, lipid-lowering medication and blood pressure.
Results: The mean follow-up time was 7.2 (±1.44) years, with 194 (128 men)
events, of these 31 (18 men) with DM. Overall diabetic subjects had a 2.5fold
higher risk for CVD. However, in women with a CAC above the 75th age- and
sex-specific percentile we observed a 3-fold increased risk for CVD (ageadjusted HR 3.02; 95% CI: 1.36–6.70, full model: 3.61;1.59–8.21) for diabetic
women compared to women without diabetes, whereas the HR attenuates to
1.99 (0.73–5.41, full model: 2.34;0.89–6.13) in diabetic women with less CAC.
These high risks and differences are not observed in diabetic men with high
CAC or with CAC to a lesser extent (full model: HR 1.28;0.64–2.54, respective
1.59;0.75–3.36).
Conclusions: Our study confirms the high risk for CVD in subjects with
Diabetes mellitus. Moreover, we found that diabetic women exposed to a high
CAC are especially susceptible for CVD.
292 A POSSIBLE LINK BETWEEN ATHEROSCLEROTIC PLAQUE
INFLAMMATION AND VISCERAL FAT INFLAMMATION
S.E. Kim1 , E.J. Kim2 , J.S. Yeo3 , H.S. Seog2 . 1 Nuclear Medicine,
2
Cardiovascular Center, Korea University Guro Hospital, Seoul, 3 Nuclear
Medicine, Dongkuk University Ilsan Hospital, Goyang-si, Republic of Korea
Background: We hypothesized that active atherosclerotic change in the major
arteries would accompany increased inflammation within visceral fat and it could
be detected in humans using F-18 FDG PET/CT.
Methods: We observed 44 consecutive subjects with cardiovascular disease.
For all of them, an one-hour PET/CT was performed after injection of FDG
(370–555 MBq). FDG uptake in the aorta or its major branches was evaluated
visually and semiquantitatively. Maximal standard uptake values (SUV) of the
highest regions of interest were calculated in the subcutaneous fat and visceral
fat area, separately.
Results: Significant FDG uptake in the arterial wall was noted in 21 patients
(plaque positive; PP group), all of whom have experienced acute cardiovascular
events (acute coronary syndrome or ischemic stroke) within a week. The
other 23 patients (plaque negative; PN group) had chronic stable angina or
asymptomatic carotid stenosis. Visceral fat SUV was significantly higher as
compared to subcutaneous fat SUV (0.49±0.15 vs. 0.15±0.05, p < 0.001) in
PP group, whereas there was no significant difference in PN group (0.18±.07
vs. 0.16±.03, p = 0.622). When we compared two groups, PP group showed
higher visceral fat SUV than PN group (p < 0.001). In terms of subcutaneous
fat SUV, the results were similar in two groups (p = 0.773).
Conclusions: We demonstrated that atherosclerotic plaque inflammation was
associated with increased inflammation within visceral fat. Further evaluation to
determine whether metabolic activity of visceral adipose tissue is a marker or
mediator of vascular inflammation is also needed.
293 UROKINASE PLASMINOGEN ACTIVATOR (UPA) AND
ATHEROSCLEROSIS
B. Fuhrman, J. Khateeb, Y. Lati, M. Aviram. Lipid Research Laboratory,
Technion Faculty of Medicine/Rambam Medical Center, Haifa, Israel
Background: The urokinase plasminogen activator (uPA) and its receptor
uPAR, play a role in the pathogenesis of atherosclerosis. Increased plasma
levels of uPA were shown to be associated with plaque rupture. The
atherosclerotic plaque is characterized by cholesterol-loaded macrophage-foam
cells and oxidized lipids. The goal of our project is to study in detail the functions
of the uPA/uPAR system in atherosclerosis, in relation to macrophage-foam cell
formation.
Results: Our key findings show that uPA increases macrophage atherogenicity.
We have shown that uPA increased cellular cholesterol accumulation, resulting
from an increase in cellular cholesterol biosynthesis. This effect required the
64
binding of uPA to its receptor, uPAR, and was mediated via activation of
SREBP-1 through PI3K and MEK signal transduction pathways. Furthermore,
uPA increased macrophage oxidative stress by enhancing NADPH oxidase
activation. In turn, uPA initiated an oxidative stress-response by upregulating
the expression of macrophage paraoxonase 2 (PON2) through the PI3KNADPH oxidase-ROS-MEK-SREBP2 signaling cascade activation. In addition,
we have shown that uPA binding to uPAR on hepatocytes stimulates PPARg
nuclear export, resulting in down-regulation of paraoxonase 1 (PON1) gene
expression, and decreased PON1 secretion. Reduced levels of PON1 in serum
may lead to reduced protection of LDL and of HDL from oxidation, reduced
HDL-mediated cholesterol efflux from macrophages, and increased oxidative
stress in atherosclerotic lesions.
Conclusions: Taken together, our work represents a new paradigm by which
uPA is implicated in atherogenic processes beyond its role in the fibrinolytic
system, and provides new insight into the relationship between uPA and
vulnerable plaque.
294 ACCELERATED ATHEROSCLEROSIS IN FEMALE MICE LACKING
THE ANDROGEN RECEPTOR
J. Bourghardt1 , A. Wilhelmson1 , C. Alexanderson1 , K. De Gendt2 ,
G. Verhoeven2 , A. Holmäng3 , A. Krettek4,5 , S.-O. Olofsson1 , C. Ohlsson6 ,
Å. Tivesten1 . 1 Wallenberg Laboratory, University of Gothenburg, Gothenburg,
Sweden, 2 Katholieke Universiteit Leuven, Leuven, Belgium, 3 Institute of
Neuroscience and Physiology, Unversity of Gothenburg, 4 Nordic School of
Public Health, 5 Institute of Medicine, University of Gothenburg, 6 Center
for Bone and Arthritis Research, University of Gothenburg, Gothenburg,
Sweden
Objective: In women, elevated androgen levels have been reported to
associate with the metabolic syndrome and atherosclerosis. However, the
physiological role of androgens in metabolism and cardiovascular health in
females is unclear. We evaluated the impact of physiological, androgen receptor
(AR)-dependent actions of androgens on atherosclerosis using female ARdeficient (AR−/−) mice on an apolipoprotein E-deficient background.
Methods and Results: After 8 weeks on high-fat diet, atherosclerosis assessed
en face was increased by 59% (P < 0.01) in female AR−/− mice compared with
control mice. Female AR−/− mice had increased body weight and relative fat
mass (+62%, P < 0.001), increased hepatic triglyceride levels (+33%, P < 0.05)
and reduced insulin sensitivity. In addition, the female AR−/− mice had elevated
serum levels of cholesterol (+52%, P < 0.001) and triglycerides (+44%, P < 0.05)
compared with control mice.
Conclusions: Female AR−/− mice display accelerated atherosclerosis
associated with obesity, hepatic steatosis, insulin resistance and dyslipidemia.
These results suggest that AR-mediated effects of androgens are crucial for
metabolism and cardiovascular health in females.
295 ATHEROSCLEROSIS REGRESSION IN A MOUSE MODEL WITH
HUMAN-LIKE HYPERCHOLESTEROLEMIA
J. Björkegren1 , S. Hägg1 , S. Maleki1 , M. Shang1 , T. Michoel2 , J. Skogsberg1 .
1
Karolinska Institutet, Stockholm, Sweden, 2 University of Freiburg, Freiburg,
Germany
Atherosclerosis is a lifelong, progressive disease that becomes clinically
significant in a large fraction of the population, leading to myocardial
infarction (MI) and stroke. Statin based lipid-lowering regimens reduce
morbidity and mortality from both MI and stroke. To fully exploit the
beneficial effects of lipid lowering, we need a better understanding of
the transcriptional changes induced by lowering plasma lipoproteins. We
have recently performed transcriptional profiling at 10-week intervals in
atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic
switch to lower plasma lipoproteins (Ldlr−/− Apob100/100 Mttpflox/flox Mx1-Cre).
Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and
plateaued after advanced lesions formed. Analysis of lesion expression profiles
indicated that accumulation of lipid-poor macrophages reached a point that
led to the rapid expansion phase with accelerated foam-cell formation and
inflammation, an interpretation supported by lesion histology. In the current
study, we preformed genetic lowering of plasma cholesterol (i.e. lipoproteins)
at the rapid lesion expansion start point which prevented the formation of
advanced plaques. In addition, lowering of plasma cholesterol in plaques that
already had been formed not only halted the atherosclerosis lesion development
it also substantially regressed the lesion size. By performing transcriptional
profiling of the atherosclerotic arterial wall in the plasma cholesterol lowered
mice important gene networks responsible for atherosclerosis regression were
identified. These networks will be validated in relevant cell culture systems and
in human association studies. The identified networks should be of interest for
the development of novel atherosclerosis therapies.
296 TRANSCRIPTOMIC ANALYSIS OF MACROPHAGE POLARIZATION:
ROLE OF PPARY IN ALTERNATIVE ACTIVATION
E. Derlindati, D. Ardigò, I. Zavaroni. Internal Medicine, University of Parma,
Parma, Italy
Introduction: Human macrophages (HM) undergo different forms of activation
in response to cytokines. Classical activation (M1) displays pro-inflammatory
properties, and can be induced by IFNg and/or LPS. Alternative activation (M2a)
shows immuno-regulatory properties and is promoted by IL-4, whereas IL-10
produces a “deactivated” state, with immunosuppressing characteristics (M2c).
The PPARg system appears to be implicated in M2a activation enhancing their
anti-inflammatory properties.
Aim: Evaluation of the biological processes involved in the different types of HM
activation compared to the effects induced by exposure to PPARg-agonists.
Methods: HM were stimulated with INFg+LPS (M1), IL-4 (M2a), IL-10 (M2c), or
a PPARg-agonist (GW1929-Sigma). Phenotypic changes for each experimental
condition were investigated through whole-genome transcriptomic analysis
by microarrays (Agilent-Technologies). Gene ontology (GO) analysis was
performed using Gorilla tool. Validation was obtained by RT-PCR on the most
differentially expressed genes.
Results: After characterization of M1, M2a, and M2c genomic profiles,
we investigated the differences between PPARy-stimulated HM and M1,
observing a significant decrease in immunitary and inflammatory processes
(corresponding GO-term’s p-value = 2.00×10−35 and 1.20×10−20 ) and a great
increase in cell cycle activity (p = 1.51×10−35 ). Similar, but quantitatively lower,
results were observed also in comparison with M2a and M2c, although these
two subpopulations are usually regarded as anti-inflammatory.
Discussion: Exposure of HM to a PPARg-agonist induces a transcriptomic
phenotype closer to M2a/M2c than to M1. Nevertheless, PPARg-agonist
activation decreases inflammatory cytokine production and increases cellcycle gene expression also compared to cytokine-induced M2a and M2c
activation, suggesting a specific role of PPARg in the regulation of macrophage
differentiation.
297 DARBEPOETIN ENHANCES ENDOTHELIUM-DEPENDENT
VASOMOTOR FUNCTION IN PATIENTS WITH STABLE
CORONARY ARTERY DISEASE ONLY AFTER PRECEDING
ISCHAEMIA-REPERFUSION
L. Tilling, J. Hunt, A. Donald, B. Clapp, P. Chowienczyk. King’s College
London, British Heart Foundation Centre, London, UK
Objective: To examine whether the erythropoietin analogue darbepoetin
improves flow mediated dilatation (FMD), a measure of endothelium-derived
NO, and whether this is influenced by preceding ischaemia-reperfusion.
Background: Vasoprotective effects of erythropoietin in animal models are
mediated by endothelium-derived nitric oxide (NO) and/or mobilization of
endothelial progenitor cells (EPC) and may be enhanced by ischaemia. Whether
they are present in humans is unknown.
Methods: 36 patients (50−75 years) with stable coronary artery disease were
randomized to receive a single dose of darbepoetin (300 mg) or saline placebo.
FMD was measured at the brachial artery using high resolution ultrasound
before and at 24h, 72h and 7 days after darbepoetin/placebo. At 24h FMD was
repeated after 20 minutes ischaemia-reperfusion of the upper limb. A further
group of 11 patients were studied according to the same protocol, all receiving
darbepoetin, with omission of forearm ischaemia-reperfusion at 24h.
Results: Immunoreactive erythropoietin peaked at 24h and remained elevated
at approximately 500 fold baseline at 72h. FMD did not differ significantly
between groups at 24h (before ischaemia-reperfusion). At 72h, (48h after
ischaemia-reperfusion) FMD was greater (by 2.3±0.5% in the darbepoetin
compared to the placebo group, a 66% increase over baseline, P < 0.001)
and greater than FMD at the same time point without preceding ischaemiareperfusion (P < 0.01). There was no increase in CD133+ /CD34+ /VEGFR2+
cells after darbepoetin treatment.
Conclusions: Preceding ischaemia-reperfusion is required for darbepoetin
to enhance endothelial function, possibly by increasing expression of the
erythropoietin receptor and by a mechanism likely to involve Akt/NO rather
than circulating EPC.
298 RELATION OF MATRIX METALLOPROTEINASES (MMPS)-POSITIVE
MACROPHAGES TO CORONARY PLAQUE FRAGILITY AND
CORONARY ARTERIAL OUTWARD REMODELING
T. Ito1 , S. Yamada1 , N. Hirayama-Kuniyoshi1 , M. Shiomi1,2 . 1 Institute for
Experimental Animals, Kobe Univesity Graduate School of Medicine, 2 Section
of Animal Models for Cardiovascular Diseases, Division of Cardiovascular
Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
Objectives: The purpose of this study is to examine the influence of MMPspositive macrophages on coronary plaque fragility and coronary outward
remodeling.
Methods: Coronary arteries of myocardial infarction-prone Watanabe heritable
hyperlipidemic (WHHLMI) rabbits aged 15−24 months were fixed with 10%
79th EAS Congress
neutral buffered formalin solution and were embedded in paraffin. Coronary
segments were sectioned serially and stained immunohistochemically using
antibodies specific for rabbit macrophages (RAM-11), MMP-1, MMP-12, and
for a-actin (1A4). Sections were also stained with elastic van Gieson staining
and Azan-Mallory staining.
Results: Various types of atherosclerotic lesions were observed in the coronary
arteries, such as plaque with large lipid core covered with thin fibrous cap,
fibroatheroma, fibromuscular lesion, macrophage-rich lesion, and plaque with
calcification or intraplaque hemorrhage. Macrophages were observed beneath
the fibrous cap and at a bottom of the intimal plaques where the tunica media
was attenuated. At a plaque surface, macrophages were positive for MMPS;
the density of SMCs and collagen fibers were decreased in the fibrous cap; the
fibrous cap was attenuated partly. At a bottom of the intimal plaque where the
tunica media was attenuated, macrophages was positive for MMPs; the elastic
lamina was disappeared, the layer of SMCs in the tunica media was reduced.
Conclusions: These observations suggest that MMP-positive macrophages at
the plaque surface may relate to plaque fragility and those observed at a bottom
of the intimal plaques may relate to arterial outward remodeling.
299 VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR
2-INHIBITION PREVENTS ENDOTHELIAL SUSCEPTIBILITY TO
TNF-a UNDER CHRONIC NON-UNIFORM SHEAR STRESS
K. Urschel, W.G. Daniel, C.D. Garlichs, I. Cicha. Department of Cardiology
and Angiology, University of Erlangen-Nuremberg, Erlangen, Germany
Introduction: Non-uniform shear stress contributes to the development of
atherosclerotic lesions by activating inflammatory pathways in endothelial cells
(ECs). VEGFR2 is a key receptor for endothelial mechanotransduction. We
investigated the role of VEGFR2 in ECs activated by non-uniform shear
stress, with focus on monocyte recruitment and endothelial adhesion molecule
expression.
Methods: ECs seeded in bifurcating flow-through cell culture slides were
exposed to shear stress overnight, followed by 2 hours stimulation with TNF-a.
Subsequently, ECs were perfused for 1 hour with medium containing THP-1
monocytes to study cell adhesion. During flow, cells were incubated with a
specific VEGFR2-antagonist (ZM 323881). For siRNA approach, cells were
transfected with siRNAs knocking down VEGFR2 prior to shear stress exposure.
Endothelial protein expression was determined by immunofluorescence and cell
adhesion by light microcopy.
Results: siRNA-mediated VEGFR2 knockdown resulted in a decrease of
VEGFR2 protein expression by 40%. This knockdown was accompanied by
a marked reduction of TNF-a-induced NFú-B translocation from cytoplasm to
the nucleus in ECs. Either siRNA-mediated receptor knockdown, or treatment
with the specific inhibitor, significantly reduced TNF-a-induced monocytic cell
recruitment to endothelium under non-uniform shear stress conditions. The
decrease in monocytic cell adhesion via inhibition of VEGFR2 signaling was
accompanied by a reduction of E-selectin and VCAM-1.
Conclusion: The inhibition of pro-atherogenic mechanical signaling by blocking
VEGFR2 decreased endothelial activation and monocytic cell recruitment in
response to inflammatory cytokines under in vivo-like non-uniform shear stress
conditions. Localized targeting of VEGFR2 can therefore represent a useful
approach in the treatment of atherosclerosis.
300 THE GUT MICROBIOTA MODULATES ATHEROSCLEROSIS
PROGRESSION IN MALE APOE−/− MICE
F. Fåk1 , C. Reinhardt2 , F.-P. Martin3 , F. Bäckhed2 . 1 Dept. of Medicine,
Gothenburg University, 2 University of Gothenburg, Gothenburg, Sweden,
3
Nestlé Research Center, Lausanne, Switzerland
Introduction and Objective: Bacterial infections appear to correlate with
cardiovascular disease but the precise role of bacteria in disease initiation or
progression has yet to be elucidated.
Design: To investigate the role of bacteria in atherosclerosis, we re-derived
atherosclerosis-prone apolipoprotein E-deficient mice (Apoe/−) as germ free.
The mice were kept on a high fat, Western diet supplemented with cholesterol
for 12 weeks and were sacrificed at 20 weeks of age. Atherosclerosis was
assessed in the aortic root region of the heart and entire aortas were pinned
and stained with Oil Red O. In addition, blood cholesterol, triglycerides, lipid
metabolites and cytokines were analyzed.
Results: Male germ free Apoe/− mice exhibited significantly decreased lesion
area in the aortic root along with lower blood cholesterol and TG triglyceride
levels, as compared to conventionally raised mice. Female mice, however,
developed lesions of the same size as the control mice.
Conclusions: Our results indicate that bacteria are important for atherosclerosis progression through a gender-dependent mechanism.
65
301 ALCOHOL INHIBITS NOTCH SIGNALING IN VASCULAR SMOOTH
MUSCLE CELLS AT THE LEVEL OF g-SECRETASE
D. Morrow1 , J. Cullen1 , P. Cahill2 , E. Redmond1 . 1 Surgery, University of
Rochester, Rochester, NY, USA, 2 Vascular Health Research Center, Dublin
City University, Dublin, Ireland
Moderate alcohol consumption is associated with reduced cardiovascular
disease. We recently reported an inhibitory effect of alcohol (ethanol, EtOH)
on Notch signaling and subsequently on vascular smooth muscle (SMC)
proliferation, a key process in atherosclerotic plaque development. The object
of this study was to test the hypothesis that EtOH inhibits Notch signaling in
SMC at the level of g-secretase, a protease that catalyzes the release of the
intracellular domain of Notch (NICD) necessary for signaling.
Human coronary artery SMC were treated with a recombinant Notch
ligand, DLL4 (2 mg/ml), or transfected with/without constitutively active Notch
1ICD (N1ICD), in the absence or presence of EtOH (25 mM). Ethanol
treatment inhibited DLL4-stimulated CBF-1/RBP-Jk dependent promoter activity
(determined by luciferase assay) and DLL4-stimulated downstream target gene
HRT3 mRNA expression. In contrast, ethanol had no effect on N1ICD-driven
CBF1/RBP-Jk dependent promoter activity. These data suggest that EtOH
inhibits Notch signaling at, or prior to, NICD generation. g-secretase activity
was determined in solubilized membrane preparations from SMC treated
with/without EtOH (25 mM) or the g-secretase inhibitor DAPT (20 mM) using
(i) a fluorometric assay kit and (ii) western blot detection of cleavage products
using a Flag-tagged Notch based substrate, N100Flag. In both assays, Ethanol
inhibited g-secretase activity, to the same extent as DAPT. In contrast, ethanol
had no effect on a-secretase (TACE/ADAM 17) activity, determined using a
fluorometric assay kit.
In conclusion, EtOH inhibits Notch signaling in SMC via inhibition of
the g-secretase mediated intramembrane proteolysis necessary for NICD
generation and subsequent transcriptional activation.
302 CB2 RECEPTOR SIGNALING DOES NOT MODULATE
ATHEROGENESIS IN MICE
F. Willecke, K. Zeschky, D. Wolf, M. Moser, I. Hilgendorf, C. Bode, A. Zirlik.
Universitätsklinik Freiburg, Freiburg im Breisgau, Germany
Strong evidence supports a protective role of the cannabinoid receptor 2 (CB2 )
in inflammation and atherosclerosis. However, direct proof of its involvement in
lesion formation is lacking. The present study aimed to characterize the role of
the CB2 receptor in Murine atherogenesis.
Methods and Results: Low density lipoprotein receptor-deficient (LDLR−/− )
mice subjected to intraperitoneal injections of the selective CB2 receptor agonist
JWH-133 or vehicle 3x/week consumed a high cholesterol diet for 16 weeks.
Surprisingly, intimal lesion size did not differ between both groups in sections
of the aortic roots (0.316±0.038 mm2 vs. 0.312±0.044 mm2 , P = 0.94) and
arches (0.091±0.024 mm2 vs. 0.066±0.013 mm2 , P = 0.41), suggesting that
CB2 activation does not modulate atherogenesis in vivo. Plaque content of
lipids, macrophages, smooth muscle cells, T cells, and collagen was also
−/−
similar in both groups. Accordingly, CB−/−
mice developed lesions of
2 /LDLR
similar size in roots (0.261±0.038 mm2 vs. 0.223±0.023 mm2 , P = 0.40) and
−/−
arches (0.095±0.022 mm2 vs. 0.075±0.022 mm2 , P = 0.54) as CB+/+
2 /LDLR
controls consuming HCD for 16 weeks. With exception of an increase in lipid
and macrophage content, plaque composition was also similar between these
two groups. Neither genetic deficiency nor pharmacologic activation of the
CB2 receptor altered inflammatory cytokine expression or peritoneal leukocyte
recruitment in vivo or inflammatory cell adhesion in the flow chamber in vitro.
Conclusion: Our study demonstrates that activation and deletion of the CB2
receptor do not modulate atherogenesis in mice. Our data do not challenge the
multiple reports involving CB2 in other inflammatory processes. However, in the
context of atherosclerosis, CB2 does not appear to be a suitable therapeutic
target.
303 MICRORNA 143–145 DEFICIENCY IS ASSOCIATED WITH IMPAIRED
VASCULAR FUNCTION
G.D. Norata1 , C. Pinna1 , F. Zappella1 , L. Elia2 , G. Condorelli3 , A. Catapano1 .
1
University of Milan, Milan, Italy, 2 University of California San Diego, La Jolla,
CA, USA, 3 Consiglio Nazionale delle Ricerche, Segrate, Italy
Background: MicroRNAs are necessary for vascular smooth muscle growth,
differentiation and function. MiR143–145 modulate cytoskeletal dynamics
and acquisition of the contractile phenotype by smooth muscle cells. Loss
of this miRNA cluster results in deregulated blood pressure and reduced
vasoconstriction. As all these observations point to a key role for miR143–145
in the vasculature, we therefore investigated whether miR143–145 deficiency
is associated with impaired vascular tone.
Methods and Results: Vasocontraction was assessed in isolated aortic
rings from miR143–145 KO and wild type animals incubated with increasing
concentrations of phenylephrine (10-9 M to 10-5 M) or KCl 0.3M. In both cases,
aortic vessel contraction was dramatically reduced in miR143–145 KO animals
compared to controls. Next, aortic rings were pre-contracted with phenylephrine
66
(10-7 M) and concentration responses for acetylcholine were obtained. A
significantly reduced vasodilatation was observed in miR143–145 KO animals
compared to controls and similar results were obtained when an exogenous
donor of nitric oxide (sodium nitroprusside) was used. Isoprenaline mediated
vasodilation was significantly reduced in miR143–145 KO animals compared to
controls in the absence or in the presence of the guanylate cyclase inhibitor
ODQ (10-4 M), suggesting that also beta adrenergic vasodilatation is impaired
following miR143–145 deficiency. Finally the effect of a stable prostacyclin
mimetic, namely iloprost, was investigated and again a reduced vasodilatation
was observed in miR143–145 KO animals.
Conclusion: miR143–145 deficiency is associated not only with altered
vasocontraction but also with impaired vasodilatation which probably reflects
the impaired VSMC differentiation phenotype reported in miR143–145 KO
animals.
304 THE INFLUENCE OF INTRACORONARY INJECTION OF
MONONUCLEAR BONE MARROW CELLS ON PROTHROMBOTIC
MARKERS IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
S. Solheim1 , I. Seljeflot1 , K. Lunde2 , S. Aakhus2 , V. Bratseth3 , K. Forfang2 ,
H. Arnesen1 . 1 Center for Clinical Heart Research and Department of
Cardiology, Oslo University Hospital, Ullevål, 2 Department of Cardiology, Oslo
University Hospital, Rikshospitalet, 3 Center for Clinical Heart Research, Oslo
University Hospital, Ullevål, Oslo, Norway
The effects of intracoronary injection of mononuclear bone marrow cells
(mBMC) on hemostasis are not clarified. The aim of the present substudy of the
autologous stem cell transplantation in acute myocardial infarction (ASTAMI)
trial was to investigate the influence of intracoronary injection of mBMC on
selected circulating prothrombotic markers.
Methods: One hundred patients with acute ST-elevation myocardial infarction
(AMI) treated with percutaneous coronary intervention (PCI) on the left
descending coronary artery were randomized to receive autologous mBMC
(median 6 days after the AMI) or to a control group. Fasting blood samples
were drawn the day before stem cell transplantation (Tx-1, 4−5 days after the
AMI), and 1 day (Tx+1), 3 days (Tx+3), 2−3 weeks and 3 months after the
transplantation.
Results: No between group differences in change from Tx-1 to the subsequent
time points could be detected regarding circulating levels of tissue factor (TF),
prothrombin fragment 1+2 (F1+2), D-dimer, endogenous thrombin potential
(ETP) and plasminogen activator inhibitor-1 (PAI-1). However, TF and F1+2
decreased from Tx-1 to the subsequent time points in both groups, except from
a small increase of TF at 3 months in the control group. D-dimer, ETP and
PAI-1 decreased from Tx-1 to 2−3 weeks and 3 months in both groups.
In conclusion, intracoronary injection of mBMC did not influence on selected
prothrombotic markers in patients with AMI treated with PCI. However, a
decrease of the circulating markers from Tx-1 to 2−3 weeks and 3 months
could be demonstrated in both groups.
305 SOLUBLE FORM OF THE ENDOTHELIAL ADHESION MOLECULE
CD146 BINDS PREFERENTIALLY PROINFLAMMATORYPROATHEROSCLEROTIC CD16+ MONOCYTE SUBSET
C. Barisione, G. Ghigliotti, P. Fabbi, P. Altieri, C. Brunelli, P. Spallarossa,
A. Barsotti, S. Garibaldi. Internal Medicine, University of Genoa, Genoa,
Italy
The cell adhesion molecule CD146 is normally located at the endothelial cellto-cell junction level and colocalizes with actin cytoskeleton. The soluble form
of CD146 (sCD146) has been identified in the endothelial cell supernatant
and in normal human plasma, and is increased in pathologic conditions
with altered endothelial function. Monocytes do not express CD146, but can
bind the sCD146, which promotes the transendothelial migration, a central
step in the atherosclerotic plaque development. Since circulating monocytes
can be distinguished in different subsets for phenotypic and functional
heterogeneity and transendothelial migration capacity, we hypothesized that
monocyte subsets differently bind sCD146. Based on surface CD14 and
CD16 expression, monocytes were distinguished by FACS into three subsets:
CD14++(bright)CD16−, CD14++(bright)CD16+ and CD14+(dim)CD16+. CD16+
monocytes have been found to possess higher transendothelial migration
ability.
Analysis of blood monocytes from 30 healthy subjects revealed that
higher percentages of CD14++(bright)CD16+ (2.26 (1.62–3.87)%) (median,
first and third quartile) and CD14+(dim)CD16+ (2.59 (1.28–4.80)%) than
CD14++(bright)CD16− (0.66 (0.47–1.01)%) were positive for CD146 (both
p < 0.01). Moreover, in vitro treatment of ficoll separated monocytes with
recombinant CD146 showed that both CD16+ subsets increased their
percentage of CD146-positive events compared to CD16− monocytes
(p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples
of subjects from our study group and showed a correlation with percentage of
CD146-positive CD14+(dim)/CD16+ monocytes.
These results suggest that sCD146 differentially binds to monocyte subsets;
this may account for their different endothelial adhesion and transmigration
ability and might represent a potential target to limit atherosclerotic plaque
development.
306 ISOPRENOID PATHWAY INHIBITORS BLOCK INNATE PATHWAYS
OF IMPORTANCE FOR EARLY ATHEROGENESIS
H. Loppnow1 , M. Buerke2 , A. Schlitt2 , U. Müller-Werdan2 , K. Werdan2 .
1
KIM-III, Forschungslabor, 2 KIM-III, Martin-Luther-Universität Halle-Wittenberg,
Halle (Saale), Germany
The first steps in human atherogenesis are not finally resolved. Activation
of innate pathways by atherosclerosis-risk-factor-related compounds, such
as microorganisms or cholesterol, may initiate the inflammatory cascade.
Cytokine-mediated interaction of vessel wall cells and invading monocytes
may further trigger local inflammatory processes. Anti-inflammatory effects of
statins may counteract inflammation. In an atherosclerosis-related inflammatory
coculture model consisting of vascular smooth muscle cells (SMC) and
monocytes, lipopolysaccharide-treatment resulted in synergistic IL-6 production.
Interestingly, 25-hydroxycholesterol induced an even higher synergistic IL-6
production. The standard anti-inflammatory drugs aspirin and indomethacin
(Indo) lowered the IL-6 production by 60%. Sim-, ator-, flu- or pravastatin
also reduced it (53%, 50%, 64% and 60%, respectively). Studies with the
geranylation inhibitor GGTI extended these investigation. GGTI also reduced
the IL-6 synergism, although less potent than the statins. Combination of
statins with aspirin/Indo completely inhibited the synergistic IL-6 production.
The inhibitors blocked STAT3 phosphorylation, providing evidence for an
autocrine role of IL-6 in the synergism. In summary, the data show that innate
pathways can be potently activated by pro-atherogenic compounds and that
this inflammatory response can be limited by anti-inflammatory drugs. The data
implicate that statins have the capacity to influence atherogenesis by regulating
cytokine-mediated innate inflammatory pathways in the vessel wall.
307 INFLAMMATORY MARKERS AND DISEASE SEVERITY IN PATIENTS
WITH CHRONIC HEART FAILURE. LIMITED EFFECTS OF EXERCISE
TRAINING
R. Byrkjeland1 , B.B. Nilsson2 , A.S. Westheim2 , H. Arnesen1 , I. Seljeflot1 .
1
Center for Clinical Heart Research, Department of Cardiology, 2 Department
of Cardiology, Oslo University Hospital, Ullevål, Oslo, Norway
Introduction: Chronic heart failure (CHF) is associated with increased
inflammation, and exercise training has in some studies shown to have antiinflammatory effect, although controversies exist. We investigated the effects
of exercise training in CHF-patients on markers of inflammation, and further
explored any associations between inflammation and the severity and etiology
of CHF.
Methods: Eighty patients in stable CHF (mean age 70.1±7.9) were randomized
to a 4 month group-based exercise training program or control group. Functional
capacity was measured by 6-min walking test and a bicycle ergometer test.
Fasting blood samples were drawn at baseline, after 4 and 12 moths for
analyses of a range of biomarkers.
Results: Measures of functional capacity were significantly inversely related
to CRP, IL-6, VCAM-1 and TGF-b, and NT-proBNP levels were significantly
correlated to CRP, TNF-a, IL-6, VCAM-1, ICAM-1 and TGF-b. Patients with
hypertension as etiology of CHF showed higher levels of CRP (p < 0.01), IL-6
(p = 0.05) and TNF-a (p = 0.02) compared to other etiologies. No significant
differences in changes between the exercise training group and the control
group were obtained in any of the measured variables, except in patients
with idiopathic dilated cardiomyopathy, where significant reductions in CRP
(p = 0.029), ICAM-1 (p = 0.013), TGF-b (p = 0.02) and TNF-a (p = 0.014) were
observed.
Conclusions: Measures of CHF severity were significantly correlated with
several markers of inflammation. We could not demonstrate anti-inflammatory
effect of exercise in this older population of CHF patients, however, the etiology
of CHF affected this result and also the inflammatory profile in the patients.
308 SPECIFIC LP(A) APHERESIS FOR CAROTID ATHEROSCLEROSIS
STABILIZATION IN CHD PATIENTS WITH ELEVATED LP(A) LEVELS
M. Safarova1 , M. Ezhov1 , O. Afanasieva1 , T. Balakhonova1 , I. Adamova2 ,
G. Konavalov3 , S. Pokrovsky1 . 1 Russian Cardiology Research Center,
2
POCARD Ltd., 3 Medsi Group of Companies, Moscow, Russia
Objective: To evaluate the impact of lipoprotein(a) [Lp(a)] apheresis on the
carotid intima-media thickness (CIMT) in CHD patients with Lp(a) levels
50 mg/dl and LDL-C concentration 2.5 mmol/l.
Methods: We recruited 33 patients (22 men, 11 women, 54.2±7.4 years)
with angiographically verified coronary atherosclerosis. Patients were divided
into two groups: group I (n = 15) received Lp(a) apheresis plus atorvastatin,
group II (n = 18) − atorvastatin. Initially groups were comparable on clinical and
biochemical characteristics. The CIMT was measured at baseline and after the
6-month therapy by two independent blinded operators on the distal 1 cm of
right and left common carotid arteries before the bifurcation. Specific Lp(a)
apheresis was performed with “Lp(a) Lipopak”® columns once a week.
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Results: By the single Lp(a) apheresis procedure Lp(a) decreased by an
average of 66±7%, LDL-C levels did not significantly change. Currently data on
CIMT changes were obtained in 8 patients from apheresis group. We revealed
reduction in the mean CIMT on Lp(a) apheresis from 0.912±0.340 mm to
0.882±0.265 mm vs lack of changes in control group: from 0.882±0.212 mm to
0.882±0.162 mm. Lp(a) apheresis had greater efficacy regarding the amount
of regressed segments of carotid artery than atorvastatin alone: 10 of 16
segments (63%) vs 14 of 36 segments (38%), p = 0.14, respectively.
Conclusion: Our preliminary data have shown that specific Lp(a) lowering could
stabilize CIMT in CHD patients. This is the first study providing the evidence
in using Lp(a) as a therapeutic target for achieving a beneficial effect on a
surrogate marker of atherosclerosis.
309 CATECHOL-O-METHYLTRANSFERASE (COMT) IS DISPENSABLE
FOR VASCULAR PROTECTION BY ESTRADIOL IN MICE
A.S.K. Wilhelmson1 , J. Bourghardt1 , P. Fogelstrand1 , J.A. Gogos2 ,
Å. Tivesten1 . 1 Wallenberg Laboratory for Cardiovascular Research,
Sahlgrenska University Hospital, Gothenburg University, Gothenburg, Sweden,
2
Department of Physiology and Cellular Biophysics and Department of
Neuroscience, Columbia University, New York, NJ, USA
The endogenous estradiol metabolite 2-methoxyestradiol is formed from
estradiol via the activity of the enzyme catechol-O-methyltransferase
(COMT). Like estradiol, 2-methoxyestradiol reduces experimental atherosclerosis and neointima formation. Moreover, COMT-dependent formation of
2-methoxyestradiol has been suggested to mediate the antimitogenic effect of
estradiol on smooth muscle cells in vitro. This study evaluates whether the
endogenous production of 2-methoxyestradiol mediates the vasculoprotective
actions of estradiol in vivo.
Male and female COMT knockout and wild-type (WT) mice on apolipoprotein
E-deficient background were gonadectomized and treated with estradiol
(0.83 mg/day) or placebo. Atherosclerosis development was evaluated after 8
weeks on atherogenic diet. In addition, neointima formation was evaluated in
female COMT knockout and WT mice 4 weeks after ligation of the carotid
artery. Atherosclerotic lesion formation was reduced by estradiol treatment in
both males (COMTKO −53% and WT −48%) and females (COMTKO −82%
and WT −78%), and estradiol was equally effective in COMT knockout and
WT mice. Accordingly, estradiol treatment reduced intimal hyperplasia to a
similar extent in female COMT knockout and WT mice (COMTKO −77% and
WT −80%). Contrary to our hypothesis, female COMT knockout mice with intact
endogenous hormone production developed less atherosclerosis than their WT
littermates (−25%).
In conclusion, our results do not support the hypothesis of COMT-generated
2-methoxyestradiol being a mediator of the vascular actions of estradiol
since the COMT enzyme is dispensable for vascular protection by estradiol.
Instead, COMT-deficiency in female mice is associated with protection against
atherosclerosis.
310 LONG-TERM TREATMENT WITH LOW-DOSE METOPROLOL CR/XL
IS ASSOCIATED WITH INCREASED PLAQUE ECHOGENICITY:
THE BETA-BLOCKER CHOLESTEROL-LOWERING ASYMPTOMATIC
PLAQUE STUDY (BCAPS)
G. Östling1 , I. Gonçalves1 , J. Wikstrand2 , G. Berglund1 , J. Nilsson1 ,
B. Hedblad1 . 1 Department of Clinical Sciences, Malmö, Lund University,
Malmö, 2 Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska
Academy, Gothenburg University, Gothenburg, Sweden
Objectives: To examine whether the decrease in IMT progression rate in
the carotid bulb induced by metoprolol CR/XL treatment (25 mg once daily)
observed in the b-blocker Cholesterol-lowering Asymptomatic Plaque Study
(BCAPS) was accompanied by an effect on carotid plaque echogenicity.
Methods: Gray scale median (GSM) in carotid plaques, used as a score of
echogenicity, was measured at baseline and after 36 months in those 341
subjects (aged 49 to 69 years) with an asymptomatic moderate- to largesized carotid plaque present at baseline and at follow-up. Participants were
in a factorial design assigned to treatment with metoprolol CR/XL (25 mg once
daily), fluvastatin (40 mg once daily) or corresponding placebo.
Results: After 36 months plaques were more echogenic in participants
treated compared to those not treated with metoprolol CR/XL (57.3±16.8
versus 51.8±20.0, p = 0.006). GSM had increased more from baseline in the
metoprolol CR/XL treated subjects (25±15 versus 18±20, p < 0.001), and
plaques that had become more echolucent were less frequent in the metoprolol
CR/XL treated subjects (3.6 versus 17.0%, p < 0.001).
Conclusions: Long-term treatment with low dose metoprolol CR/XL in clinically
healthy subjects with moderate-sized carotid plaques was associated with
increase in plaque echogenicity, suggesting a potential beneficial effect of the
b-blocker treatment on plaque stability.
67
311 INFLAMMATION, LEUKOCYTE ACTIVATION, AND SURROGATE
ATHEROSCLEROSIS MARKERS IN RHEUMATOID ARTHRITIS
PATIENTS ARE RELATED TO MITOCHONDRIAL DEPOLARISATION
AND OXIDATIVE STRESS
C. Lopez-Pedrera1 , C. Perez-Sanchez1 , P. Ruiz-Limón1 , M.A. Aguirre1 ,
R.M. Carretero1 , N. Barbarroja1 , A. Rodriguez-Ariza1 , E. Collantes-Estevez2 ,
J.M. Villalba3 , F. Velasco4 , M. Khamashta5 , M.J. Cuadrado5 . 1 Research
Unit, 2 Rheumatology Service, Reina Sofia Hospital/IMIBIC, 3 Cell Biology,
Immunology and Physiololgy, Universidad de Córdoba, 4 Haematology, Reina
Sofia Hospital/IMIBIC, Córdoba, Spain, 5 Lupus Research Unit, St Thomas
Hospital, London, UK
The aim of this study was to assess the mitochondrial integrity of circulating
leukocytes from 20 RA patients, their overall oxidative status, and their
relationship with leukocyte activation, chronic inflammation, hypercoagulable
state, and early atherogenesis.
Increased expression of TF and PAR2 was found in neutrophils from AR
patients, with higher plasma levels of VEGF, tPA, MCP1, MIP1a, TNFa, IL8,
IL17A and IL23. AR monocytes and neutrophils had higher peroxides and
peroxynitrite levels and more depolarised mitochondria, while intracellular GSH
was significantly decreased. Plasmatic TAC and NO were reduced in AR
patients, with increased N-Tyr levels. Both peroxide/peroxinytrite production in
neutrophils and N-Tyr plasma levels showed positive correlations with TF and
PAR2. Peroxide levels further correlated with inflammatory markers. Association
studies showed direct relationship between positivity for both RF and anti-CCP,
and increased expression of pro-thrombotic, pro-inflammatory and neutrophils
oxidative stress markers. Direct association was found between increased
intimate media thickness, and prothrombotic/proinflammatory molecules and
markers of oxidative stress in neutrophils. CD11b on monocytes and CD11B
and CD66B on neutrophils were higher in AR patients, and positively associated
to the presence of RF and anti-CCP. Those leukocyte activation markers
positively correlated with peroxides and negatively with intracellular GSH.
Conclusions:
1. RA autoantibodies (RF and CCP) might contribute to hypercoagulable/proinflammatory state through the induction of mitochondrial depolarisation and TF/PAR2 over-expression in neutrophils.
2. Increased activation of leukocytes in RA would confer them improved
adhesive capacity to endothelium, contributing to higher cardiovascular
risk.
Supported by JA0246/2009, PS09/01809, and Spanish Society of Rheumatology.
312 IN VITRO DIFFERENTIATION OF BONE MARROW-DERIVED
PORCINE MESENCHYMAL STEM CELLS INTO ENDOTHELIAL
CELLS: POTENTIAL ROLE OF CANONICAL WNT SIGNALING
PATHWAY
D.K. Agrawal, D. Pankajakshan, V. Kansal. Center for Clinical & Translational
Science, Creighton University School of Medicine, Omaha, NE, USA
Mesenchymal stem cells (MSCs) hold immense potential for the regeneration of damaged tissues in cardiovascular diseases. In this study,
we investigated the potential of porcine MSCs to differentiate into
endothelial cells (ECs) in vitro. The cultured bone marrow-derived cells
were CD11b− CD34− CD44+ CD45− CD90+ and showed mesodermal lineage
differentiation which are characteristic of MSCs. The MSCs were induced
to differentiate to ECs using endothelial growth media (EGM) and EGM
media containing high concentrations of VEGF (EGM+VEGF; 50ng/ml). The
endothelial basal medium (EBM) without growth factors served as the control.
The EC differentiation was assessed by the presence of vWF, ability to
take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and
qPCR of EC markers vWF, VE-cadherin, PECAM-1, CD34, VEGF-R1 and
VEGF-R2 after 10days of stimulation. The cells cultured in EGM+VEGF
media demonstrated higher amount of DiI-AcLDL positive cells, and enhanced
presence of vWF (90%), VE-Cadherin (60%) and PECAM-1 (48%) positive
cells, compared to EGM. They showed profuse sprouting of capillary tubes
and closed polygon formation in the angiogenesis assay. There was a 1.5−2
fold increase in the mRNA expression for the endothelial markers in the cells
stimulated with EGM+VEGF media when compared to control. RT2 Profiler
PCR array showed upregulation of genes involved in canonical wnt signaling
pathway in cells cultured in EGM+VEGF which is a potential mechanism
involved in EC differentiation. The results demonstrate the ability of porcine
MSCs to differentiate into ECs in in vitro inducing conditions. The differentiated
cells may provide new options for cardiovascular disease therapy and tissue
engineering.
68
313 EFFECTS OF LOW-DOSE TNF-a-ADMINISTRATION ON
OXIDATIVE/NITROSATIVE STRESS, THE AKT/ENOS/NO PATHWAY
AND VIABILITY IN CARDIAC ENDOTHELIAL CELLS
H. Strijdom, A. Genis, M. Mudau, C. Westcott, A. Lochner. Division of Medical
Physiology, University of Stellenbosch, Tygerberg, South Africa
Low-dose TNF-a-administration (5ng/ml) has been shown to be cardioprotective; paradoxically, TNF-a is also associated with progression from endothelial
activation and dysfunction to atherosclerosis.
Aims: Therefore, our study aimed to investigate the effects of low-dose
TNF-a-treatment on various parameters in cardiac microvascular endothelial
cells (CMECs):
i. intracellular NO, ROS and peroxynitrite levels;
ii. Akt/eNOS signalling;
iii. markers of oxidative/nitrosative stress (p22phox ; nitrotyrosine); and
iv. apoptosis/necrosis.
Methods: Adult rat CMECs were treated with 5ng/ml TNF-a for 2h, 6h and
24h. Intracellular ROS and NO production was measured by FACS-analysis of
appropriate sensitive fluorescent probes. Expression/phosporylation of eNOS,
iNOS, PKB/Akt, p22phox , nitrotyrosine, heatshock protein-90 and caveolin-1
were determined by Western blotting and/or proteomic analysis. Necrosis and
apoptosis were determined by propidium iodide and Annexin-V fluorescence.
Results:
i. NO-specific DAF-2/DA fluorescence increased significantly over the 24h
period, whereas a transient increase followed by a significant decrease
was observed in DHR-123 fluorescence. DHE-fluorescence was significantly
reduced after 24h.
ii. Phosphorylated eNOS increased significantly after 24h, associated with
reduced Hsp90 and unchanged caveolin-1 and increased total and
phosphorylated PKB/Akt. No iNOS expression was detected.
iii. Markers of oxidative/nitrosative stress showed inconsistant patterns after
24h ( ↑ p22phox and ↓ nitrotyrosine).
iv. TNF-a-treatment was not associated with increased necrosis/apoptosis.
Conclusions: The study shows that low-dose TNF-a-treatment stimulates modest increases in NO-production likely due to activation of the Akt/eNOS-pathway,
with no apparent partcipation from iNOS. Markers of oxidative/nitrosative stress
showed mixed patterns: ↑ p22phox and ↓ nitrotyrosine expression, associated
with reduced fluorescence of ROS and peroxynitrite probes. No signs of
apoptosis/necrosis were observed.
314 AN ASSOCIATION OF MUTATIONS OF MITOCHONDRIAL DNA
WITH AORTIC ATHEROSCLEROTIC LESIONS
I. Sobenin1,2,3 , M.A. Sazonova2,3,4 , A.Y. Postnov2 , A.N. Orekhov3,4 . 1 Laboratory
of Cellular Mechanisms of Atherogenesis, Institute of General Pathology and
Pathophysiology, 2 Russian Cardiology Research-and-Production Complex,
3
Institute for Atherosclerosis Research, 4 Institute of General Pathology and
Pathophysiology, Moscow, Russia
Background and Aims: Heteroplasmic mtDNA defects are an important cause
of human diseases that primarily involve post-mitotic tissues. The majority
of deleterious mtDNA point mutations are heteroplasmic, and mutant load
can vary significantly among tissues, even in the same subject. Mutations of
human mitochondrial genome can be a possible determinant of atherosclerotic
lesions. To test this hypothesis, mitochondrial mutations observed in various
pathologies were analyzed to determine which mutations can be associated
with atherosclerosis.
Material and Methods: The level of heteroplasmy in fresh autopsy samples
of human aortic intima was determined by pyrosequencing method adopted
for conditions where both mutant and normal allele were present in the same
specimen.
Results: Ten mitochondrial mutations belonging to genes MT-RNR1, MT-TL1,
MT-TL2, MT-ND1, MT-ND2, MT-ND5, MT-ND6, and MT-CYB were associated
with atherosclerosis, since 29−86% of aortic samples had a significant
difference between atherosclerotic plaques and unaffected tissue, with the
respect to the level of heteroplasmy. Further, the homogenates of affected
and normal intimae of 10 aortas were compared to reveal the average level
of heteroplasmy for these mutations. For five mutations (G12315A, G14459A,
C5178A, A1555G, and G14846A), the mean level of heteroplasmy was higher in
atherosclerotic intimal homogenates in comparison with the unaffected tissue.
Conclusion: Thus, at least five mitochondrial mutations are associated with
atherosclerosis. Such mutations may result in defects in the protein chains
of respiratory enzymes and tRNAs, therefore producing oxidative stress and
increasing the probability of plaque formation. Acknowledgement: This study
was supported by Russian Ministry of Education and Science.
315 THE JAK2 INHIBITOR-TYRPHOSTIN AG490 DOWN-REGULATES
NAPDH OXIDASE ACTIVITY AND EXPRESSION IN THE AORTA
OF HYPERCHOLESTEROLEMIC APOE-DEFICIENT MICE
M. Raicu1 , I.M. Fenyo1 , I.C. Florea1 , A. Manea1,2 . 1 Molecular Biology and
Pharmacology, Nicolae Simionescu Institute of Cellular Biology and Pathology,
Bucharest, 2 Petru Poni Institute of Macromolecular Chemistry, Iasi, Romania
Objective: Oxidative stress-induced vascular injury represents a major contributor to the pathoetiology of atherosclerosis. Elevated NADPH oxidase (Nox)
activity promotes oxidative injury of the cardiovascular cells. Janus-tyrosinekinases (Jak) regulate various aspects of the atherosclerotic process. Here, we
investigated the potential of Jak2 inhibition to counteract Nox-dependent O•−
2
formation in atherogenesis in hypercholesterolemic apolipoprotein E-deficient
−/−
(ApoE ) mice.
Methods and Results: Male ApoE−/− mice fed a high-fat, cholesterol-rich diet
were treated for 5 weeks with either vehicle or tyrphostin AG490 (1 mg/kg),
a specific Jak2 inhibitor. Lucigenin-enhanced-chemiluminescence assay, realtime PCR and Western-blot analysis revealed that Nox-derived O•−
2 generation,
Nox1, Nox2, and Nox4 mRNA and protein levels were significantly increased
in the aortas of ApoE−/− mice fed a high-fat diet compared to ApoE−/− mice
fed a normal diet. Treatment with tyrphostin AG490 significantly reduced the
up-regulated Nox activity, the expression of each Nox subtype, as well as the
protein level of CD68, a macrophage-specific marker. Morphometric analysis
showed a marked reduction of atherosclerotic lesions in the aorta of AG490treated animals.
Conclusions: These data provide new insights into the regulation of vascular
Nox by tyrphostins in the cardiovascular system. Pharmacological manipulation
of Jak2-related signaling pathway may represent a novel strategy to reduce
oxidative stress in atherosclerosis.
Work supported by Romanian Ministry of Education, and Research (PNII-TE
65/2010) and by an EFSD New Horizons grant. Adrian Manea acknowledges
the financial support of European Social Fund − “Cristofor I. Simionescu”
Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral
Operational Programme Human Resources Development 2007–2011.
316 RESISTIN UP-REGULATES FRACTALKINE AND CX3CR1
EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS BY
MAPK–NF-úB PATHWAY
A.-M. Gan1 , I. Manduteanu1 , V. Simion1 , D. Stan1 , M.-M. Pirvulescu1 ,
M. Calin1,2 , E. Butoi1 , M. Simionescu1 . 1 Institute of Cellular Biology and
Pathology “N. Simionescu”, 2 Institute of Macromolecular Chemistry “Petru
Poni”, Bucharest, Romania
The expression of the chemokine fracktalkine (Fk) and its receptor, CX3CR1 is
elevated in atherosclerotic lesions in humans, particularly at the level of smooth
muscle cells (SMC). The aim of this study is to determine whether resistin, a
cytokine with an important role in the inflammatory process associated with
atherosclerosis, induces the expression of Fk and CX3CR1 in human SMC and
to investigate the potential signaling pathways involved.
To this purpose cultured human SMC were exposed to 50, 100 and 200 ng/ml
resistin and then the expression of Fk and CX3CR1 was assayed by quantitative
RT-PCR, flow cytometry and Western blot. The signaling mechanisms involved
in the process were investigated by Western blot using specific MAPKs inhibitors
and transient transfection of SMC with siRNA for NF-úB. The evaluation
of the functional role of resistin-induced Fk expression was determined by
monocyte adhesion and chemotaxis assays. The experiments revealed that
resistin induces SMC activation by up regulating the expression of Fk and
CX3CR1 and increases monocyte adhesion and transmigration. Induction of
Fk and CX3CR1 by resistin was reduced by blocking of NF-úB transcription
factor and by using inhibitors of p38, ERK1/2 and JNK MAPKs pathways. The
results indicate that resistin up-regulates Fk and CX3CR1 in human SMC by
mechanisms involving p38, ERK1/2 and JNK MAPKs and the transcription factor
NF-úB and highlight new possible mechanisms involved in the pro-inflammatory
effects of resistin in atherogenesis.
This work was supported by Romanian Academy and the Romanian Ministry
of Education and Research-CNMP Grant No.41037.
317 RELATION OF EPICARDIAL ADIPOSE TISSUE TO CORONARY
ATHEROSCLEROSIS IS REGION-SPECIFIC AND INDEPENDENT
OF CONVENTIONAL RISK FACTORS AND INTRA-ABDOMINAL
ADIPOSITY
T.-D. Wang1 , W.-J. Lee2 , W.-J. Chen1 , M.-F. Chen1 . 1 Internal Medicine
(Cardiology), 2 Medical Imaging, National Taiwan University Hospital, Taipei
City, Taiwan R.O.C.
Objective: To elucidate which measurement of epicardial adipose tissue (EAT)
best reflects its atherogenic risk, we examined the associations between
different EAT measurements and various atherosclerotic parameters of the
entire coronary tree and individual coronary arteries.
Methods: This study included 224 patients underwent multidetector computed
tomography before diagnostic coronary angiography. Regional thickness, cross-
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sectional areas, and total volume of EAT were measured. 4 atherosclerotic
parameters, including severity score, extent score, calcium volume score, and
number of coronary arteries with 50% luminal stenosis, of the entire coronary
tree and individual coronary arteries were assessed.
Results: Both total EAT volume and thickness of EAT in the left atrioventricular
groove were unanimously associated with the presence of coronary
atherosclerosis dichotomously defined by the 4 scoring systems. However,
only EAT thickness in the left atrioventricular groove, but not total EAT volume,
was significantly associated with all 4 parameters of coronary atherosclerosis
in a dose-dependent manner, even after adjustments for conventional risk
factors, body-mass index, waist circumference, C-reactive protein, and intraabdominal visceral fat area. Using the receiver-operating-characteristic analysis,
12.2 mm was the optimal cutoff point for left atrioventricular groove thickness to
predict the presence of significant coronary stenosis. Among the three coronary
arteries, left atrioventricular groove thickness was most strongly correlated with
50% diameter stenosis in the embedded left circumflex artery by multivariate
regression model.
Conclusions: Thickness of EAT in the left atrioventricular groove provides a
more accurate assessment of its atherogenic risk and is therefore a better
coronary risk factor than total EAT volume.
318 PREDICTION OF LDL TRANSPORT AND PLAQUE FORMATION
IN THE CAROTID ARTERY
N. Filipovic1 , N. Meunier2 , M. Rosic1 , I. Tanaskovic1 , D. Fotiadis3 . 1 University
of Kragujevac, Kragujevac, Serbia, 2 University Paris Descartes, Paris, France,
3
University of Ioannina, Ioannina, Greece
Atherosclerosis develops from oxidized low-density lipoprotein molecules (LDL).
Computer modeling can help in prediction of accumulation of LDL as well as
plaque formation and development.
In this study, experimental and computer model of plaque formation and
developing are compared. Experiment setup considered the LDL transport
(uptake) into the blood vessel wall in the isolated rabbit carotid artery under
physiologically relevant constant pressure and perfusion flow with changing the
outflow resistance and blood vessel diameter consequently leading to shear
stress alteration.
Twenty rabbits, treated with a high-fat diet were allocated into early
and late atherosclerosis groups. Intima-media thickness was assessed by
intravascular ultrasound (IVUS). Plaque severity was identified and analyzed
histopathologically. Plaque formation is simulated numerically using a specific
animal data obtained from IVUS and histological data. The three-dimensional
blood flow is governed by the Navier-Stokes equations, together with the
continuity equation. Mass transfer within the blood lumen and through the
arterial wall is coupled with the blood flow and is modeled by the convectiondiffusion equation. LDL transport in lumen of the vessel is described by KedemKatchalsky equations. The inflammatory process is solved using three additional
reaction-diffusion partial differential equations. We found a strong correlation of
the oxidized LDL, concentrations in the intima of macrophages and cytokines
with low shear stress computer simulation. Also intima-media thickness was
fitted with specific procedure in computational model.
319 A ROLE FOR OXIDATIVE STRESS IN CIGARETTE SMOKE-INDUCED
ELEVATION OF OSTEOPONTIN IN CULTURED ENDOTHELIAL
CELLS
E.L. Bishop, I.M. Fearon. BioAssessment, British American Tobacco,
Southampton, UK
Objective: To determine whether the potential inflammatory cardiovascular
biomarker osteopontin can be detected in cultured human vein endothelial cells
(HUVECs) exposed to cigarette smoke total particulate matter (TPM) and to
ascertain the role of oxidative stress in this response.
Methods: Human umbilical vein endothelial cells (HUVECs) were exposed for
24 hours to non-cytotoxic concentrations of cigarette smoke TPM (12−48 mg/ml).
mRNA levels were examined by PCR microarray. Osteopontin and MMP-3
protein levels were measured in media by electrochemiluminescence detection
(Meso Scale Discovery; MSD). Western blots were performed in which proteins
were run on a 12% SDS-page gel and specific proteins detected using an
anti-osteopontin antibody (Abcam). Immunohistochemistry studies were also
performed using this antibody. In experiments with the antioxidant ascorbate
(200 mM), this was added to the HUVECs for 5 hours prior to TPM exposure.
Results: In HUVECs treated with TPM osteopontin gene and protein levels
were elevated in a dose-dependent manner. The increase in osteopontin
expression seen was reduced when cells were pre-treated with ascorbate.
Western blotting for the C-terminal 32 kDa fragment further showed that
osteopontin can be functionally cleaved by MMP-3. MMP-3 was up regulated in
TPM-exposed cells but this effect was insensitive to ascorbate pre-treatment.
Conclusions: HUVEC osteopontin expression is up-regulated in response to
cigarette smoke extract exposure in cultured endothelial cells, an effect which
is oxidative stress-dependent. This response may be regulated by cleavage by
MMP-3, an effect which may occur independently of oxidative stress.
69
320 PLEIOTROPIC EFFECTS OF NIACIN THERAPY IN ADDITION TO
ATORVASTATIN IN CORONARY HEART DISEASE PATIENTS WITH
ELEVATED LIPOPROTEIN(A) LEVELS
M. Safarova, E. Trukhacheva, M. Ezhov, O. Afanasieva, M. Tripoten,
S. Pokrovsky. Russian Cardiology Research Center, Moscow, Russia
Objective: To evaluate the effects of niacin and atorvastatin combination on
risk factors of atherosclerosis in men with CHD and high lipoprotein(a) [Lp(a)]
levels.
Methods: Sixty men (54±6 years) with angiographic evidence of CHD and
Lp(a) 30 mg/dl (mean 80±33 mg/dl) were randomized into two groups: active
group (n = 30) received niacin 1500 mg plus atorvastatin, control group (n = 30) −
atorvastatin alone. Blood samples were collected for lipid profile, hsCRP, LpPLA2, PAI-1, tPA/PAI-1 complex. Carotid intima-media thickness (CIMT) and
pulse-wave velocity (PWV) were measured at baseline and after 6 months by
investigators blinded to randomization.
Results: Treatment groups were comparable at baseline. Median percent
change from baseline decreased for Lp(a) levels by 23%, LDL-C by 25%,
triglycerides by 20%, tPA/PAI-1 by 25% and increased for HDL-C by 16% in
active group (p < 0.01). Combination treatment produced statistically significant
reduction in the mean CIMT (right: 0.83±0.17 vs 0.77±0.18 mm, p = 0.02;
left: 0.86±0.21 vs 0.79±0.16, p = 0.02). Atorvastatin treatment showed no
progression of carotid atherosclerosis (right: 0.83±0.19 vs 0.80±0.17 mm,
p = 0.5; left: 0.86±0.31 vs 0.84±0.20, p = 0.6). The Lp-PLA2 levels decreased
up to 32% regardless the treatment group. Concentration of hsCRP and PWV
remained unchanged during the study in both groups. In active group significant
correlation was observed between the changes in CIMT and Lp(a), and TG
levels (r = 0.42, p = 0.049; r = 0.37, p = 0.02, respectively).
Conclusion: In CHD patients with Lp(a) excess niacin in addition to atorvastatin
results in regression of carotid atherosclerotic disease in 6 months, mainly due
to the reduction in Lp(a) levels.
321 ENHANCED EXPRESSION OF SIRT1 IN ATHEROSCLEROTIC
PLAQUES IS INVOLVED IN THE PATHOGENESIS OF ACUTE
CORONARY SYNDROMES
K. Hatakeyama1 , Y. Sato2 , T. Tsuruda3 , Y. Sekita3 , K. Nishihira4 , Y. Shibata4 ,
Y. Asada1 . 1 Department of Pathology, Faculty of Medicine, University of
Miyazaki, 2 University of Miyazaki, 3 Department of Medicine, Faculty of
Medicine, University of Miyazaki, 4 Miyazaki Medical Association Hospital,
Miyazaki, Japan
Objective: To examine the expression and localization of SIRT1 in human
atherosclerotic plaques and coronary thrombi obtained from patients of acute
myocardial infarction (AMI).
Background: Chronic inflammation plays an important role in the pathogenesis
of acute coronary syndromes. SIRT1, which belongs to the class III
histone deacetylases (HDACs), has been reported to regulate diverse
biological processes, including cell survival, apoptosis, gluconeogenesis, stress
resistance and inflammatory response. Therefore, SIRT1 in the arterial wall
may play a role for the progression of atherosclerotic lesions. However, SIRT1
expression in atherosclerotic plaques have not been reported.
Methods and Results: Coronary arteries and abdominal aortas were obtained
from 10 autopsy cases. Coronary thrombi were obtained from 10 AMI
patients who had undergone thrombus removal using aspiration catheter.
Localization of SIRT1 was examined by immunohistochemistry using antibodies
for SIRT1, macrophages, smooth muscle cells (SMCs) and endothelial cells.
SIRT1 immunoreactivity was detected in advanced atherosclerotic plaques and
coronary thrombi, especially in macrophages and SMCs, but undetectable in the
diffuse intimal thickening/adaptive lesions. In coronary thrombi, SIRT1-positive
macrophages are found in the disrupted plaque. SIRT1 expression assessed by
Western blotting was markedly enhanced in advanced atherosclerotic plaques,
compared with early lesions.
Conclusions: These results suggest that SIRT1 is involved in the pathogenesis
of acute coronary syndromes.
322 EXPOSURE OF HUMAN MACROPHAGES TO TOBACCO
SMOKE INDUCES MMP14 EXPRESSION AND GENERATION OF
PROTEOLYTICALLY ACTIVE MICROVESICLES
C. Li1 , Y. Liu1 , D. Yu2 , K.J. Williams1 , M.-L. Liu1 . 1 Endocrinology/Medicine,
Temple University School of Medicine, Philadelphia, PA, USA, 2 Tianjin Medical
University, Tianjin, China
Cigarette smoking increases the risk of atherosclerosis, plaque rupture,
and intravascular thrombosis. The underlying mechanisms, however, are
not well understood. We recently reported that tobacco smoke induces
human monocyte/macrophages to generate tissue factor-positive, procoagulant
microvesicles (MVs, or microparticles) (Li M et al. ATVB 2010;30:1818). We now
sought to investigate whether smoke-induced MVs could contribute to plaque
instability. Our focus was the membrane type 1-matrix metalloproteinase (MT1MMP), also known as MMP14. This molecule appears on MVs shed by invasive
tumor cells, degrades extracellular matrix, and activates MMP2.
70
We prepared stock tobacco smoke extract (TSE) by bubbling smoke from
5 research cigarettes through 10ml RPMI/BSA. Exposure of differentiated
human THP-1 macrophages to 2.5−5.0% TSE significantly increased cellular
MMP14 detected by immunoblots. Next, the MVs were isolated from conditioned
medium of human THP-1 macrophages after 20h treatment with 0 (control) or
5% TSE. Immunoblots revealed abundant MMP14 on the TSE-induced MVs.
Moreover, TSE-induced MVs were proteolytically active, shown by cleavage
of a fluorogenic peptide (substrate 1, R&D Systems; control MVs: 100±2.3,
TSE-MVs: 254.6±24 fluorescence units, mean±SEM, n = 4, p < 0.05) and
gelatinolytic activity on zymography at the molecular weight of MMP14. We and
others reported MAPKs activation after TSE exposure. Here, pre-treatment of
macrophages with the JNK inhibitor SP600125 (10 mM) significantly attenuated
TSE-induced MMP14 expression as well as the proteolytic activity of the
released MVs.
Overall, proteolytically active MVs induced by tobacco smoke may be novel
mediators of matrix destruction in a variety of locations, including the fibrous
cap of atheromata.
323 OVEREXPRESSION OF ABCA1 IN HUMAN PLAQUES EXPOSED TO
HYPERCHOLESTEROLEMIA: ROLE OF MICRORNA MODULATION
C. Mandolini1 , D. Santovito1 , F. Buttitta2 , V. De Nardis1 , A. Marchetti2 ,
L. Felicioni2 , M. Bucci1 , S. Ucchino3 , A. Mezzetti1 , F. Cipollone1 . 1 Center of
Excellence on Atherosclerosis, Hypertension and Dyslipidemia, 2 Oncological
Molecular Medicine, 3 Division of Vascular Surgery, ‘G. d’Annunzio’ University −
Chieti-Pescara, Chieti, Italy
Background: ABCA1 is expressed in cholesterol-loaded macrophages and
is modulated by intracellular cholesterol levels. Futhermore it cooperates with
ABCG1 and LXR-alpha in reverse cholesterol transport. However, mechanisms
linking dyslipidaemias and ABCA1 expression is still largely unknown.
MicroRNAs represent an emerging class of gene regulators implicated in
cardiovascular diseases. By using bioinformatical prediction tools, we identified
some microRNAs that may regulate ABCA1 expression (miR-145, miR-33,
miR-384, miR-27). Thus, the study aim was to investigate the expression
of ABCA1, ABCG1, LXR-alpha, and microRNAs targeting ABCA1-pathway in
atherosclerotic plaques of hypercholesterolemic patients.
Material and Methods: Atherosclerotic plaques obtained from 32 patients
undergoing carotid endarterectomy were subdivided into hypercholesterolemic
(n = 13) and control (n = 19) groups according to presence or absence of
hypercholesterolemia (as defined by LDL-C >130 mg/dL). ABCA1, ABCG1,
LXR-alpha mRNAs as well as regulating miRNAs were analyzed by realtime PCR, protein levels by Western blot, and ABCA1 localization by
immunohistochemistry.
Results: ABCA1 mRNA was more expressed in hypercholesterolemic than
in control plaques (13.93±3.76 vs 46.78±6.43 fold induction, p = 0.002).
Furthermore, a direct correlation was found between plasma LDL-cholesterol
levels and plaque ABCA1 mRNA expression (R: 0.412; p = 0.019). This
difference was not observed at the protein level, thus suggesting a posttranscriptional regulation. Finally, a trend supporting an inverse correlation
between ABCA1 mRNA and miRNA145 was observed (R: −0.137).
Conclusions: To the best of our knowledge, this is the first demonstration that
ABCA1 mRNA is overexpressed in plaques of hypercholesterolemic patients.
Immunohistochemistry and proteins’ preliminary data suggest a strong posttranscriptional modulation, possibly due to microRNAs.
324 CHOLESTEROL CONTENT OF ERYTHROCYTE MEMBRANES IS
ASSOCIATED WITH LIPID CORE SIZE IN ANIMAL MODEL OF
ATHEROSCLEROSIS
D. Tziakas1 , A. Kapelouzou2 , G. Chalikias1 , D. Stakos1 , S. Apostolakis1 ,
A. Agapaki2 , A. Kostakis2 , H. Boudoulas2 , S. Konstantinides1 . 1 Democritus
University of Thrace, Alexandroúpolis, 2 Biomedical Research Foundation
Academy of Athens, Athens, Greece
Introduction: Total cholesterol content of erythrocyte membranes (CEM) has
emerged as a novel marker of clinical instability in Coronary Artery Disease
(CAD). CEM may contribute to the growth of the lipid core of atherosclerotic
plaques and thus to plaque instability. We assessed the association between
CEM levels and the size of lipid core in aortic atherosclerotic lesions in an
established animal model of atherosclerosis.
Methods: Eight white New Zealand rabbits placed on a diet containing
0.75% cholesterol for 5 months comprised the study group, whereas 6
rabbits on normal diet formed the control group. At the end of the
study period, atherosclerotic plaque area and lipid core size (%) were
analyzed histomorphometrically in segments of the thoracic aorta. Lesions
measurements were determined on transverse sections taken from two sites
along the length of thoracic aorta (extending from just above the aortic valve to
the level between the third and fourth intercostals arteries).
Results: After 5 months on cholesterol-rich diet, CEM levels were significantly
higher (p = 0.005) in the study (median 89.5 mg/mg, interquartitile range (IQR)
63–101.3) compared to control group (29.5 mg/mg, IQR 26.3–40.3). CEM levels
at 5 months were associated with lipid core size (r = 0.714, p = 0.047) whereas
no association was found with atherosclerotic plaque area (r = 0.214, p = 0.610).
Cholesterol blood levels were neither associated with lipid core size (r = 0.286,
p = 0.493) nor with atherosclerotic plaque area (r = 0.429, p = 0.289).
Conclusions: These findings suggest that cholesterol load of erythrocyte
membranes is associated with lipid core size in cholesterol-fed animal models
of atherosclerosis.
325 THERAPEUTIC REGRESSION OF ATHEROSCLEROSIS
A. Ali, E. Grinevich, A. Mansharipova. Cardiology, Scientific Research Institute
of Cardiology and Internal Diseases, Almaty, Kazakhstan
Aim: NO replacement therapy by NO donors could restore NO deficits. HDLs
are carriers of excessive cellular cholesterol by reverse cholesterol transport
(RCT) pathway. Plasma lipoproteins enrichment by anionic phosphatidylinositol
(PI) increases the negative surface potential and stimulates RCT (by
ABC1receptors). PI stimulates rapid flux and clearance of plasma cholesterol by
increased hepatic uptake (by SR-B1receptors). Regression of atherosclerosis,
stimulating RCT pathway was the aim.
Methods: 30 male Chinchilla rabbits, fed with 2% cholesterol diet for 6 months.
After 6 months, were divided into 3 groups with continued feeding. Group 1:
without treatment. Group 2: treated with isosorbide dinitrate. Group 3: treated
with PI encapsulated ISDN (nanobiotechnology). Control: 5 animals at regular
diet. Animals were sacrificed after 10&20 days of treatment. (5 animals/group).
Abdominal aortas were fixed for Oil Red O (ORO) staining, complex morphology
and electron microscopy.
Results: ORO: Lipid infiltration in group 1 (38.2±3.6%), 27.2 times > intact
animals 1.4±0.2%, p < 0.001. Group 2 (24.3±3.6%), 1.6 times < group 1,
p < 0.001. Group 3 (2.3±0.6%), 16.6 times < group 1, p < 0.001. Microscopy
(×100–×27000): Group 3: express reduction of lipid vacuoles and foam cells,
elastic fibers restoration, endothelium regeneration.
Conclusion: Obtained data provides evidence of endothelial cells regeneration,
restoration of elastic fibres (probably due to regulation of fibulin 5 by PI3K/Akt),
clearance of foam cells and regression of atherosclerosis by PI+ISDN. PI+ISDN
provide antiatherogenic properties of NO donor and PI, probably due to
reduction of cytokines production, decrease in adhesion molecules expression,
inhibition of monocyte-endothelium adhesion.
326 IMPACT OF LOWER LIMB REVASCULARIZATION ON AORTIC
AUGMENTATION INDEX AND SUBENDOCARDIAL VIABILITY RATIO
IN PATIENTS WITH PERIPHERAL ARTERIAL DISEASE
V. Jacomella, A. Shenoy, K. Mosimann, M. Kohler, C. Thalhammer,
B. Amann-Vesti, M. Husmann. Clinic for Angiology, University Hospital Zürich,
Zürich, Switzerland
Background: Aortic augmentation index (AIx) and subendocardial viability ratio
(SEVR) are surrogate markers for arterial stiffness and cardiovascular morbidity
and mortality. Peripheral arterial disease (PAD) is associated with an increased
cardiovascular risk. In patients with claudication the effect of balloon angioplasty
(PTA) on AIx and SEVR has not been determined so far.
Patients and Methods: In a non-randomized, prospective, controlled, singlecentre study AIx and SEVR were assessed in 109 patients with PAD Rutherford
stage I-III. Measurements, including ankle brachial pressure index (ABI) were
performed before and three months after PTA and compared to age- and sexmatched PAD patients under best medical treatment.
Results: The effect of PTA on AIx, SEVR and ABI was assessed in 61 patients
(44% female, mean age 68 years) undergoing lower limb PTA and compared
to 48 conservatively treated patients (38% female, mean age 68 years). ABI
significantly improved after PTA (from 0.73±0.02 to 0.85±0.03; p = 0.001)
but remained unchanged in the control group (0.85±0.23 to 0.80±0.21;
p = 0.16). Revascularization was associated with a significant reduction of AIx
(31.5±1.1% to 28.8±1.1%; p = 0.01) and a significant improvement of SEVR
(132.2±2.9% to 141.4±3.7%, p = 0.01). There were no changes observed from
baseline to follow-up in the conservatively treated group.
Conclusion: Revascularization in patients with Rutherford stage I-III is
associated with an improvement of markers for arterial stiffness and endocardial
perfusion.
327 PHARMACOLOGICAL INHIBITION OF JAK/STAT SIGNALING
PREVENTS HIGH-GLUCOSE-INDUCED UP-REGULATION OF
ENDOTHELIN-1 EXPRESSION IN HUMAN ENDOTHELIAL CELLS
S. Manea1 , A. Manea1,2 , C. Heltianu1 . 1 Institute of Cellular Biology and
Pathology ‘Nicolae Simionescu’, Bucharest, 2 ‘Petru Poni’ Institute of
Macromolecular Chemistry, Iasi, Romania
Objective: Accelerated atherosclerosis represents a major complication
of diabetes mellitus. Emerging evidence demonstrates the involvement
of endothelin-1 (ET-1) in plaque formation. The molecular mechanisms
accountable for the increased production of ET-1 are not entirely deciphered.
Our aim has been to investigate the role of Jak/STAT signaling, an essential
79th EAS Congress
pathogenic mechanism leading to endothelial cell dysfunction, in the regulation
of ET-1 synthesis in human endothelial cells (EAhy926 cells line).
Methods and Results: EAhy926 cells were exposed to normal (5 mM) or high
(25 mM) glucose concentrations in the presence/absence of Jak/STAT inhibitors.
Using real-time PCR, ELISA, and gene reporter assay, we found that Jak/STAT
inhibitors significantly diminished the high-glucose-dependent up-regulation of
ET-1 mRNA, peptide synthesis, and promoter activity. In silico analysis of the
human ET-1 promoter revealed the presence of typical STAT1/GAS elements.
Transient overexpression of STAT1 indicated an up-regulation of ET-1 promoter
activity. Chromatin immunoprecipitation demonstrated the physical interaction
of STAT1 proteins with the predicted GAS sites.
Conclusions: Regulation of ET-1 synthesis by the Jak/STAT pathway thus
represents a novel mechanism by which high glucose induces endothelial cell
dysfunction in diabetes. In this context, the modulation of this system and the
subsequent decrease in ET-1 level may represent a key pharmacological target
in diabetes-associated cardiovascular disorders.
Work supported by Romanian Ministry of Education, and Research (PNII-TE
65/2010) and by an EFSD New Horizons grant. Adrian Manea acknowledges
328 METOPROLOL REDUCES PRO-INFLAMMATORY CYTOKINES AND
ATHEROSCLEROSIS IN APOE−/− MICE
M.E. Johansson1 , E. Bernberg2 , M.A. Ulleryd1 , G. Bergström2 . 1 Institute
of Neuroscience and Physiology, Unversity of Gothenburg, 2 Wallenberg
Laboratory, University of Gothenburg, Gothenburg, Sweden
Introduction: A few studies in animals and humans suggest that metoprolol
(b1-selective adrenoceptor antagonist) may have a direct anti-atherosclerotic
effect. Surprisingly, no studies have yet been performed in atherosclerosis prone
ApoE−/− mice. The aim of the present study was thus to evaluate the effect
of metoprolol on development of atherosclerosis in ApoE−/− mice, and also to
investigate its effect on pro-inflammatory cytokines.
Methods: In a dose-finding study we selected metoprolol at a dose of 2.5 mg/kg
per hour since it reduced heart rate slightly but left the circadian rhythm and
stress induced increase in heart rate intact. Male ApoE−/− mice were treated
with metoprolol or saline for 11 weeks via osmotic minipumps. Atherosclerosis
was assessed en face in thoracic aorta. Total cholesterol levels and a panel of
Th1/Th2 cytokines were analyzed in serum samples.
Results: Metoprolol significantly reduced atherosclerotic plaque area in thoracic
aorta (p < 0.05 vs. Control) and also reduced serum levels of tumor necrosis
factor-a (TNF-a) and the chemokine CXCL1 (p < 0.01 vs. Control for both). Total
cholesterol levels were not affected by metoprolol treatment.
Conclusions: In this study we found that a moderate dose of metoprolol
significantly reduced atherosclerotic plaque area in thoracic aorta of ApoE−/−
mice. As a possible mechanism we also found that metoprolol lowered serum
levels of pro-inflammatory cytokines TNF-a and CXCL1, which both have known
pro-atherogenic effects.
329 THE LEGACY OF METABOLIC MEMORY AND THE ACTIVATION
OF PRO-INFLAMMATORY GENES ARE ASSOCIATED WITH
EPIGENETIC PERSISTENCE
A. El-Osta. BakerIDI Heart and Diabetes Institute, Melbourne, VIC, Australia
Diabetes and accelerated vascular complications is a major clinical problem.
Of particular interest is the phenomenon of metabolic memory where diabetic
patients, despite improved glycemic control, develop complications as a result of
prior poor glycemic control. In recent clinical evaluates from the large Diabetes
Control and Complications Trial (DCCT), and its follow-up, the prospective
Epidemiology of Diabetes Interventions and Complications (EDIC) trial, diabetic
subjects have continued to develop ongoing end-organ injury, as a result
of previous periods of poor glycemic control. This has been interpreted as
indicating that there is a memory of past glycemia known as metabolic
memory, which is a major determinant of subsequent development of vascular
complications. It remains unknown as to the molecular mechanism whereby
hyperglycemic memory leads to a program of diabetic vascular complications.
We have postulated that epigenetic pathways participate in this phenomenon.
Furthermore, since mitochondrial ROS generation has been demonstrated to
play a central role in diabetic vascular complications, there role in mediating
these epigenetic modifications was also examined. We show that specified
histone methylases and demethylases are central determinants in the methylwriting and methyl-erasing events that cause persistent epigenetic changes that
are functionally associated with gene regulation. The distinct changes we have
identified in the histone code defines a new component of epigenetic control,
which could explain why transient elevations of glucose often lead to progressive
diabetic vascular complications. We also discuss the fate the epigenome may
have on metabolic syndrome and future vasculature memories that often end
in organ injury.
71
330 EARLY CHANGES IN VASCULAR SMOOTH MUSCLE CELLS GENE
EXPRESSION PROFILE IN RESPONSE TO VASCULAR INJURY
A. Fedorov1,2 , A. Razuvaev3 , A. Kostareva1 , U. Hedin3 , J. Raud4 , J. Roy3 .
1
Inst of Molecular Biology and Genetics, Almazov Ctr, Saint Petersburg,
Russia, 2 Ariadne Genomics, Inc., Rockville, MD, USA, 3 Karolinska Instt,
4
Cardoz AB, Stockholm, Sweden
Introduction: Early genome wide molecular events happened in vascular
smooth muscle cells (vSMC) in response to vascular injury regulate vSMC
phenotype, which in turn determine severity of later vascular reactions during
atherosclerosis or restenosis progression. In the present work, we investigated
the early alterations in vSMC gene expression profile in response to balloon
injury in the rat carotid artery model.
Results: Total RNA was isolated from intact rat carotid arteries and
5, 10 and 20 hours after balloon injury. Gene expression profiling was
performed using Affymetrix microarrays. Hierarchical clustering revealed several
temporal expression patterns of vSMC genes. Subnetwork enrichment analysis
using Pathway Studio program (www.ariadnegenomics.com) demonstrated that
differentially expressed genes involved in regulation of cell processes specific
for vascular cells as well as inflammation and ossification. Q-PCR confirmed
that vSMC significantly upregulate expression of inflammatory cytokines (IL-6,
Mcp1, Cxcl1, Aimp1) at the early phase (5hours) of response to injury. IHC
showed that within the vessel wall, inflammatory proteins CD44 and Mcp1
are expressed by vSMC and the expression increased in response to injury.
Simultaneously intracellular localization of key repressor of vSMC contractile
phenotype myocardin changed from nucleus to cytoplasmic.
Conclusion: Early changes in vSMC gene expression profile in response to
injury are characterized by upregulation of genes involved in inflammatory
response which could control of neointima formation by regulation vSMC
behavior and immune cells recruitment.
Acknowledgements: The work was supported by grants from Swedish research
Council and Karolinska Institutet, Swedish Heart and Lung foundation, MES of
Russia (project P1308).
331 KYNURENINE PATHWAY − A NEW LINK BETWEEN ENDOTHELIAL
DYSFUNCTION AND CAROTID ATHEROSCLEROSIS IN CHRONIC
KIDNEY DISEASE PATIENTS
K. Pawlak1 , M. Mysliwiec2 , D. Pawlak1 . 1 Monitored Pharmacotherapy,
2
Nephrology and Clinical Transplantation, Medical University, Bialystok,
Poland
Objective: The endothelium dysfunction is an important component of
atherosclertic cardiovascular disease. It has been also suggested that
kynurenine pathway activation may be involved in the pathogenesis of this
disease.
Methods: This is a cross-sectional study in chronic kidney disease (CKD)
patients (n = 106; 60 Males). The plasma markers of endothelial dysfunction and
kynurenine (KYN), kynurenic acid (KYNA), anthranilic acid (AA) and quinolinic
acid (QA) were measured in relation to an early indicator of the systemic
atherosclerosis − intima-media thickness (IMT).
Results: Kynurenines, von Willebrand factor (vWF), thrombomodulin (TM),
soluble adhesion molecules (sICAM-1, sVCAM-1) and IMT in each uraemic
group were significantly higher than in healthy people. In contrast, no significant
differences in sE-selectin and sP-selectin concentrations were observed
between CKD patients and controls. Kynurenines were positively associated
with vWF, TM, sICAM-1 and sVCAM-1, whereas sP-selectin was inversely
associated with the most of kynurenines. IMT was positively correlated both
with KYN, QA as well as with endothelial markers: TM, vWF, sICAM-1 and
sVCAM-1 (all p < 0.01). Finally, multiple regression analysis identified age, vWF,
sVCAM-1 and QA levels as the independent variables significantly associated
with increased IMT in this population (adjusted r2 = 0.51).
Conclusions: This study suggests a relationship between kynurenine pathway
activation, endothelial dysfunction and the progression of atherosclerosis in
CKD patients. It opens a new idea that the inhibition of kynurenine pathway
may provide an effective strategy to slow down endothelial dysfunction and
thereby the prevalence of atherosclerosis in this population.
332 EXTRACORONARY ATHEROSCLEROSIS: RELATIONSHIP WITH
VASCULAR ALTERATIONS AND LIPOPROTEIN (A)
R. Toro1 , P. Gomez1 , M. Virseda2 , I. Tinoco2 , C. Rodriguez Leal2 , A. Mangas1 .
1
Medicine, 2 Cadiz University, Cadiz, Spain
Introduction: Several studies have shown that a high serum level of
lipoprotein (a), Lp(a), may be an independent risk factor for atherosclerosis
disease; Little is known about the relation of serum of Lp(a) to extracoronary
disease. Futhermore, atherosclerotic risk factors leads to vascular remodelling,
creating arterial stiffnes within aorta and other large arteries
Aim: To investigate the relationship between early extracoronary atherosclerosis, vascular alterations and serum lipid profile.
Material and Methods: This study was conducted on 107 patients with
ecographic plaques, 60 carotid (Group A) and and 47 femoral (Group B)
72
plaques respectively, in addition to 15 healthy controls. All of them were
subjected to complete examination, and measurement of total cholesterol
(CT);TG; LDL-c; HDL-c; Lp(a). Radial applanation tonometry (AT) and intimamedia thickness (IMT) and plaque formation were assessed in both carotid and
femoral arteries.
Results: The serum Lp(a) concentration was significantly higher in subjects of
group A and B than in control group (p < 0.01) but was not significantly different
among group with carotids and femoral plaques. Mean values of serum CT,
TG, and LDL-c revealed significant elevation in both group compared to controls
(p < 0.005). PWV was significantly higher in both groups than in healthy controls
(p < 0.002).
Table 1.
Age
Hypertension
Diabetes mellitus
CT (mg/dl)
TG (mg/dl)
LDL-c (mg/dl)
HDL-c (mg/dl)
Lp(a)
Group A
Group B
Control group
56 (38−71)
58 (83.3%)
32 (53.3%)
208±52.2
188±77.3
144.9±49.2
41.6±8.4
45±45
54 (41−69)
30 (63.8%)
21 (44.7%)
202±50.3
162±66.1
136±47.9
41.4±8.8
44±43
54 (37−65)
11 (73.3%)
6 (40%)
163.5±43
158±60
83.3±31
45±12
20±27
Conclusion: Serum Lp(a) level has a clear influence on extracoronary
atherosclerosis. The patients with atherosclerosis extracoronary have increased
of PWV
Such alterations are independent of the presence of the alterations of lipids
included Lp(a) and the plaque ubication.
333 SUPPRESSOR OF CYTOKINE SIGNALING-3 AND INTIMAL
HYPERPLASIA IN PORCINE CORONARY ARTERIES FOLLOWING
CORONARY INTERVENTION
D.K. Agrawal, G.K. Gupta, M.G. DelCore, G.I. Hatzoudis. Center for Clinical &
Translational Science, Creighton University School of Medicine, Omaha, NE,
USA
Purpose: Inflammation may initiate, progress, and destabilize atherosclerotic
lesions. However, the role of inflammation in neointimal hyperplasia and
restenosis after mechanical injury to arteries during coronary intervention
(balloon angioplasty/stenting) is not well established. The growth and
differentiation of cells is regulated by cytokines. Suppressor of cytokine signaling
(SOCS)-3 is a negative regulator of cytokines. The purpose of this study was to
examine the expression of SOCS-3 in proliferating smooth muscle cells (SMCs)
of neointimal thickenings after coronary artery intervention in a swine model.
Methods: Six female Yucatan miniswine were fed special high cholesterol diet
for 8 months. At 4 months of high cholesterol diet, 4 miniswine underwent
coronary intervention (angioplasty or stenting) and 2 animals were kept as
control. At the end of 8 months animals were euthanized, coronary arteries
were isolated and morphological and histological studies were performed.
Results: Results from morphological studies, including H&E and elastic van
giesson stain, show typical lesions with significant neointimal proliferation.
Histological evaluation showed expression of SMa-actin and significantly
increased proliferating cell nuclear antigen in neointimal lesion. There was
significantly decreased expression of SOCS-3 in SMCs of neointimal region
than control.
Conclusion: These data suggest that expression of SOCS-3 is decreased in
proliferating SMCs of neointimal lesions. This leads to uncontrolled growth of
vascular SMCs in injured arteries leading to restenosis. Therefore, local delivery
of SOCS-3 gene at the site of injury after coronary artery intervention could
regulate the proliferation of vascular SMCs and help in preventing the neointimal
formation and restenosis.
334 EFFECTS OF CIGARETTE SMOKE AND ITS INTERACTION
WITH LDL IN A 3-DIMENSIONAL COCULTURE MODEL OF PLAQUE
DEVELOPMENT
Y. Steffen1 , D. Weiss1 , S. Lebrun2 , K. Stolle1 , M. Lietz1 , J. Schueller1 ,
T. Wallerath1 . 1 PHILIP MORRIS Research Laboratories GmbH, Cologne,
Germany, 2 PHILIP MORRIS International, Neuchatel, Switzerland
Cigarette smoke (CS) is an acknowledged risk factor for cardiovascular events.
However, the mechanisms by which inhaled or second hand CS drive plaque
progression in coronary or peripheral vessels are still largely unknown. In the
present study, physiologically relevant co-cultures of vascular and monocytic
cells have been used to identify potential mechanisms by which extracts of
CS (CSE) can lead to key steps (proliferation, adhesion, transmigration, and
foam cell formation) of atherogenesis. To further advance the relevance of our
3-D model, the interaction of CSE with another known risk factor (LDL) was
investigated. CSE was shown to have an effect on the activity of the prooxidant
and proinflammatory enzyme myeloperoxidase (MPO). Through generation of
its oxidation product, nitrogen dioxide, MPO has the ability to oxidize LDL
resulting in cholesterol deposition and, finally, foam cell formation, an important
step in atherosclerotic plaque initiation and progression. We propose that
the enhanced proliferation of smooth muscle cells as well as accelerated
adhesion, transmigration and foam cell formation of monocytic cells in the 3-D
co-culture model are caused by the interaction of CSE with LDL. The same
pro-atherogenic effects were also observed with oxLDL, indicating that the proatherogenic features of CSE are mainly dependent on the interaction with LDL.
Our findings are in line with the in vivo situation, where evidence suggests that
oxLDL and MPO-activity is increased in smokers. In summary, a growing body
of evidence suggests that CSE may promote atherosclerosis through oxidative
modification of LDL.
335 THE APOE−/− MOUSE AS A CIGARETTE SMOKE-RESPONSIVE
ATHEROSCLEROSIS MODEL SHOWING REVERSIBILITY UPON
SMOKING CESSATION
K. Stolle1 , M. Lietz1 , Y. Steffen1 , H. Weiler1 , M. Möhring1 , A. Berges2 ,
S. Lebrun3 . 1 Philip Morris International R&D, Philip Morris Research
Laboratories GmbH, Cologne, Germany, 2 Philip Morris International R&D,
Philip Morris Research Laboratories, bvba, Leuven, Belgium, 3 Philip Morris
International R&D, Philip Morris Products S. A., Neuchâtel, Switzerland
ApoE−/− mice develop a pronounced hyperlipidemia and also form plaques that
show some similarities to those that are seen in man. We have previously shown
that mainstream smoke (MS) exposure accelerates atherosclerotic plaque
formation in male ApoE−/− mice. In this study, female ApoE−/− mice showed
enhanced atherosclerosis after 6 months of exposure to MS when compared
with exposure to fresh air (sham). When exposure to MS was discontinued
(cessation) after 3 months of MS exposure this appeared to slow down plaque
development. This finding is in line with the observation that the lipid profile was
changed to a more pro-atherogenic profile in the MS-group, whereas the lipid
profile in the cessation group was comparable to sham.
Determination of arachidonic pathway metabolites and inflammatory cells in the
plaque suggest that oxidative stress and inflammation may play an important
role in the progression (and regression) of MS-dependent atherosclerosis
development. The reversibility of a number of endpoints after cessation supports
the use of the ApoE−/− mouse as a cigarette smoke-responsive atherosclerosis
model to study mechanisms of cardiovascular disease development and
regression.
336 DOUBLE NEGATIVE T CELLS IN ANGIOTENSIN II DEPENDENT
HYPERTENSION
T. Mikołajczyk1 , R. Nosalski1 , A. Sagan1 , D. Skiba1 , D. Ludew1 , P. Matusik1 ,
R. Korbut2 , T. Guzik1 . 1 Department of Internal and Agricultural Medicine,
2
Department of Pharmacology, Jagiellonian University School of Medicine,
Cracow, Poland
Hypertension is associated with T cell activation. In particular double negative
T cells (CD4-CD8-CD3+; DN) are increased. They comprise 1−3% of
particularly pro-inflammatory T cell subset in peripheral blood. Their tissue
distribution is unknown. RANTES chemokine has been implicated as possible
mechanism for T cell recruitment in hypertension. Accordingly we cytometrically
studied the characteristics of T cells infiltrating perivascular space, particularly
adipose tissue (AT) with emphasis on the mechanisms of DN T cell infiltration
in a model of chronic (14 day) angiotensin II minipump infusion.
Results: Percentage of total leukocytes infiltrating perivascular adipose tissue
was higher in AngII-mice than in controls (13±4% vs. 4.5±1.5% p < 0.01). Total
number of aortic CD3+ cells increased in hypertension (1055±565 vs. 419±207
cells/mg; p < 0.01) with particular increase in aortic DN T cells (227±52
vs. 96±40 cells/mg; p < 0.01). Expression of chemokine receptor CCR5 on
circulating and aortic T cells was increased after 14 days of angiotensin II
infusion. And was the highest on aortic DN T cells. The mean intensity of
fluorescence of CCR5 was ~10-fold higher on aortic T cells in comparison to
circulating T cells. Chemotaxis assays confirmed that RANTES leads to T cell,
but not B cell migration and that Ang II infusion increases chemotactic potential
by two fold. DN T cells migrated most efficiently towards RANTES.
Conclusion: In hypertension, the double negative cells are distinct subset of
T lymphocytes with the highest migratory properties towards RANTES due to
their expression of CCR5.
79th EAS Congress
337 PERIPHERAL ARTERIAL DISEASE IN METABOLIC SYNDROME
(METS): PREDOMINANT LOCALIZATION OF ATHEROSCLEROSIS,
HORMONE ABNORMALITY AND CARDIOVASCULAR
COMORBIDITY
V. Andreev, V. Zelinsky. Vascular Surgery, Saint-Petersburg State Mechnikov
Medical Academy, Saint Petersburg, Russia
Objective: The aim of our study is to assess prevalence of MetS in patients
with peripheral arterial disease (PAD) and its correlation with localization of
atherosclerosis, androgen profile and cardiovascular comorbidity.
Methods: The presence of MetS was assessed in 98 PAD male patients. Mets
was defined by the criteria of IDF (2005). All patients had undergone peripheral
angiography. Level of total testosterone 11 nmol/L was considered normal.
Results: The prevalence of the metabolic syndrome in the study population
was 59.2%. The mean age of patients with MetS was 61.4±5.2 (mean±SD)
years compared with 68.1±6.7 years for patients without MetS (P = 0.032).
26.5% of PAD patients presented total testosterone below normal. In patients
with and without MetS, respectively, levels of plasma total testosterone were
8.2±0.7 versus 15.9±2.1 nmol/L (P < 0.05). A previous myocardial infarction
was documented in 45.9% of patients with MetS and in 25.5% of those without
MetS (P < 0.05). 27 (27.6%) patients with Mets had cerebrovascular accident
in anamnesis (versus 17.3% in subjects without MetS, P < 0.05). Severe
atherosclerosis of infrainguinal arteries was detected in 79.6% of patients with
MetS (versus 35.7% in control, P < 0.05). In our study there were no patients
with MetS with isolate occlusion of iliac arteries, but only in association with
atherosclerosis of runoff vessels.
Conclusion: MetS is present in >50% of PAD patients and strongly associated
with hypoandrogenaemia, severe atherosclerosis of infrainguinal arteries and
high cardiovascular comorbidity.
338 CARDIOVASCULAR RISK IN YOUNG WOMEN WITH PREMATURE
OVARIAN FAILURE AND SURGICAL MENOPAUSE
N. Sharashkina, O. Tkacheva, N. Latysheva, I. Novikova, L. Butoreva. V.I.
Kulakov Research Center for Obstetrics, Gynecology and Perinatology,
Moscow, Russia
Introduction: Recent studies indicated increasing cardiovascular morbidity in
young women. This may be due to an increase in traditional and non-traditional
risk factors among women of reproductive age. We studied premature ovarian
failure (POF) and surgical menopause (SM) in young women and assessed the
impact of these factors on endothelial function, atherosclerotic changes and
lipid metabolism.
Methods: we analyzed serum levels of total cholesterol (TC), HDL-C, LDL-C
and triglycerides (TG). Endothelium-dependent vasodilation and carotid artery
intima-media thickness (CA-IMT) were evaluated in 16 patients with POF, 14
with SM and 17 healthy controls (CN). Inter-group differences were calculated
using Student’s t-test.
Results: We found a worse lipid profile among women with POF and SM.
LDL-C was increased significantly in POF and SM (POF: 3.36 mmol/l [SD 0.51]
and SM: 3.15 mmol/l [SD 0.46] vs CN: 2.82 mmol/l [SD 0.36], p < 0.05); TC and
TG were higher in the POF and SM groups, but not significantly (p > 0.05).
Compared to controls, endothelium-dependent vasodilation was significantly
reduced in POF and SM patients (10.5% [SD 4.3] and 8.8% [SD 3.1] vs 14.5%
[SD 3.6], p < 0.05). The mean combined CA-IMT was significantly higher in POF
and SM patients (0.76 mm [SD 0.08] and 0.73 mm [SD 0.09], respectively vs
0.52 mm [SD 0.04], p < 0.05).
Conclusions: We conclude that impaired endothelial vasoreactivity and
increased CA-IMT are prevalent in POF and SM patients and are associated
with traditional risk factors that strongly suggest that POF and SM could be a
risk factor for atherosclerosis.
339 CROSS-TALK OF HOMOCYST(E)INE AND THIOREDOXIN IN
CORONARY ARTERY DISEASE
L. Zhong1 , Y. Wu1 , L. Yang2 . 1 College of Life Sciences, Graduate University of
Chinese Academy of Sciences, 2 Department of Endocrinology, Chinese PLA
General Hospital, Beijing, China
Objective: The aim of our study is to investigate clinical implications and
molecular mechanisms underlying a negative relationship between thioredoxin
(Trx) and total homocysteine (tHcy) in patients with coronary artery disease
(CAD). Trx system, including thioredoxin reductase (TrxR), Trx and NADPH, is
critical for cell functions.
Methods and Results: Data from 120 CAD patients were retrospectively
analyzed. Of these, coronary angiography was performed in 59 patients.
Coronary injury was inversely and significantly associated with the ratio
of serum Trx activity/tHcy (P = 0.009) or Trx activity (P = 0.004). Human
endothelial cells treated with homocysteine had decreased transcription
and activity of cytosolic Trx (hTrx1). The same cells exhibited activated
apoptosis signal-regulating kinase 1-p38 MAPK but not JNK pathway, which
might stem from Trx falling or modification rather than oxidative stress,
because homocysteine-treated cells retained increased antioxidant components
73
Nrf2/TrxR. Homocysteine and homocystine acted differently: homocysteine
could reduce intermolecular disulfide bonds within hTrx1 homodimer or ASK1
dimer/polymer, whereas homocystine reacted with thiol group, such as hTrx1Cys73, to inhibit its activity. Efficiency of homocystine as hTrx1 inhibitor was
about 7-fold higher than that of homocysteine as disulfide reductant.
Conclusions: Homocystine is a negative regulator of hTrx1. In CAD patients,
high homocystine cooperates with low hTrx1 to drive coronary artery lesion.
340 ROLE OF PERI-RENAL FAT (PRF) IN ASSOCIATION WITH RISK
FACTORS FOR ATHEROSCLEROSIS: A NEW METHODOLOGY
L. Borges Roever1 , E.S. Resende1 , N.P. Silva2 , A.D. Debs3 , L.H. Resende4 ,
Atherosclerosis. 1 Cardiology, 2 Biochemistry, 3 Ultrasound, 4 Medicine, Federal
University of Uberlândia, Uberlândia, Brazil
Introduction: The PRF has been associated with hypertension but its
measurement and association with risk factors is not well described.
Objective: To correlate the thickness of the PRF with risk markers for
atherosclerosis.
Patients and Methods: A total of 48 volunteers of both sexes, 24 women (F), 15
controls and 9 with MS, and 24 men (M), 12 in each group, selected with basis
in their clinical history and physical examination underwent clinical evaluation
and ultrasound (US). We did not include those who were using drugs that could
interfere with metabolic and hemodynamic profile, and those who drank more
than 15 g/day of ethanol. The thickness of the PRF was measured by the
distance between the inner face of the transverse muscle of the abdomen and
the posterior face of the right kidney.
Results: The correlation was positive (p < 0.05) with risk factors in men
starting with the TGP (0.24 cm), LDL-C (0.34 cm), total cholesterol (0.36 cm),
triglycerides (0.65 cm), waist circumference (0.66 cm), SBP (0.65 cm), Glucose
(0.67 cm), DBP (0.68 cm) and Gamma-GT (1.23 cm), and in women with the
Gamma-GT (0.14 cm), triglycerides (0.24 cm), SBP (0.60 cm) and the waist
circumference (0, 79 cm).
Conclusion: The thickness is correlated with PRF crhonology risk factors for
atherosclerosis.
341 b2 -GLYCOPROTEIN I ENHANCES ENDOTHELIAL NITRIC OXIDE
SYNTHASE EXPRESSION AND NITRIC OXIDE GENERATION IN
HUMAN AORTIC ENDOTHELIAL CELLS
A.-N. Chiang1 , W.-C. Chiu1 , W.H.-H. Sheu2 , S.-Y. Lin2 . 1 Institute of
Biochemistry and Molecular Biology, National Yang-Ming University, Taipei,
2
Department of Education and Research, Taichung Veterans General Hospital,
Taichung, Taiwan R.O.C.
The object of the present study was to investigate the role of b2 -glycoprotein
I (b2 -GPI) in the regulation of endothelial nitric oxide synthases (eNOS)
and nitric oxide (NO) production in human aortic endothelial cells (HAECs).
From the immunohistochemical analysis, b2 -GPI was highly expressed in the
endothelium of human atherosclerotic plaque. Positive immunostaining of b2 GPI was neutralized by annexin A2 antibody, indicating that annexin A2 may
bind b2 -GPI on the membrane of HAECs. To elucidate the effect of b2 -GPI on
eNOS expression, we determined mRNA and protein levels of eNOS by realtime quantitative PCR and Western blot analysis, respectively. It shows that
eNOS expression and NO production were all increased in HAECs treated
with 200 mg/ml b2 -GPI. Knockdown of eNOS gene using small interfering
RNAs (siRNAs) indicates that b2 -GPI-activated NO generation was attenuated
by eNOS siRNA. We also investigated the modulation of protein expression
in HAECs infected with adenovirus expressing b2 -GPI by 2-dimension gel
electrophoresis. We hypothesized that b2 -GPI may target some intracellular
pathways. Our findings suggest that b2 -GPI stimulates MAPK signaling pathway
via phosphorylation of p38, extracellular signal-regulated kinase (ERK), and
c-Jun N-terminal kinase (JNK). The effect of b2 -GPI on the expression of
some inflammatory or chemoattractant factors, such as interleukin (IL)-6, IL-8,
vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1
(ICAM-1) and monocyte chemoattractant protein 1 (MCP-1) has also been
assessed in this study. However, more research remains to be done to address
the roles of b2 -GPI in the pathogenesis of cardiovascular diseases.
342 CORRELATION BETWEEN SERUM PARAOXONASE AND
HOMOCYSTEINE THIOLACTONASE ACTIVITY, ADIPOKINES
AND ASYMMETRIC DIMETHYLARGININE LEVELS IN RENAL
TRANSPLANT PATIENTS
L. Locsey1 , I. Seres2 , F. Sztanek2 , L. Szabo1 , L. Asztalos1 , G. Paragh2 .
1
Institute of Surgery, 2 I. Dept. Med., University of Debrecen, Debrecen,
Hungary
HDL-associated paraoxonase (PON1) prevents LDL-C from oxidation thus
providing protection against atherosclerotic disease. Our aim was to investigate
the connection between serum paraoxonase and homocysteine thiolactonase
(HTLase) activity, renal function, adipokines and asymmetric dimethylarginine
(ADMA) levels in renal transplant recipients. 79 transplanted patients (38 males,
41 females, age:49.01±14.00 years) were included in the study. PON1, HTLase
74
activity was determined spectrophotometrically, adiponectins and ADMA levels
were measured by ELISA method. Our patients have hypercholesterolaemia,
high LDL and ApoB levels and parallel with improved renal function,
decreased cystatinC and homocysteine levels (p < 0.001). In obese patients
(BMI > 30kg/m2 , n = 14) LDL (p < 0.05) and leptin concentrations (58.06 vs.
15.16ng/ml, p < 0.01) were significantly higher than in malnourished group
(n = 9). We did not find significant difference between serum adiponectin levels
(16.81 vs. 21.61ug/ml) and PON1 activity (91.45 vs. 101.20U/l) in obese and
malnourished renal transplant patients. HTLase activity was 438.20 vs. 385.99
U/l) in obese and malnourished transplated patients..After transplantation a
significant negative correlation was found between serum PON1 activity and
improved renal function (p < 0.01). Between PON1 activity and adiponectin
levels there was a significant correlation (p = 0.0276) and between serum PON1
activity and ADMA levels there was a negative, non-significant correlation
(p = 0.2302).
Between HTLase and PON1 activity and HTLase and PON/HDL ration there
was a positive linear correlation (p < 0.03). Dyslipidaemic, obese transplanted
patients have high LDL, leptin levels and PON1 activity does not correlate
significantly with leptin, ADMA and CRP levels. With improved renal function
significant increased in PON1 activity can found. Between serum PON1 and
adiponectin there was a positive correlation, between HTLase and ADMA levels
we found a significant, negative correlation.
343 3-HYDROXYKYNURENINE − A NEW FACTOR ASSOCIATED
WITH CAROTID ATHEROSCLEROSIS, OXIDATIVE STRESS AND
INFLAMMATION IN URAEMIA
D. Pawlak1 , M. Mysliwiec2 , K. Pawlak1 . 1 Monitored Pharmacotherapy,
2
Nephrology and Clinical Transplantation, Medical University, Bialystok,
Poland
Objective: Increased oxidative stress (SOX), inflammation and carotid
atherosclerosis have been reported in chronic kidney disease (CKD) patients,
but their associations with kynurenine (KYN) pathway activation remain
unknown.
Methods: We determined the plasma concentrations of tryptophan (TRP), KYN,
3-hydroxykynurenine (3-HKYN); SOX markers: Cu/Zn superoxide dismutase
(Cu/Zn SOD), total peroxide, high sensitivity C-reactive protein (hs CRP)
as a indicator of inflammation, and an early indicator of the systemic
atherosclerosis − intima-media thickness (IMT) in 146 CKD patients and healthy
controls.
Results: The plasma concentrations of TRP was decreased by 40−60% in
CKD patients compared to controls, whereas KYN and 3-HKYN levels were
increased by 32−96% and 184–306%, respectively. These changes were
accompanied by significant increase in the KYN/TRP ratios by 140–240%, and
3-HKYN/KYN ratios by 40–154% in uremics compared to controls. CKD patients
showed a significant increase in Cu/Zn SOD, total peroxide, hs CRP and IMT
values compared to controls. KYN and 3-HKYN were positively associated with
inflammation, SOX markers and IMT in uraemics. Finally, multiple regression
analysis identified age and 3-HKYN levels as the independent variables
significantly associated with increased IMT in this population (adjusted r2 =
0.304).
Conclusions: The results of the present study suggest a relationship between
kynurenine pathway activation and increased oxidative stress, inflammation and
progression of atherosclerosis in CKD patients. It suggests a new idea that the
disturbances of the endogenous kynurenine pathway can play a role in the
pathogenesis of arterial damage in this population.
344 LEPTIN TRIGGERS DISTURBED CA2+ TRANSPORT IN MONOCYTES
OF OVERWEIGHT PATIENTS
J. Tamás Padra, I. Seres, G. Fóris, Z. Balogh, G. Kónya, Z. Karányi,
G. Paragh. University of Debrecen, Debrecen, Hungary
The increased [Ca2+ ]i in monocytes of overweight subjects may be the cause
of many concomitant pathological symptoms. The aim of the present study
was to elucidate the relationship between the leptin induced NADPH oxidase
activation and [Ca2+ ]i homeostasis in human monocytes. Our results are as
follows: 1. The basal level of [Ca2+ ]i in resting monocytes of overweight patients
(OW monocytes) was higher than that in control cells, whereas the leptininduced peak of Ca2+ signal was lower and the return to basal level was
delayed in the OW monocytes. 2. When Ca2+ signals were calculated as
areas under time curves (AUC), the signals were more pronounced in OW
monocytes than in control cells, as a consequence of injured Ca2+ extrusion
from cells. 3. Using different inhibitors, we proved that in control cells the
Ca2+ signals originated from intracellular pools, whereas in OW cells they
were generated predominantly by Ca2+ -influx from medium. Finally, we found
correlation between the leptin induced superoxide anion generation and Ca2+
signals (in AUC). The disturbed [Ca2+ ]i homeostasis in OW monocytes was fully
restored in the presence of NADPH oxidase inhibiting fluvastatin. Therefore,
statins through decrease in free radical generation have beneficial effect on
elevated [Ca2+ ]i and consequently on the obesity.
345 CHLOROGENIC ACID REDUCE PLASMA LIPIDS AND
INFLAMMATORY-RELATED GENE EXPRESSION IN
HYPERLIPIDEMIC RABBITS
Y.-M. Yu1 , W.-C. Chang2 . 1 Nutrition and Health Sciences, 2 Graduate Institute
of Medical Sciences, Chang Jung Christian University, Tainan City, Taiwan
R.O.C.
Atherosclerosis is a major cause of coronary heart disease. Chlorogenic acid
(CA) is a phenolic compound present in fruits, vegetables and some drinks,
including apples, pears, berries, black tea and coffee. It has been found to have
antioxidant, antimutagenic, and immunomodulatory properties. In this study,
we investigated the effects of chlorogenic acid on oxidative stress and antiinflammatory gene expression in hyperlipidemic rabbits. Twenty-four NZW male
rabbits were assigned randomly into four dietary groups and were sacrificed
after eight weeks. The normal group was fed regular rabbit chow and the control
group was fed a high fat and cholesterol diet. The probucol group and the CA
group were fed the same diet as the control group plus 0.1% (w/w) probucol or
chlorogenic acid. The result indicated that the serum levels of total cholesterol,
LDL-cholesterol, triglyceride, MDA, luminol-CL and intimal thickening of thoracic
aorta were significantly increased in the control group compared with the normal
group, and decreased in the probucol and the CA group. The lag time of LDL
oxidation in the control group was shorter than the normal group, and longer
in the probucol and the CA group. The mRNA expression of VCAM-1 (vascular
cell adhesion molecule-1) and MCP-1 (monocyte chemotactic protein-1) were
significantly increased in the control group compared with the normal group,
and decreased in the probucol and the CA group. In conclusion, chlorogenic
acid suppresses the progression of atherosclerosis by lowering serum lipid,
lipid oxidation and inflammatory-related gene expression including VCAM-1and
MCP-1 in hyperlipidemic rabbits.
346 IMPACT OF CIGARETTE SMOKE ON THE TRANSMIGRATION OF
MONOCYTES THROUGH AN ENDOTHELIAL CELL LAYER
I. Gallitz1 , R. Janssens1 , K. Stolle1 , Y. Steffen1 , S. Wagner1 , S. Lebrun2 ,
M. Lietz1 . 1 PHILIP MORRIS Research Laboratories GmbH, Koeln, Germany,
2
PHILIP MORRIS International, Neuchatel, Switzerland
The transmigration of monocytes through the vascular endothelium is one of the
key steps in the development of atherosclerosis. Cigarette smoking is a known
risk factor for the development of atherosclerosis, but the mechanisms involved
are not completely understood. To investigate the impact of cigarette smoke on
the transmigration of monocytes through an endothelial cell layer, we developed
a transmigration assay. Human coronary artery endothelial cells (HCAEC) were
grown on transwell filter inserts and treated either directly, i.e., with cigarette
smoke bubbled through phosphate-buffered saline (sbPBS) or total particulate
matter (TPM), or indirectly, i.e., with the supernatant of sbPBS- or TPMexposed monocytes. The transmigration rate of THP-1 cells (human acute
monocytic leukemia cell line) was determined by flow cytometry. Blocking
antibodies against surface molecules (e.g., E-selectin, ICAM-1, VCAM-1) were
used to investigate the molecular mechanisms involved in the cigarette-smokeenhanced transmigration of THP-1 cells through the endothelial cell layer.
Direct treatment showed no effect. Indirect treatment showed a cigarette-smokeenhanced transmigration rate which was higher for TPM than for sbPBS.
347 INVOLVEMENT OF PROTEIN TYROSINE PHOSPHATASE
HYPERHOMOCYSTEINEMIA AND HYPERCHOLESTEROLEMIA
INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION
S. Sharma, M. Singh, P.L. Sharma. Cardiovascular Division, Department of
Pharmacology, ISF College of Pharmacy, Moga, India
Introduction: Protein tyrosine phosphatase (PTPase) regulates processes
involved in pathological vessel wall functions viz. inhibition of survival pathways
and reduce the phosphorylation status of enzymes.
Aim/Objective: The Present study is designed to investigate involvement
of PTPase in vascular endothelium dysfunction (VED) associated with
Hyperhomocysteinemia (Hhcy) and hypercholesterolemia (Hch). Furthermore
possible downstream mechanisms inhibited by PTPase were also elaborated in
both the conditions.
Methods and Purpose: Hhcy was induced in a group of male wistar rats
by administration of L-Methionine (1.7% w/w., p.o.) while Hch was produced
by feeding high cholesterol diet. After 4 weeks of Methionine and Cholesterol
rich diet administration, Hhcy was confirmed by estimating serum Hhcy conc.
(Hhcy > 12 mm/l) and Hch was confirmed by increase in serum conc. of
cholesterol, TG-c and decrease in HDL-c level. VED was assessed, in terms of
attenuation of endothelium-dependent relaxation (EDR), a decrease in serum
nitrate/nitrite (N/N) level, as well as mRNA expression of eNOS (rtPCR) and
disruption of integrity of vascular endothelium (Electron microscopy).
Results: Sodium orthovanadate (SOV), (8 mg/kg, p.o., 16 mg/kg, p.o and
24 mg/kg, p.o) and atorvastatin (30 mg/kg, p.o) (positive control) treatment
significantly improved EDR, Serum N/N, mRNA expression of eNOS and
integrity of vascular endothelium. However, this ameliorative effect of SOV was
79th EAS Congress
75
significantly blocked by Wortmannin (PhosphatidylInositol-3-Kinase inhibitor)
and L-NAME (Inhibitor of eNOS).
Conclusion: Therefore, it may be concluded that sodium orthovanadate, a
specific inhibitor of PTPase, may stimulate PI3K and eNOS and consequently
improve vascular endothelium dysfunction. Thus, inhibition of PTPase might be
a useful approach in the therapy of vascular diseases.
processes. Numbers of HLA-DR+ cells were found to be significantly larger in
the middle third of the intima than in the superficial and deep intimal portions.
We speculate that a widespread distribution of HLA-DR-expressing cells in
the intima of normal human aorta might play a role in the surveillance and
maintenance of vascular homeostasis.
Supported by Russian Ministry of Science and Education.
348 VASOPROTECTIVE ACTIVITY OF SUPRAMOLECULAR SUPEROXIDE
DISMUTASE–CHONDROITIN SULFATE–CATALASE CONJUGATE
A.V. Maksimenko, A.V. Vavaev, E.G. Tischenko. Institute of Experimental
Cardiology, Russian Cardiology Research-and-Production Complex, Moscow,
Russia
351 RELATIONSHIP BETWEEN INSULIN SENSITIVITY AND LIPID
PROFILE IN EUTHYROID TYPE 1 DIABETIC PATIENTS
T. Bulum, L. Duvnjak, I. Prkačin. University Hospital Merkur, Vuk Vrhovac Clinic
for Diabetes, Endocrinology and Metabolic Diseases, School of Medicine,
University of Zagreb, Zagreb, Croatia
Bienzyme superoxide dismutase-chondroitin sulfate-catalase conjugate was
obtained by covalent connection one another of superoxide dismutase (SOD)
with catalase (CAT) via endothelial glycocalyx glycosaminoglycans − chondroitin
sulfate (CHS). This conjugate (SOD-CHS-CAT) possessed vasoprotective
activity in respect to platelet interactions, tonus of the ring arterial fragment of
rat vessel, normalization of hemodynamic rat and rabbit indices changed with
hydrogen peroxide administration as oxidative stress model. The SOD-CHSCAT conjugate had antiplatelet potential due to its antiaggregation action by
means of combined enzyme activities and acquired supramolecular structure.
The influence on arterial fragment tonus was equal for SOD and CAT in native
and connected in conjugate form. Blood pressure and heart rate were significant
and effective normalized with SOD-CHS-CAT conjugate in rats and rabbits (after
hydrogen peroxide administration as perturbance stimulus). At the first time
we have found in vivo the chronic prophylaxis action for antioxidant bienzyme
conjugate. It is important for real development of peroral form of SOD-CHSCAT conjugate. These results indicate the universal approach to development
of enzyme nanoconjugates of medical destination.
This research was supported by RFBR grants 06−08–00011, 06−04–08002-ofi,
09−08–00023, and Roszdrav, Rosmedtechnology, and Minzdravsocrazvitiya of
Russian Federation.
Introduction: Insulin resistance may contribute to the high risk for
cardiovascular disease in patients with type 1 diabetes (DM1). The major
quantitative change associated with the insulin resistance syndrome is
an elevation in triglycerides and small dense LDL cholesterol particles,
accompanied by a decreased HDL cholesterol level. The aim of this study was
to explore impact of insulin sensitivity on serum lipids in patients with DM1.
Patients and Methods: We divided 304 patients with normal thyroid
function according to median estimated glucose disposal rate (eGDR =
9.72 mg kg−1 min−1 ) into lower (n = 153) and higher-insulin sensitivity (n = 151)
group. None showed signs of adrenal, thyroid, renal or cardiovascular disease
and received drugs apart from insulin that could attenuate lipid metabolism or
insulin sensitivity.
Results: Patients with lower insulin sensitivity in comparison to patients with
higher insulin sensitivity showed significantly higher total, LDL, VLDL cholesterol
(5.23 vs 4.83; 2.98 vs 2.64; 0.5 vs 0.35 mmol/L, p < 0.001), and triglycerides
level (1.1 vs 0.76 mmol/L, p < 0.001) and lower HDL cholesterol level (1.58 vs
1.76 mmol/L, p < 0.001). Multivariate logistic regression models have found
significant association between total (OR: 1.6, 95% CI: 1.2−2.2), LDL (OR:
1.9, 95% CI: 1.3−2.7), HDL (OR: 0.4, 95% CI: 0.3−0.8), VLDL cholesterol (OR:
35.2, 95% CI: 10.3–120.3), and triglycerides (OR: 4.9, 95% CI: 2.8−8.6) and
progression to insulin resistance in our patients.
Conclusion: Our study showed that patients with DM1 and lower insulin
sensitivity are at higher risk of cardiovascular disease because of abnormalities
in lipid and lipoprotein metabolism due to insulin resistance.
349 CELL PROLIFERATION IN DIFFERENT ATHEROSCLEROTIC
LESIONS OF HUMAN ARTERIES
A.N. Orekhov1,2 , E.R. Andreeva3 , I.V. Adrianova3 , Y.V. Bobryshev4 .
1
Institiute for Atherosclerosis Research, 2 Institiute of General Pathology and
Pathophysiology, 3 Intitute of Experimental Cardiology, Russian Cardiology
Research Center, Moscow, Russia, 4 Faculty of Medicine, University of New
South Wales, Sydney, NSW, Australia
In the present work, we studied intima thickness and proliferation of newly
“infiltrated” hematogenous and resident cells in atherosclerotic lesions of aorta,
carotid and coronary arteries. Hematogenous cells in lipofibrous plaques of the
coronary and carotid arteries were found to account for a third and almost for
a half of the total cell population, respectively, while atherosclerotic lesions in
the aorta contain no more than 15% of hematogenous cells. This suggests that
the contribution of hematogenous cells to the development of atherosclerosis
in the carotid and coronary arteries appears to be more significant than in the
aorta. Despite the differences in the numbers of accumulating hematogenous
cells in the intima, a similar “bell-shaped” dependence of cell numbers on
the lesion type, involved the following sequence: unaffected intima − initial
lesions − fatty streaks − lipofibrous plaques − fibrous plaques, was detected
in the coronary and carotid arteries. The visualization of PCNA-positive cells
in atherosclerotic and unaffected zones of the coronary and carotid arteries
revealed similar patterns of the distribution of proliferating cells. The maximum
numbers of PCNA-positive resident cells were identified in lipofibrous plaques.
The changes in the total cell numbers were found to be accompanied by the
changes in the numbers of both proliferating resident cells and proliferating
hematogenous cells.
Supported by Russian Ministry of Science and Education.
352 ASSOCIATION BETWEEN EXTRACELLULAR MATRIX PROTEINS
AND MATRIX METALLOPROTEINASE ACTIVITY IN PATIENTS WITH
ATHEROSCLEROTIC AND NON-ATHEROSCLEROTIC THORACIC
AORTIC ANEURYSMS
O. Irtyuga1 , I. Voronkina2 , L. Smagina2 , V. Uspensky1 , N. Tsoy1 , E. Shlyakhto1 ,
O. Moiseeva1 . 1 Almazov Heart, Blood and Endocrinology Centre, 2 Research
Institute of Experimental Medicine, Saint Petersburg, Russia
Objectives: It is well known that matrix metalloproteases (MMP) are involved in
the pathogenesis of aortic aneurysms through extracellular matrix degradation.
However, the question remains whether etiology of thoracic aorta aneurysm
(TAA) is associated with specific changes in activity of MMP’s and in
composition of extracellular matrix proteins.
Methods: We examined 26 pts with TAA (age 54.7±2.2 yrs;m:f 2:1):12 pts
with bicuspid aortic valve (BAV), 6 pts with tricuspid aortic valve (TAV),
8 pts with an atherosclerosis, and 6 pts IHD and without aortic dilatation
(age 56.5±3.5 yrs;m:f 2:1). Pts with infective endocarditis and rheumatic
disease were excluded. MMP2 and MMP9 activities were assessed in
aortic tissue homogenates by substrate-specific zymographic analysis. The
collagen I, elastin and fibrillin were determined by quantitative immunoblotting
techniques.
350 DISTRIBUTION OF HLA-DR-EXPRESSING CELLS IN THE INTIMA
OF HUMAN AORTA: POSSIBLE ROLE IN SURVEILLANCE AND
MAINTENANCE OF VASCULAR HOMEOSTASIS
Y.V. Bobryshev1,2 , M.M. Moisenovich3 , O.L. Pustovalova3 , I.I. Agapov4 ,
A.N. Orekhov1,5 . 1 Institute for Atherosclerosis Research, Russian Academy
of Natural Sciences, Moscow, Russia, 2 Faculty of Medicine, University of
New South Wales, Sydney, NSW, Australia, 3 Biological Department, Moscow
State University, 4 Shumakov Federal Research Center of Transplantology
and Artificial Organs, 5 Institute of General Pathology and Pathophysiology,
Russian Academy of Medical Sciences, Moscow, Russia
The architectonics and cell composition of the human large arteries are not
sufficiently understood. The present study is the first to undertake an analysis of
the distribution and quantities of HLA-DR-expressing cells in grossly undiseased
human intima using immunohistochemical and immunofluorescent analysis,
complemented by the advantages of confocal microscopy. The study revealed
a widespread distribution of HLA-DR-expressing cells throughout the intimal
space where the cells were integrated into continuous networks via long cell
Figure: Collagen, elastin, fibrillin content in aortic tissue.
Results: Maximum diameter of the aorta was detected in pts with
atherosclerosis: 54.6±4.2 mm vs 43.4±2.8 mm in pts with BAV and
46.1±4.2 mm in pts with TAV. MMP-9 activity was increased in all pts with
TAA compared with the control group (283.49±82 OD). Greater MMP-9 activity
76
was measured in pts with BAV (563.9±79.8;p = 0.04 compared control) and
atherosclerosis (427.5±116.1 OD) compared with TAV (367±92.1 OD). MMP-2
activity did not significantly differ in groups. An abundance of MMP-9 in the
atherosclerotic and BAV groups was associated with increased collagen/elastin
ratio (1.5:1).
Conclusion: Abnormal collagen/elastin ratio in patients with atherosclerosis
and BAV may lead to increased activity of MMP9 and play an independent role
in pathogenesis of aorta aneurysm.
353 PURE TOCOTRIENOL ISOMERS REDUCE PROTEIN AND
GENE EXPRESSION OF LPS-INDUCED INFLAMMATION AND
ENDOTHELIAL ACTIVATION IN ENDOTHELIAL CELLS
S. Muid1 , G.R.A. Froemming2 , H. Nawawi1 . 1 Centre for Pathology Diagnostics
and Research Laboratories (CPDRL), Faculty of Medicine, Sungai Buloh
Campus, University Teknologi MARA, 2 Institute for Medical Molecular
Biotechnology (IMMB), Faculty of Medicine, Universiti Teknologi MARA,
Sungai Buloh, Malaysia
Objectives:
i. To investigate the effects of pure tocotrienol (TCT) isomers and a-Tocopherol
(TOC) on protein and gene expression of inflammatory and endothelial
activation markers and
ii. To determine the optimal TCT isomers and a-TOC concentrations on
inhibition of these markers in stimulated endothelial cells.
Methods: Human umbilical vein endothelial cells (HUVEC) were incubated
with various TCT isomer concentrations (0.3−10 mM) together with lipopolysaccharide, LPS (1 mg/ml). Soluble protein expression of IL-6 and ICAM-1 were
measured by ELISA (Bender MedSystems, Vienna, Austria). Gene expression
of these markers was measured using a SYBR-Green Quantitative real-time
PCR assay (Bio-Rad Laboratories, USA).
Results: For IL-6, a-, b-, g- and d-TCT isomers but not a-TOC led to significant
reduction in IL-6 soluble protein concentration, [optimal concentration]
(% inhibition); a-TCT:10mM (31%), b-TCT:10mM (47%), g-TCT: 0.6mM (29%),
d-TCT:2.5mM (27%);p < 0.05 and a-TOC:0.3mM (0.6%), p > 0.05. For sICAM-1,
a-, b-, g- and d-TCT isomers and a-TOC led to significant reduction in sICAM-1;
a-TCT:5mM (37%), b-TCT:5mM (25%), g-TCT:0.6mM (62%), d-TCT:0.6mM (66%)
and a-TOC:0.6mM (17%) respectively, p < 0.05. All TCT isomers but not a-TOC
led to downregulation of IL-6 and sICAM-1 gene expression compared to
controls.
Conclusion: Pure TCT isomers have more potent anti-inflammatory effects
than a-TOC. g and d-TCT are more potent at lower concentration than a and
b-TCT isomers for both IL-6 and sICAM-1 inhibition.
to endothelial dysfunction. Recently, the turnover of endothelial cells has been
better understood with a crucial role of endothelial progenitor cells (EPC). In
addition, increased amount of microparticles derived from endothelial apoptotic
cells (EMP) has been reported in patients with uncontrolled risk factors. We
aimed to examine the effects of two hypolipidemic strategies (atorvastatin 80 mg
or atorvastatin 20 mg plus ezetimibe 10 mg, for achieving of comparable lipid
goals) in subjects with coronary artery disease (CAD) with cIMT more than
1.0 mm on EPC, EMP and platelet microparticles (PMP) levels.
Methods: This was an open label, randomized, parallel-designed study with
blinded endpoints. The patients were accompanied for quantification of EPC,
EMP and PMP at baseline and after 6 months of treatment.
Results: For the group treated with atorvastatin plus ezetimibe, no differences
were seen for the three sub-populations of EPC analyzed. However, there were
higher EMP levels after treatment (p = 0.03 vs. baseline), as well as in the
PMP levels (p = 0.02 vs. baseline). In the group treated with atorvastatin 80 mg,
there was a significant reduction in the CD34+/KDR+ EPC levels (p = 0.004)
and higher levels in the PMP (p = 0.001).
Conclusion: Hypolipidemic therapy with both strategies affects the turnover of
endothelial cells and is associated with increased levels of PMP and EMP.
356 CORONARY-HEART DISEASE (CHD), PERIPHERAL-VASCULAR
DISEASE (PVD) OR BOTH (C&PD): PROGNOSTIC IMPLICATIONS
FOR RECURRENT EVENTS. THE AIRVAG COHORT EXTENDED
FOLLOW-UP
M.L. Peralta1 , C. Aranda2 , A.I. Huelmos3 , I. Gonzalez Anglada2 , E. Puras4 ,
B. Herreros2 , C. Sanchez5 , V. Castilla2 , L. Lopez Bescos3,6 , C. Guijarro2,6 ,
The AIRVAG Investigators. 1 Family Medicine, 2 Internal Medicine, 3 Cardiology,
4
Vascular Surgery, 5 Neurology, Hospital Universitario Fundacion Alcorcon,
6
Medicine, Universidad Rey Juan Carlos, Alcorcon, Spain
Objectives: To evaluate the prognostic implications of the presence of CHD,
PVD or both for new ischemic events
Patients and Methods: Prospective AIRVAG cohort: 183 patients (<70 yearold), with symptomatic CHD (96, 52%), PVD (32, 18%) or C&PD (55, 30%)
followed for >9 years. Time to new ischemic events was analyzed by the
Kaplan Meier method (univariate, log-rank test) and Cox regression models
(multivariate).
Results: New ischemic events occurred in 25% CHD, 50% PVD, 47% C&PD
(p = 0.001 CHD vs others).
354 RELATIONSHIP BETWEEN ENDOTHELIAL AND RENAL FUNCTION
IN PATIENTS WITH CHRONIC HEART FAILURE AND PRESERVED
EJECTION FRACTION
O. Mytrokhina, O. Kuryata, A. Miroshnichenko. Dnepropetrovsk State Medical
Academy, Dnepropetrovsk, Ukraine
Renal disorders (RD) is predicts progression of chronic heart failure (CHF).
Endothelial dysfunction (ED) is identified as a marker of the progression of
atherosclerosis however the relationship between renal and endothelial function
in patients with CHF and preserved ejection fraction remains debatable.
Objectives: We aimed to evaluate relationship between renal and endothelial
function in patients with CHF and preserved ejection fraction.
Methods: 56 pts (27 M, 29 F, mean age − 60.16±3.54 years) with CHF NYHA
I−III class and preserved ejection fraction (LVEF > 45%) were enrolled. Pts were
divided into two groups: 35 (62.5%) pts (1 group) with glomerular filtration rate
(GFR) 60−90 ml/min and 21 (37.5%) pts (2 group) with GFR >90 ml/min. The
serum creatinine (SCr) levels were determined in the blood. GFR was calculated
by Cockcroft-Gault formula. Flow-mediated dilation (FMD) was assessed during
reactive hyperemia.
Results: Among 56 patients 75% had ED. 94.3% of patients in the 1 group
had ED, since ED had been matched in 83.5% of patients of the 2 group. The
FMD and SCr levels correlated to NYHA functional class (r = −0.30; p < 0.05
and r = 0.42; p < 0.05 respectively) and GFR (r = 0.51; p < 0.01 and r = −0.56;
p < 0.01 respectively).
Conclusion: Our results provide suggestive evidence that endothelial
dysfunction is not only a powerful predictor of CHF and preserved ejection
fraction but also renal dysfunction in in this group of patients.
Fig. 1. All-events survival.
355 EVALUATION OF ENDOTHELIAL PROGENITOR CELLS AND
MICROPARTICLES AFTER HIPOLIPEMIANT TREATMENT IN
PATIENTS WIHT SUBCLINICAL ATHEROSCLEROSIS
C. Nunes França, C.E.S. Ferreira, M.C.O. Izar, F.A. Helfenstein Fonseca.
Medicine, Federal University of São Paulo, São Paulo, Brazil
Introduction: The use of statins for LDL-cholesterol reduction has been
established in the literature. The association with ezetimibe allowed a
new mechanism for reducing the cholesterol levels. Atherosclerosis is an
inflammatory disease complicated with thrombotic events, a condition related
Fig. 2. CHD survival.
79th EAS Congress
PVD was associated with a hazard ratio [HR] of 2.6 (95% conficence interval
[CI] 1.5−4.3, p < 0.001) of new events regardless of the simultaneuos presence
of CHD, and remained as a strong predictor after adjusting for age and sex
(HR 2.0, 05% CI 1.1−3.5, p < 0.05) and traditional risk factors (diabetes, blood
pressure, tobacco, LDL, HDL, TG, microalbuminuria; HR 1.9, 95% CI 1.1−3.3,
p < 0.05). New coronary events occurred in a similar proportion for all three
groups (CHD 22%, PVD 22%, C&PD 31%, p = 0.4).
Thus other CV events (stroke, peripheral) were more common in PVD or C&PD
than in CHD.
Conclusions: Patients with PVD have about a 2-fold increased risk for new
ischemic events than CHD patients (similar risk for coronary events and
increased risk for other CV events). CHD on top of PVD has no relevant
prognostic implications. Intensive risk factor control is mandatory for PVD
patients
357 VISFATIN AND HSCRP IN RELATION TO ABDOMINAL OBESITY
IN MORBID OBESE SUBJECTS
Z. Karbaschian1 , A. Golpaie1 , M.J. Hosseinzadeh-Attar1 , M. Hosseiny2 ,
M. Talebpor3 , N. Karbaschian4 . 1 Nutrition, 2 Biostatistics, 3 Surgery, Tehran
University of Medical Sciences, 4 Nutrition, Azad University, Tehran, Iran
Introduction: Adipose tissue produces numerous cytokines which make
interests of investigators to find the associations between these adipokines,
CRP and CVD risk factors and their possible role in the pathogenesis of
disease. The purpose of this study was to investigate serum levels of visfatin,
a new adipokine, and hsCRP in relation to abdominal obesity in morbid obese
patients.
Methods: 46 subjects with morbid obesity aged 36±10.4 yr were studied. BMI,
waist circumference, visfatin, hsCRP, HDL, LDL, TG and total cholesterol were
measured. Pearson’s correlation was used to evaluate the relationship between
variables. P values <0.05 were considered as significant.
Results: Mean BMI, waist circumference and waist to hip ratio were 45.3±5.3,
126±14.8 cm, and 0.91±0.1 respectively. The levels of serum visfatin,
hsCRP, LDL, HDL, TG, total cholesterol were 5.14±3.2 ng/ml, 8.9±7.4 mg/l,
105.2±29.4, 40.05±10, 182±107.4, and 184.2±37.7 mg/ml respectively. In
these patients we also found a positive significant correlation between visfatin
and hsCRP (p < 0.0001).
Conclusion: Since hsCRP, an inflammatory factor is a well-known CVD risk
factor, the association between visfatin and hsCRP may suggest that visfatin
plays a role in inflammatory process in obesity complications such as CVD and
diabetes.
358 THE ROLE OF EXTRACELLULAR HYALURONAN IN DEVELOPMENT
OF ATHEROSCLEROSIS AND ENGINEERED BEE HYALURONIDASE
AS A POTENTIAL TOOL FOR THE TREATMENT
B. Greiderer-Kleinlercher, A. Iliás, S. Reitinger, G. Lepperdinger. Extracellular
Matrix Research, Institute for Biomedical Aging Research, Innsbruck, Austria
Atherosclerotic cardiovascular disease is the major cause of death in Western
countries and its incidence is rapidly growing. Atherosclerosis is a chronic
inflammatory disease. The main components of atheromatous lesions in the
arterial tunica intima are excess fat, collagen, elastin and hyaluronan. For
the reduction of hyaluronan in early lesion development Hyaluronidase from
honey bee (Apis mellifera) is a potential tool. It shares 30% of sequence
identity with human hyaluronidases, which are involved in fertilization and the
turnover of hyaluronan. The advantage in contrast to human hyaluronidases
is that bee hyaluronidase exhibits activity at neutral pH as well. Recently, bee
venom hyaluronidase has been produced in large quantity in Pichia pastoris,
and its biochemical characterization has been performed. The aim of our
study is to utilize engineered hyaluronidase to remove hyaluronan from the
developing lesions within arteries and in this way, to prevent further progression
of atherosclerosis. Wild-type bee hyaluronidase and engineered form of bee
hyaluronidase, targeted to developing lesions were constructed and produced
in large quantity in Pichia pastoris. To study the transport of the recombinant
enzymes through activated endothelium an in vitro model was established. Our
results show that the targeted version of bee hyaluronidase can bind to the
activated endothelium and both enzymes can be transported through bEnd.3
cell monolayer. Our hypothesis is tested in vivo, using ApoE-deficient mice
maintained on Western-type diet, administering the recombinant enzymes by
tail vein injection.
359 RELATIONSHIPS BETWEEN BLOOD PRESSURE AND LIPID
MARKERS OF ATHEROSCLEROSIS
E. Vitalyevna Pello, S.K. Malyutina, G.I. Simonova, Y.P. Nikitin. Department of
Internal Medicine, Institute of Internal Medicine of SB RAMS, Novosibirsk,
Russia
Purpose: In anticipation of the derivation of profitability concerning concept
of the risk factors predisposing to the ensuing manifestation of stable and
vulnerable pathophysiological features of atherosclerosis (AT), in our search
77
were assessed the ties between blood pressure (BP) and indicators of lipid
homeostasis.
Methods: We collected data of 324 subjects (EPOGH study).
Results: Hypertension (HT) is considered as one of the most important factors
contributing to the subsequent occurrence of AT. Hence, according to the
preliminary represented definition, we revealed that clinic, home, 24-h, day, and
night systolic/diastolic BP were brightly positively associated with TC, TG, and
LDL-C, but were negatively integrated with HDL-C. Partial correlation analysis
controlling for age showed straightforward associations of the overwhelming
majority of BP parameters with TG and opposite direction of their correlations
with HDL-C. Nevertheless, a lesser proportion of BP components was partially
related to the LDL-C. The interaction of BP with TC merely disappeared. Partial
correlations with joint inclusion of age and BMI were approximately identical.
The inter-group comparison reaped benefits of the existing testimony of either
gender or BMI significance. Actually, the current observations are congruent
with the descriptions of prior epidemiological studies, demonstrate that clinic
BP is more strongly integrated with lipid abnormalities than ambulatory BP, and
unveil that an incremental influence of the combination of several agents from
the constellation of risk factors presumably performs a preferential role in the
determination of AT.
Conclusions: The findings of the conducted comprehensive investigation
confirm the impact of BP on the atherogenesis.
360 OSTEOCALCIN AND METABOLIC SYNDROME
P. Diez Romero, M.A.A. Rodriguez, C.D. Romero, I.C. Estevanez,
C.L. Paredes, F.D. Serrano, M.T. Valdeperez, J.S.F. Rubio, J.M. Nuñez-Cortes.
Medicina Interna, Hospital Universitario Gregorio Marañón, Madrid, Spain
Osteocalcin (linear peptide hormone synthesized by osteoblasts) may play
a role as an endocrine signal and thus participate in the control of energy
metabolism and glucose homeostasis through its effects on adipocytes and
pancreatic beta cells. Therefore, other metabolic disorders associated with high
cardiovascular risk may be accompanied by changes of osteocalcin plasmatic
levels.
Objectives: The aim of this study was to determine the presence of
metabolic abnormalities associated with high cardiovascular risk according
to the osteocalcin concentration, in order to assess whether low levels of
osteocalcin may be involved or contribute to the development of metabolic
syndrome.
Material and Methods: We studied 29 patients who had lower serum levels of
osteocalcin to 2.7 ng/dl. In this population has given the following cardiovascular
risk profile: LDL, HDL, glucose, triglycerides, BMI and presence of metabolic
syndrome. The data are processed with SPSS version 16.0. Statistical tests
are used chi square and Mann-Whitney.
Results: The metabolic syndrome was present in 55.2% of the sample.
Hypertension was the most frequent component trait, present in 51.7%. The
proportion of low levels of osteocalcin in patients with metabolic syndrome was
60% (p = 0.68).
Conclusions: Low levels of osteocalcin are related, but not significantly, with
an increased incidence of metabolic syndrome. This feature could be related as
an indirect marker of cardiovascular risk. However, further studies are needed
to confirm this hypothesis.
361 EFFECTS OF QUINAPRIL AND TELMISARTAN ON MATRIX
METALLOPROTEINASE-2 AND ITS TISSUE INHIBITOR IN PATIENTS
WITH CAD UNDERWENT PTCA
V. Naumov, A. Partigulova, Y. Kosenkov, E. Orlova, V. Kuharchuk, V. Masenko.
Cardiology Research Center, Moscow, Russia
Introduction: Matrix metalloproteinases are crucial players in vascular
inflammation in atherosclerosis. There’s certain data that ACE inhibitors and
ARBs have anti-inflammatory properties.
Aim: To study the influence of 6-month therapy with quinapril or telmisartan
on blood concentrations of matrix metalloproteinase-2 (MMP-2) and its tissue
inhibitor (TIMP-2) in patients underwent PTCA.
Methods: 28 patients with stable angina pectoris in addition to standard
therapy (atorvastatin, aspirin, clopidogrel, beta-blockers) were treated with
quinapril 20 mg − group 1; 26 patients were treated with telmisartan 40 mg
daily − group 2. The average level of total cholesterol was 4.5±1.2 mmol/l in
group 1 and 4.4±1.1 mmol/l in group 2. The MMP-2 and TIMP-2 levels in serum
were measured before PTCA, on the second day after procedure and after
6-month administration of quinapril and telmisartan.
Results: The baseline levels of MMP-2 and TIMP-2 were 162.3±27.2 ng/ml
and 63.3±8.1 ng/ml in group 1 and 147.5±32.9 ng/ml and 63.8±9.6 ng/ml in
group 2. There were no significant changes of these values on the second day
after PTCA. Levels of MMP-2 and TIMP-2 increased on 15.3% (p = 0.000017)
and 21.2% (p = 0.0002) after 6-month quinapril administration and on 12.2%
(p = 0.002) and 26.8% (p < 0.0001) after 6-month telmisartan administration
compared to baseline levels. It was no significant difference in characteristic
alterations in both groups.
78
Conclusion: Thus, 6-month treatment with quinapril and telmisartan almost
equally increase serum levels of MMP-2 and TIMP-2 in patients with stable
angina pectoris. This may indicate to mediated influence of renin-angiotensin
system on extracellular vascular matrix.
362 ATHEROGENIC LIPID PROFILE AND DEVELOPMENT OF
MULTIVESSEL CORONARY ARTERY DISEASE IN WOMEN
R. Saito, Y. Kawai, M. Watanabe, A. Motoyama, R. Ishida, M. Kitayama,
K. Kajinami. Dept of Cardiology, Kanazawa Medical University, Uchinada,
Japan
Background: Considerable sex differences exist in clinical characteristics of
coronary artery disease (CAD), and worse prognosis in women relative to men
has been considered, at least in part, as a result of greater prevalence of
multivessel disease at their first diagnoses.
Methods: To explore potential determinant of CAD severity in women,
we enrolled consecutive 70 patients (mean 72 yr), who received coronary
intervention for the first time from Jan. 2006 to Dec. 2009, and analyzed their
risk profiles according to their age.
Results: As a whole subjects, no significant differences were observed in the
prevalence of diabetes, hypertension, smoking, family history of CAD, body
mass index, and lipid profiles except for HDLC levels across single, double,
and triple vessel disease groups. Notably, in older women (>80 yr) (n = 15),
cholesterol levels in LDL (97 to 148 mg/dl) and in nonHDL (115 to 169 mg/dl)
significantly (p < 0.01) increased and decreased, respectively, according to
diseased vessel number. This association was not observed in younger women
group (<80 yr), nor other age-separated subgroups. Also in male patients
treated in the same time period (n = 187, mean 69 yr), association between
lipid profiles and disease severity could not be observed.
Conclusion: Atherogenic lipid profiles in women may confer sex-specific
development of multivessel CAD especially in older age.
363 RELATIONSHIP BETWEEN THE NUMBER OF STENOSED
CORONARY ARTERIES AND RENAL FUNCTION IN PATIENTS
WITH ISCHEMIC HEART DISEASE
N. Bardachenko1 , O. Kuryata1 , L. Bardachenko2 . 1 Dnipropetrovsk State
Medical Academy, 2 Dnipropetrovsk Regional Clinical Hospital, Dnipropetrovsk,
Ukraine
Background: There are several risk-prediction models for renal disease in
patients with ischemic heart disease (IHD) but models for relationship between
the number of stenosed coronary arteries (SCA) and renal dysfunction (RD)
are not used and remains debatable.
Objectives: we aimed to evaluate the relationship between the number of SCA
and RD in pts with IHD.
Methods: 220 pts (180 M, 40 F, mean age − 57.65±2.75 years) with IHD after
percutaneous coronary intervention (PCI) were enrolled. All pts were divided
into two groups: 110 (50%) pts (1 group) with glomerular filtration rate (GFR)
60−90 ml/min (mean level − 76.12±3.75 ml/min) and 110 (50%) pts (2 group)
with GFR >90 ml/min (mean level − 101.30±2.68 ml/min). The serum creatinine
(SCr) levels were determined in the blood and Cockcroft-Gault formula was used
to determine GFR.
Results: After PCI in 1 group 12.7% pts hadn’t SCA, 19.1% pts had 1 SCA,
30% pts had 2 SCA and 38.2% pts had 3 and more SCA when in 2 group 21.8%
pts hadn’t SCA, 24.5% pts had 1 SCA, 26.4% pts had 2 SCA and 27.3% pts had
3 and more SCA respectively. GFR correlated to the number of SCA (r = −0.22;
p < 0.05).
Conclusion: Our study showed a negative relationship between renal function
and the number of SCA, which indicates the need to assess renal function in
patients with IHD.
364 HUMAN PLASMA PHOSPHOLIPID TRANSFER PROTEIN (PLTP)
EXPRESSION IN TRANSGENIC RABBITS IS PROATHEROGENIC
D. Masson1,2 , V. Deckert1 , T. Gautier1 , A. Klein1 , C. Desrumaux1 , C. Viglietta3 ,
J.-P. Pais de Barros1 , N. Le Guern1 , J. Grober1 , J. Labbé1 , F. Ménétrier1 ,
P.-J. Ripoll4 , M. Leroux-Coyau3 , G. Jolivet3 , L.-M. Houdebine3 , L. Lagrost1,2 .
1
INSERM-Université de Bourgogne, UMR866, 2 CHU Dijon, Dijon, 3 INRA,
4
BioProtein Technologies, Jouy-en-Josas, France
Objectives: Plasma phospholipid transfer protein (PLTP) is involved in
intravascular lipoprotein metabolism. PLTP is known to act through two
main mechanisms: by remodelling HDL and by increasing apoB-containing
lipoproteins. The aim was to generate a new model of human PLTP transgenic
(HuPLTPTg) rabbit and to determine whether PLTP expression modulates
atherosclerosis in this species that, unlike humans and mice displays naturally
very low PLTP activty.
Methods and Results: In HuPLTPTg rabbits, the human PLTP cDNA was
placed under the control of the human eF1-a gene promoter, resulting in
a widespread tissue expression pattern and in increased plasma PLTP. The
HuPLTPTg rabbits showed a significant increase in the cholesterol content
of the plasma apoB-containing lipoprotein fractions, with a more severe trait
when animals were fed a cholesterol-rich diet. In contrast, HDL cholesterol
level was not modified in HuPLTPTg rabbits. Formation of aortic fatty streaks
was increased in hypercholesterolemic HuPLTPTg animals as compared to nontransgenic littermates.
Conclusion: Human PLTP expression in HuPLTPTg rabbit worsens atherosclerosis as a result of increased levels of atherogenic apoB-containing lipoproteins,
but not of alterations in their antioxidative protection or in cholesterol content of
plasma HDL.
365 RECOMBINANT ANTI-PHOSPHORYLCHOLINE IGG ANTIBODIES
PREVENT POST-INTERVENTIONAL ACCELERATED
ATHEROSCLEROTIC VASCULAR REMODELING
M.M. Ewing1,2 , S.A. Karabina3 , M. Nordzell4 , J.C. Karper2 , R. Atout3 , M.R. de
2
Vries , D. Sexton5 , H. Lettesjö6 , I. Dahlbom4 , O. Camber4 , J. Frostegard7 ,
E. Ninio3 , J.W. Jukema1 , K. Pettersson4 , P.H. Quax2 . 1 Cardiology, 2 Surgery,
Leiden University Medical Center, Leiden, The Netherlands, 3 INSERM
UMRS937, Université Pierre et Marie Curie UPMC-Paris and Faculté de
Médecine Pierre et Marie Curie, Paris, France, 4 Athera Biotechnologies AB,
Stockholm, Sweden, 5 Dyax Corporation, Boston, MA, USA, 6 Women’s and
Children’s Health, Uppsala University Hospital, Uppsala, 7 Medicine, Karolinska
University Hospital Huddinge and Karolinska Institutet, Stockholm, Sweden
Background: Human plasma contains both IgM and IgG antibodies against
phosphorylcholine (anti-PC), the dominating form being IgM anti-PC. Low levels
of IgM anti-PC are associated with increased risk for atherosclerosis-related
CVD and to poor prognosis in patients with ACS. The prototypic anti-PC,
the murine T15/E06 IgM, is shown to reduce atherogenesis, to have antiinflammatory effects and to prevent uptake of oxLDL by macrophages. Unfolded
protein response (UPR), a set of signaling pathways activated during stress, is
now emerging as an important link between inflammation and atherosclerosis.
Objective: We tested the efficacy of a recombinant human anti-PC T15 IgG1
antibody against accelerated atherosclerosis in vivo.
Methods and Results: Recombinant T15/E06 abs were used to passively
immunize ApoE*3Leiden mice on a Western-type diet undergoing femoral
arterial cuff placement to induce restenosis, using a single (3d protocol) or
twice weekly (14d protocol) injections of r-human T15 IgG1 (10 mg/kg), antistreptavidin IgG1 (anti-A2, negative control) or vehicle. T15/E06 significantly
reduced early macrophage and leukocyte infiltration in the arterial wall and
intimal thickening by >70% (p < 0.001) after 14d, accompanied by increased
relative SMC medial wall area and reduced relative leukocyte and macrophage
areas in the arterial wall. Expression of UPR markers GRP78 BiP and CHOP
in the arteries was reduced by T15/E06 treatment.
Conclusions: We showed that recombinant anti-PC IgG reduced accelerated
atherosclerosis in mice, potentially through UPR inhibition. Human IgG anti-PC
has therefore potential as a new treatment of inflammation-associated CVD.
366 THE NOVEL SPLEEN TYROSIN KINASE INHIBITOR FOSTAMATINIB
DISODIUM ATTENUATES INFLAMMATION AND ATHEROGENESIS
IN LOW DENSITY LIPOPROTEIN RECEPTOR DEFICIENT MICE
I. Hilgendorf1 , I. Remer1 , S. Eisele1 , K. Zeschky1 , C. Colberg1 ,
N. Hoppe1 , C. Bode2 , A. Zirlik1 , F. Willecke3 . 1 Atherogenesis Research
Group, Universitätsklinik Freiburg, Freiburg, 2 Department of Cardiology,
3
Universitätsklinik Freiburg, Freiburg im Breisgau, Germany
Background: Recently spleen tyrosine kinase (SYK), originally shown to
mediate immunoreceptor downstream signaling, came into focus as a potential
therapeutic target in chronic inflammatory diseases such as rheumatoid arthritis
and asthma. Interestingly, SYK was shown to be involved in the signaling of
various other receptors such as cytokine receptors and integrins. We therefore
hypothesized that inhibition of SYK would suppress the inflammatory process
underlying atherosclerosis and reduce plaque development in vivo.
Methods and Results: Low density lipoprotein receptor-deficient mice
consuming a high cholesterol diet (HCD) supplemented with two doses of
the orally available SYK inhibitor fostamatinib disodium (R788) for 16 weeks
showed a dose-dependent reduction in atherosclerotic lesion size by up
to 58.1±5.9% compared with respective control animals. Lesions of R788treated animals contained significantly less macrophages and significantly
more smooth muscle cells and collagen, characteristics associated with
more stable plaques in humans. Mechanistically, R788 treatment normalized
HCD-induced leukocytosis and inhibited inflammatory cells recruitment in
thioglycollate-induced peritonitis. Furthermore, R788 attenuated the expression
of inflammatory cytokines and chemokines both in vivo and in vitro in bone
marrow-derived macrophages and endothelial cells, cells typically resident in
atherosclerotic plaques.
Conclusion: We present the novel finding that SYK inhibition by fostamatinib
disodium, an agent that recently generated promising results in clinical phase
II trials for rheumatoid arthritis, attenuates atherogenesis in LDLR−/− mice. This
effect is most likely mediated by impairment of inflammatory cell recruitment and
reduction in inflammatory gene expression. Our data identify SYK inhibition by
fostamatinib disodium as a potential therapeutic strategy in atherosclerosis.
79th EAS Congress
367 COMPLEMENT FACTOR C5A MODULATES VEIN GRAFT
REMODELING AND ACCELERATED ATHEROSCLEROSIS VIA
INTERFERENCE IN MAST CELL ACTIVATION
M.R. de Vries1,2 , A. Wezel1,3 , A. Schepers1 , J. Kuiper3 , I. Bot3 , P.H.A. Quax1,2 .
1
Vascular Surgery, 2 Einthoven Laboratory of Experimental Vascular Medicine,
Leiden University Medical Center, 3 Biopharmaceutics, Leiden/Amsterdam
Center for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden,
The Netherlands
We previously showed that C3-activation is involved in vein graft disease (VGD).
The role of other complement-factors, including C5a, is unknown. We also
reported a role for Mast cells (MC) in atherosclerosis. MC express receptors for
complement-factor C5a, and can be activated by C5a.
We here studied the effect of C5a on MC activation in VGD and subsequent
accelerated atherosclerosis in ApoE-KO mice.
C5a-induced MC-activation in vitro resulted in increased tryptase release of
15% (p = 0.007). In murine veingrafts (n = 3−4/time point) MC tryptase mRNA
and protein levels as studied by rt-PCR and (immuno)histochemistry, decreased
after surgery and increased from 7 to 28d. C5a(R) levels increased after surgery
due to influx of inflammatory cells, than decreased and stabilized further on.
To study the effect of MC- and C5a-activation on VGD, C5a and DNP (a MCactivator) were applied locally around the vein graft during surgery (n = 10 ApoEKO/group) resulting in significant increased vein graft-thickening (VGT) at 28d
(79 and 36% resp. compared to control). This was accompanied by an increase
in perivascular MCs. Systemic application of C5aR-antagonist or Cromolyn (a
MC-stabilizer) resulted in significant decreased VGT (40 and 22% resp.). MC
were decreased by 40% and 24%. To asses the direct activation of MC by
C5a, mice were treated with C5a and Cromolyn, VGT was decreased by 54%
(p = 0.0002) compared to C5a-treated mice, to the level of Cromolyn treated
mice.
These data give a strong indication that the chemotactic potency of C5a on MC
and their subsequent activation play an important role in VGD.
368 INTERLEUKIN 15 PLAYS AN IMPORTANT ROLE IN
ATHEROGENESIS IN APOLIPOPROTEIN E KNOCKOUT MICE
O. Dadoo, A. Ashkar, C. Richards, B. Trigatti. McMaster University, Hamilton,
ON, Canada
Objective: To test the role of interleukin 15 in atherogenesis il-15 knockout
and il-15 transgenic mice were crossed to apoE−/− mice to generate apoE−/−
mice that were also either il-15−/− , il-15+/− , il-15+/+ , or overexpressing il-15transgenic.
Results: IL-15 deficiency reduced atherosclerosis in spite of increased plasma
cholesterol. Interestingly, il-15+/− apoE−/− mice exhibited intermediate levels of
atherosclerosis in the aortic sinus. Overexpression of IL-15 in il-15-transgenic
apoE−/− mice resulted in increased atherosclerosis compared to apoE−/−
littermates. Deletion of IL-15 resulted in a lack of NK cells and reduced
populations of monocytes, and CD8a+ T cells. Correspondingly, reduced levels
of CD11b+ monocytes/macrophages, and CD8a+ T cells were detected in
atherosclerotic lesions from il-15−/− apoE−/− mice. To test the role of NK cells in
atherosclerosis, NK1.1 positive cells were immune-depleted in apoE−/− mice.
Analysis revealed that atherosclerosis was reduced but to a lesser extent than
in age matched il-15−/− apoE−/− mice. Atherosclerotic plaques from these mice
were almost devoid of CD8+ T cells. In contrast, deletion of a single copy of
il-15 gene in NK cell immunodepleted apoE −/− mice resulted in a further
reduction in atherosclerosis, suggesting the involvement of NK/CD8-T cell
independent pathways participate in IL-15’s proatherogenic effects. Treatment of
macrophages with rIL-15 in vitro induced expression of pro-inflammatory targets
including iNOS, IL-6, MCP-1, and RANTES. Furthermore, il-15−/− apoE−/− mice
exhibited reduced levels of MCP-1 in plaques and reduced circulating levels
of SAA and IL-6 compared to il-15+/+ apoE−/− mice, whereas circulating levels
of SAA and IL-6 were greatly increased in il-15tg apoE KO mice compared to
apoE KO littermates.
369 NUMERICAL AND FUNCTIONAL DEFECTS OF CIRCULATING
DENDRITIC CELLS IN PATIENTS WITH CORONARY ARTERY
DISEASE
I. Van Brussel1 , E.A. Van Vré1 , G.R. De Meyer2 , C.J. Vrints1,3 ,
J.M. Bosmans1,3 , H. Bult2 . 1 Cardiology, 2 Pharmacology, University of Antwerp,
Wilrijk, 3 Cardiology, University Hospital of Antwerp, Edegem, Belgium
Background: We investigated whether activation of circulating dendritic cells
(DCs) or levels of FMS-like tyrosine kinase 3 ligand (Flt3L) and granulocytemacrophage colony stimulating factor (GM-CSF), haematopoietic growth factors
important for DC differentiation, could account for the decline of blood DC
numbers in patients with coronary artery disease (CAD).
Methods: Flt3L and GM-CSF were measured in plasma from CAD patients
(n = 15) and age-matched healthy volunteers (n = 12). Frequency and phenotype
of myeloid (mDCs) and plasmacytoid (p)DCs were analysed by multicolour flow
cytometry in fresh blood, and after overnight incubation with Toll-like receptor-4
79
(lipopolysaccharide) or -7 (imiquimod) ligands. DC function was measured by
interleukin (IL)-12 and interferon (IFN)-alpha secretion.
Results: Circulating numbers of CD11c+ mDCs and CD123+ pDCs and
frequencies of CD86+ and CCR-7+ mDCs, but not pDCs, were declined in
CAD. Also plasma Flt3L, but not GM-CSF, was lower in patients and positively
correlated with blood DC counts. In response to lipopolysaccharide, mDCs
upregulated CD83 and CD86, but CCR-7 expression and IL-12 secretion
remained unchanged, similarly in patients and controls. Conversely, pDCs from
patients displayed lower CD83 and CCR-7 expression after overnight incubation
and showed a weaker imiquimod-induced upregulation of CD83 and IFN-alpha
secretion.
Conclusions: Our data suggest that reduced blood DC counts in CAD are, at
least partly, due to impaired DC differentiation from bone marrow progenitors.
Decreased mDCs are presumably also explained by activation and subsequent
migration to atherosclerotic plaques or lymph nodes. Although mDCs are
functioning normally, pDCs from patients seemed to be both numerically and
functionally impaired.
370 LOSS OF PHOSPHOINOSITIDE 3-KINASE GAMMA ACTIVITY LEADS
TO DECREASED NEOINTIMA FORMATION AFTER VASCULAR
INJURY IN MICE
N. Smirnova1 , N. Malet1 , S. Gayral1 , A.-P. Gadeau2 , M. Wymann3 ,
L. Martinez1 , M. Laffargue1 . 1 INSERM U1048, Toulouse, 2 INSERM U1034,
Bordeaux, France, 3 Institute of Biochemistry and Genetics, University of Basel,
Basel, Switzerland
Background: Intimal hyperplasia is the narrowing of an artery in response
to an injury, characterized by fibroproliferative and inflammatory mechanisms.
Phosphoinositide 3-kinase gamma (PI3Kg) is a member of the PI3K family
abundantly expressed in immune cells and to a lesser extent in the
cardiovascular system. Regarding its important role in vessel wall inflammation
we investigated here the possible implication of PI3Kg in intimal hyperplasia
which is currently unknown.
Methods and Results: We therefore used a mouse model of mechanical
injury of the femoral artery and studied the impact of PI3Kg activity extinction
on subsequent intimal thickening. PI3Kg deficient mice and mice expressing
a catalytically inactive form of PI3Kg showed a dramatic decrease in
neointima/media ratio (about 50%) compared to wild type mice 28 days after
the lesion was performed. These changes were associated with a defect of
inflammatory cell infiltration to the lesion site at both late and early stages of
neointimal formation.
Conclusion: Our data clearly show an important role for PI3Kg in the
progression of intimal hyperplasia pinpointing PI3Kg as a good candidate for
drug development in vascular diseases.
371 LTB4 PATHWAY ACTIVATION: ONE OF THE MECHANISMS
UNDERLYING EARLY ATHEROSCLEROSIS IN OBSTRUCTIVE
SLEEP APNEA SYNDROME
F. Stanke-Labesque1 , J.-L. Pépin1 , T. de Jouvencel2 , C. Arnaud1 , J.-P. Baguet1 ,
R. Tamisier1 , J.F. Jourdil1 , P. Lévy1 , M. Bäck2,3 . 1 Grenoble University Hospital,
Grenoble, 2 Bichat University Hospital, Paris, France, 3 Center for Molecular
Medicine, Karolinska Institutet, Stockholm, Sweden
Obstructive sleep apnea (OSA) is characterized by nocturnal intermittent
hypoxia, associated with the development of early atherosclerosis. The aim
of the present study was to evaluate the inflammatory mediator leukotriene
B4 (LTB4), derived from the 5-lipoxygenase pathway of arachidonic acid
metabolism, as a potential link between OSA and atherosclerosis. To this
end, blood samples from 153 OSA patients and 45 controls were used for
isolation of polymorphonuclear neutrophils (PMN) and monocytes, followed by
mRNA and protein extraction and real time TaqMan PCR and Western blot.
Cellular LTB4 production was measured using liquid chromatography-tandem
mass spectrometry.
LTB4 production was significantly increased in PMNs derived from OSA patients
compared with controls, and correlated with the severity of disease, with mean
nocturnal O2 saturation being an independent predictor of LTB4 concentrations
(P = 0.03). PMN mRNA and protein levels of the 5-lipoxygenase activating
protein (FLAP) were significantly higher in OSA patients compared with controls,
and were significantly associated with carotid luminal diameters (r = 0.391,
P = 0.02) and intima media thickness (r = 0.352, P = 0.04). In vitro exposure of
PMNs from healthy subjects to 4-hour intermittent hypoxia induced a significant
increase in FLAP mRNA levels. Finally, stimulation of monocytes derived from
OSA patients with LTB4 significantly increased the production of IL-6 and
MCP-1.
In summary, upregulation of FLAP in PMNs in response to intermittent hypoxia,
leading to paracrine LTB4-induced effects on monocytes may participate in
early vascular remodeling in OSA patients. In conclusion, FLAP may represent
a potential therapeutic target for attenuating the cardiovascular morbidity
associated with OSA.
80
372 SELECTIVE DEPLETION OF T REGULATORY CELLS IMPACTS
EXPERIMENTAL ATHEROSCLEROSIS PROGRESSION AND VLDL
METABOLISM
R. Klingenberg1 , N. Gerdes2 , R.M. Badeau3 , D. Ketelhuth3 , M. Rudling4 ,
D. Strodthoff3 , S.K. Nilsson5 , K. Öörni6 , M. Jauhiainen7 , C. Lohmann1 ,
T. Lüscher1 , K. Lahl8 , T. Sparwasser8 , G.K. Hansson3 . 1 Experimental
Cardiovascular Research Unit, University Zurich, Irchel (ZHIP), Zurich,
Switzerland, 2 Institute for Molecular Cardiovascular Research (IMCAR),
University Hospital Aachen, Aachen, Germany, 3 Center for Molecular Medicine,
Department of Medicine, Karolinska Institutet at Karolinska University
Hospital − Solna, 4 Metabolism Unit, Department of Endocrinology, Metabolism
and Diabetes and Molecular Nutrition Unit, Department of Medicine and
Center for Biosciences and Nutrition, Karolinska Institutet at Karolinska
University Hospital − Huddinge, Stockholm, 5 Department of Physiological
Chemistry, Umeå University, Umeå, Sweden, 6 Wihuri Research Institute,
7
Department of Chronic Disease Prevention, National Institute for Health and
Welfare, Helsinki, Finland, 8 Institute for Infection Immunology, TWINCORE
Center for Experimental and Clinical Infection Research, Helmholtz Center for
Infection Research and Hanover Medical School, Hannover, Germany
Introduction: Chronic inflammation drives atherosclerosis progression.
Immunological factors play a major role in orchestrating this disease. T
regulatory cells (Treg) inhibit the activity of proinflammatory effector T cells
and are therefore thought to be atheroprotective. The impact of Tregs on lipid
metabolism and atherosclerosis was investigated.
Methods: Mice containing a transgenic diphtheria toxin receptor enhanced
green fluorescent protein fusion protein under control of the foxp3 gene
promoter (DEREG) donated bone marrow to recipient LDLr−/− mice (eGFP
Foxp3) which were previously irradiated. Diphtheria toxin treatment ablated
the Foxp3 Treg population. After challenged 8 weeks with high fat diet,
atherosclerosis parameters were assessed.
Results: Compared to Treg-containing mice (eGFP+ Foxp3+ ), Treg deficient
mice (eGFP+ Foxp3− ) had significantly: i.) larger atherosclerotic plaques; i.)
increased hypercholesterolemia; iii.) accumulated cholesterol-rich/triglyceride
poor VLDL particles; iv.) delayed chylomicron and VLDL plasma clearance;
v.) raised post-heparin plasma LPL activity; vi.) elevated hepatic LXRa and
ABCG5 gene transcription; vii.) increased proinflammatory hepatic cytokines;
viii.) amplified SRA and CD36 gene expression; ix.) heightened intestinal Mtp
gene expression. Between the two groups, triton W-1339 treatment, which
evaluates VLDL synthesis, and liver Mtp gene levels as well as both hepatic
LRP1 and syndecan 1 gene and protein levels were unaltered. Surprisingly, no
effects were registered on arterial inflammation.
Conclusion: These results indicate that Tregs protect against atherosclerosis
and suggest that they modulate lipid homeostasis. Treg deficiency affects
hepatic and intestinal lipid metabolic targets and impacts VLDL metabolism.
These data unravel connections among adaptive immunity, lipoprotein
metabolism, and atherosclerosis.
373 MACROPHAGE SUBSETS VARY IN THEIR ASSOCIATION WITH
PLAQUE STABILITY
H.J. Medbury, J. Ngo, V. James, K. Hitos, A.K. Guiffre, J.P. Fletcher. Vascular
Biology Research Centre, Surgery, University of Sydney, Westmead Hospital,
Westmead-Sydney, NSW, Australia
Background: Macrophages play a major role in atherosclerosis, contributing
to both core formation and cap degradation. However, macrophages are not a
homogenous population as, apart from the traditional inflammatory phenotype
(M1), they can also adopt various immune regulation and wound healing
phenotypes (M2). The role of these alternative macrophages in atherosclerosis
is not known, but we propose they may promote plaque stability.
Objective: To examine the association between macrophage phenotype and
plaque stability.
Methods: Human carotid atherosclerotic plaque specimens were examined by
immunohistochemistry for macrophage subset distribution and comparisons
made between stable and unstable plaques. Macrophage contribution to
plaque stability was assessed by double immunofluorescence staining of the
macrophage markers with procollagen-I.
Results: M1 and M2 macrophages were present throughout the plaque
with no significant difference between M1 and M2 levels in either the core
or cap. There was however, significantly more CD86 (M1) in the cap of
unstable compared to stable plaques (P < 0.05); whereas M2 markers were
found in equivalent densities. There was a greater polarisation towards an
M1 phenotype in the unstable plaque as evidenced by a significantly greater
ratio of (CD86:M1/CD163:M2) in the cap of unstable compared to stable
plaques (P < 0.05); a trend that was also evident in the core. Notably, collagen
production was only evident in M2 (CD163) not M1 (CD86, CD64) macrophages
(P < 0.05).
Conclusion: Macrophage subsets vary in their function in the atherosclerotic
plaque. M1 macrophages are associated with an unstable plaque while
M2 macrophages may contribute to plaque stabilisation through collagen
production.
374 IMPORTANCE OF VIMENTIN IN FOAM-CELL FORMATION AND
ATHEROGENESIS
J. Perman, J. Borén. Department of Molecular and Clinical Medicine, University
Objective: Vimentin is an intermediate filament protein important for
intracellular lipid storage and is several-fold increased in the macrophage-tofoam-cell differentiation. Vimentin has also been proposed to be important
for the macrophage defense mechanism. In this study we investigate the
importance of vimentin in foam-cell formation and atherogenesis.
Methods and Results: Mice were bone marrow transplanted with vim+/+ or
vim−/− hematopoietic stem cells, mice given bone vim−/− cells showed an
significantly reduced sub-endothelial lipid accumulation with increased intraplaque T-cells and increased plasma cytokine KC without any other significant
differences in plaque phenotype. Investigating the mechanisms for this vim+/+
and vim−/− bone-marrow derived macrophages were incubated at normoxia and
hypoxia with minimally modified LDL resulted in significantly lower accumulation
of intracellular triglycerides and uptake of LDL in vim−/− cells compared to
vim+/+ macrophages. These results depend on a reduced expression of surface
receptors important for lipoprotein uptake. Vim−/− macrophages also have an
increased secretion of T-cell recruiting cytokines KC and RANTES as well
as increased lipid peroxidation capacity and increased CD36 expression. The
increased CD36 expression is followed by increased uptake of free fatty acids.
Conclusion: These results indicate that vimentin deficiency reduces the
expression of cell surface lipoprotein receptors and thereby the lipid
accumulation in vitro and the sub-endothelial lipid accumulation in vivo.
Vimentin is also involved in the immune response where the lack of vimentin
increases the intracellular amount of free fatty acids and thereby triggering the
immune response.
375 LEUKOTIENE-INDUCED CALCIFICATION AND OSTEOGENIC
DIFFERENTIATION OF HUMAN AORTIC VALVE INTERSTITIAL
CELLS
E. Nagy, D.C. Andersson, G.K. Hansson, M. Bäck. Center for Molecular
Medicine, Karolinska Institutet, Stockholm, Sweden
Objective: Aortic valve stenosis is characterized by inflammation − akin to
early atherosclerotic lesion, a key feature preceding pronounced calcification
of the leaflets. Valvular interstitial cells (VIC) are a mesenchymal cell type that
share phenotypic similarities with vascular smooth muscle cells. The present
study investigated the hypothesis that leukotriene (LT) C4, an arachidonic acidderived proinflammatory mediator, as a potential link between inflammation and
calcification.
Methods: Primary cultures of human aortic VICs were established and
incubated in absence or presence of LTC4 (1−10 nM) for measurements
of cytosolic calcium concentrations, and reactive oxygen species (ROS).
Furthermore, RNA extraction was used for determination of transcriptional levels
of bone morphogenetic protein (BMP)-2 and -6 using TaqMan real-time PCR.
Finally, calcfication studies were performed with cell culture medium (DMEM)
supplemented with b-glycerophosphat, ascorbic acid, and CaCl2.
Results: Immunofluorescens stainings revealed expression of the CysLT1
receptor for LTC4 on VICs. In addition, LTC4 increased intracellular calcium and
enhanced ROS production (110±26%, n = 20, P < 0.001) in VICs. Moreover,
after 24h stimulation with LTC4, VIC exhibited 2.0±0.87-fold and 2.1±0.59-fold
increase in mRNA levels of BMP-2 and BMP-6, respectively (P < 0.05 compared
with untreated). Finally, after 2 weeks incubation, VICs treated with LTC4
showed enhanced cell aggregation and increase in calcium nodule formation
compared with untreated cells, evaluated by Alizarin red staining.
Conclusion: These results indicate that LTC4 induced oxidative stress,
osteogenic activation and calcification in primary culture of VICs. Collectively,
these findings suggest the LTC4-CysLT1 pathway as a potential therapeutic
target for inhibiting inflammation-associated vascular calcification.
376 INTERLEUKIN-6 REGULATES LXRa-RESPONSIVE EXPRESSION
OF ABCA1 AND ABCG1 VIA A JAK/STAT SIGNALING
PATHWAY-DEPENDENT WAY
C.-K. Tang, J. Xiao, K. Yin. Institution of Cardiovasclar Disease, University of
South China, Hengyang, China
Objective: The aim of the present study was to determine the effects and
potential mechanisms of IL-6 on ABCA1 and ABCG1 expression and cholesterol
efflux.
Methods and Results: Human THP-1 cells preincubated oxLDL served as
foam cell models. IL-6 significantly decreased the expression of ABCA1
and ABCG1 and cholesterol efflux in the THP-1 macrophage-derived foam
cells. Liver X receptor a which can regulate the expression of ABCA1 and
ABCG1 was also down-regulated by IL-6 treatment. IL-6 treatment altered the
activity of the LXRE-driven luciferase reporter in the foam cells. Other LXR
target genes such as ABCG5 and ABCG8 were also downregulated by IL-6
treatment. Furthermore, JAK inhibitor piceastannol or STAT3 siRNA reduced
phosphorylation of STAT3 and completely reversed the effect of IL-6 on LXRa.
79th EAS Congress
Under the casein-induced inflamed animal model, IL-6R signaling blockade
by tocilizumab resulted in an enhanced expression of ABCA1, ABCG1 and
LXRa in peritoneal macrophages and aortae, while a reduction in aortic root
atherosclerotic lesion area.
Conclusions: IL-6 decreased ABCA1 and ABCG1 expression and inhibited
cholesterol efflux, which was mediated by IL-6-induced JAK/STAT3 signaling
pathway and accompanying LXR inhibition.
377 EFFECTS OF TACROLIMUS-ELUTING STENTS IN A DIABETIC
RABBIT MODEL OF IN-STENT RESTENOSIS
P.W. Radke1 , A. Joost1 , A. Kaiser2 , M. Mandapathil2 , M. Basler2 ,
S. Yla-Herttuala3 , C. Weber2 , W. Ito1 . 1 Medizinische Klinik II, Universität zu
Lübeck, Lübeck, 2 RWTH Aachen, Aachen, Germany, 3 University of Kuopio,
Kuopio, Finland
Aims: Inflammation is a key pathophysiological component of restenosis
after stent implantation, especially in diabetics. We evaluated the effects of
Tacrolimus (FK506) eluting stents (173 mg, TES), a water-insoluble macrolide
immunosuppressant with anti-inflammatory properties, in diabetic and nondiabetic rabbits.
Methods: New Zeeland White rabbits (NZW, 3.0±0.2kg) were fed a cholesterolrich diet. Diabetes mellitus was induced by alloxan injections (70 mg/kg
i.v.). Animals with a fasting blood sugar 250 mg/dl were classified as
“diabetic” (n = 9). Control animals were not subjected to Alloxan injections
(n = 9). Tacrolimus-eluting (n = 18) and bare metal control stents (n = 18)
were implanted in the iliac arteries (after endothelial denudation). 8 weeks
post intervention, the extent of neointimal inflammation (macrophage marker
RAM-11 immonuhistochemistry) and neointimal proliferation (morphometric
measurements) were quantified.
Results: In diabetic animals, the degree of neointimal inflammation was
significantly lower when TES were implanted as compared to control stents
(16.8±6.4 cells/field vs. 24.3±5.9 cells/field, p < 0.05), however, the neointimal
thickness (1.13±0.31 mm2 vs. 0.88±0.32 mm2 , p=ns) as well as the intima/
media ratio (4.92±2.58 vs. 3.94±1.52, p=ns) were not different between stent
groups. In non-diabetic animals, the extent of neointimal inflammation and
proliferation did not differ between TES and control stents.
Conclusions: In Alloxan treated diabetic rabbits, but not in non-diabetic
animals, implantation of TES leads to a significant reduction in neointimal
inflammation. The extent of neointimal proliferation after TES implantation,
however, is not affected in both animal groups.
378 IMPACT OF ANTI-TNFALPHA THERAPY ON SUBCLINICAL
ATHEROSCLEROSIS IN PATIENTS WITH PSORIATIC ARTHRITIS
M. Puato, M. Zanardo, R. Ramonda, E. Faggin, M. Zanon, G. Balbi, A. Lo
Nigro, M. Rattazzi, A. Doria, P. Pauletto. Dipartimento di Medicina Clinica e
Sperimentale, Università di Padova, Padova, Italy
Objective: We evaluated the impact of 2 years of anti-TNFalpha therapy on
structural and functional properties of arteries in psoriatic arthritis (PsA) patients
stratified according to the presence/absence of hypertension.
Methods: We studied 42 PsA (23 hypertensives) who fulfilled the Moll-Wright
criteria. 44 normotensives (N-C) and 22 hypertensives (HT-C) served as
controls. We evaluated carotid intima media thickness (IMT) expressed as
mean-IMT (mean of mean-IMT measured in common, bulb, and internal carotid
artery, bilaterally) and as M-MAX (mean of maximum-IMT). Endothelial function
was evaluated by post-occlusion flow mediated dilation (FMD) of the brachial
artery by ultrasound.
Results: PsA had a higher mean-IMT compared to N-C. Hypertensive PsA
displayed higher M-MAX versus both HT-C and normotensive PsA. FMD was
significantly lower in PsA than in NC (8.9%), whereas there was no difference
between hypertensive PsA (6.1%), normotensive PsA (5.7%), and HT-C (6.3%).
The TNFa level was higher in PsA than in the other groups. IMT was related to
TNFa levels, blood pressure and lipids, whereas FMD was inversely related
to TNFa levels and blood pressure but not lipid parameters. At follow up,
although clinical status improved, IMT progressed similar in both normotensive
and hypertensive PsA (0.08 mm/year). FMD remained impaired.
Conclusions: Subclinical atherosclerosis is enhanced in PsA compared to NC.
In PsA, the hypertensive status proved to exert an additional effect on M-MAX.
FMD was reduced in PsA irrespective of hypertensives status. Thus, PsA per
se implies a pro-atherogenic remodelling which was not affected by 2 years of
anti-TNFalpha therapy.
379 THE AKITA-LDLR−/− MOUSE AS A MODEL OF DIABETIC
ATHEROSCLEROSIS
D. Engelbertsen, F. To, P. Dunér, O. Kotova, I. Söderberg, R. Alm, M. Gomez,
J. Nilsson, E. Bengtsson. Lund University, Malmö, Sweden
Objective: Diabetes is associated with detrimental macrovascular complications causing myocardial infarction and stroke. Current mouse models
of cardiovascular complications in type 1 diabetes are confounded by the
81
deleterious side-effects of diabetes induction. The Akita mouse has insulindependent diabetes and has been successfully used as a model of diabetic
microvascular complications. In this study we characterized the cardiovascular
complications in Akita-mice on an atherosclerotic background.
Results: Male and female Akita-LDLr−/− and LDLr−/− mice were fed highfat diet and sacrificed at 18 or 24 weeks of age. Akita-LDLr−/− mice were
hyperglycemic throughout the study. Both male and female Akita-LDLr−/− mice
had higher cholesterol levels than LDLr−/− control mice, whereas triglyceride
levels were only elevated in male Akita-LDLr−/− compared to control mice.
Akita-LDLr−/− males had a five-fold increased aortic lesion area compared
to LDLr−/− controls. No difference in lesion area was observed in the female
groups. Analysis of plaque composition showed that Akita-LDLr−/− male mice
had increased macrophage immunoreactivity as well as increased T-cells. In
addition, mRNA expression of IL-6 in brachiocephalic artery was positively
correlated to glucose levels in Akita-mice. Analysis of plasma cytokine levels
revealed increased IL-1b levels in Akita-LDLr−/− males, and the IL-1b levels
were positively associated to plasma glucose.
Conclusions: Hyperglycemic male Akita-LDLr−/− mice display both dyslipidemia and increased atherosclerotic burden, capturing important aspects of the
human disease. However, female Akita-LDLr−/− mice, although hyperglycemic,
do not develop larger lesions compared to non-diabetic controls. Also, we
present the first in vivo evidence of IL-1b being associated with diabetic
atherosclerosis.
380 EFFECTOR MEMORY T LYMPHOCYTES ARE ASSOCIATED
WITH CARDIOVASCULAR DISEASE IN HUMANS AND AORTIC
ATHEROSCLEROSIS IN APOE AND LDL-R KNOCK-OUT ANIMALS
G. Norata1 , E. Ammirati E2 , M. Banfi2 , V. Vacchio2 , S. Tramontana3 ,
K. Garlaschelli3 , F. Pelegatta3 , L. Grigore3 , D. Cianflone2 , A. Maseri4 ,
A. Catapano1 , A. Palini2 . 1 Department of Pharmacological Sciences,
University of Milan, 2 Università Vita-Salute San Raffaele, Milan, 3 Centro SISA
per lo Studio dell’Aterosclerosi, Bassini Hospital, Cinisello Balsamo, 4 Heart
Care Foundation, Florence, Italy
Background: Differentiation of naı̈ve CD4 T cells to effector and/or memory
cells of specialized phenotypes is a key event observed during atherogenesis.
Here, we investigated the relation between circulating T-cells subsets and
atherosclerosis in subjects with subclinical atherosclerosis, in patients with
stable and unstable coronary artery disease and in animal models of
atherosclerosis.
Methods and Results: 56 subsets of circulating CD4 T cells were
investigated by 8/color/polycromatic flow cytometry (CD3, CD4, CD45RO,
CD45RA, CCR7, CCR5, CXCR3, HLA-DR) in 183 subjects selected from
the general population (PLIC study). T effector memory (TEM), (marked
as CD3+CD4+CD45RO+CD45RA−CCR7− cells) is the subset that better
correlates with IMT even after adjustments for multiple cardiovascular risk
factors. We confirmed these findings in a cohort of patients with coronary
atherosclerosis (n = 117), where TEM levels resulted significantly increased
in patients compared to controls. In animal models, TEM circulating cells
(CD4+CD44+CD62L−) were significantly increased in LDL-R KO and ApoE KO
animals fed a western type diet for 16 weeks compared to C57BL/6J mice
fed either chow or western type diet. Of note TEM levels were significantly
correlated with the extent of atherosclerotic lesions in the aortic root.
Conclusions: In humans, among 56 different subsets of circulating CD4 T cells,
TEM levels are associated with increased atherosclerosis and coronary artery
disease independently of other cardiovascular risk factors. In animals models
TEM circulating levels are significantly correlated with the extent of aortic
atherosclerosis. T-effector memory cells could therefore represent one of the
key T cell subsets related to atherosclerosis.
381 INFLUENCE OF INDUCIBLE NITRIC OXIDE SYNTHASE IN THE
DEVELOPMENT OF ANEURYSMS IN THE ABDOMINAL AORTA
OF RATS: NEW EXPERIMENTAL MODEL
P. Prudente, K. Mata, E. Floriano, J. Junior, S. Ramos. São Paulo University,
Ribeirão Preto, Brazil
Degradation of extracellular matrix and proteins associated with inflammation
of the aortic wall is the main characteristics of the abdominal aortic aneurysms
(AAA). This research aimed to evaluate the effect of inducible nitric oxide
synthase (iNOS), and metalloproteinases (MMPs) 2 and 9, in the induction
of a new model of abdominal aortic aneurysm (AAA) in Wistar rats, using two
potential causes of MMPs release: vascular lesion in the aorta and external
local turbulence in blood flow. Animals were divided into four groups: Group
AAA (extrinsic stenosis of the abdominal aorta associated with traumatic injury
to the outer layer) and Group Sham (control). These groups were doubled: half
received PBS and half 1400W, a selective inhibitor of iNOS. Aneurysm formation
(dilation of 300%) was observed in 60−70% of the animals in group AAA by
intense remodeling of the arterial wall, characterized by severe inflammatory
response composed mainly of neutrophils and macrophages, destruction
of elastic fibers, mesenchymal proliferation with collagen deposition and
neovascularization. Immunohistochemistry showed the effective participation of
82
NO and MMP-2 and -9 in the development of aneurysms. 83% of the animals in
the group treated with 1400W did not develop aneurysms. These results point
to an effective participation of iNOS, consequently of NO and MMPs, especially
2 and 9, in the formation of AAAs.
382 IMPORTANT CYTOKINES AND CHEMOATTRACTANTS FOR
VULNERABLE ATHEROSCLEROTIC PLAQUE AND ACUTE
CORONARY SYNDROME. RESULTS OF ARTERIAL WALL AND
BLOOD STUDIES
Y.I. Ragino1 , A.M. Chernjavski2 , Y.V. Polonskaja1 , A.M. Volkov2 ,
E.V. Kashtanova1 . 1 Institute of Internal Medicine of SB RAMS, 2 Research
State of Circulation Pathology, Federal Agency of Public Health and Social
Development, Novosibirsk, Russia
Changes of concentrations of inflammatory cytokines and chemoattractantsc,
included interleukins (IL)-1-beta, IL-3, IL-5, IL-6, IL-7, IL-8, IL-12, IL-18, high
sensitive C-reactive protein (hsCRP), monocytes chemotactic protein (MCP-1),
macrophage inflammatory proteins (MIP-1-alpha and MIP-1-beta), granulocyte
macrophage colony-stimulating factor (GM-CSF), sCD40 ligand (sCD40-L),
endothelial monocytes activating protein (EMAP-II), adhesive molecules
(sICAM-1, sVCAM-1, sE-selectin) during of consecutive stages of coronary
artery atherosclerotic plaque formation and in blood of coronary atherosclerosis
men were studied. The study was included 246 men with angiographic proven
coronary atherosclerosis with (144 men) and without (102 men) of acute
coronary syndrome (ACS). Collecting of intima/media fragments was performed
during coronary artery bypass grafting operation by endarterectomy procedure.
After histological analysis of fragments the “normal intimae” was revealed in
25 cases, “lipid stain” − in 29, “early stable plaque” − in 40, “stable plaque
with fibrosis” − in 47, “vulnerable plaque” − in 51. Biomarker concentrations in
homogenate of intima/media fragments after protein measuring and in blood
were investigated by ELISAs kits. Increased levels of IL-2, IL-6, IL-8, IL-18,
hsCRP, MIP-1-alpha, sCD40-L, MCP-1, GM-CSF and EMAP-II were typical for
unstable/vulnerable atherosclerotic plaques. On the other hand, increased blood
levels of IL-6, IL-8, IL-18, hsCRP, sCD40-L, MCP-1, EMAP-II, sVCAM-1 and
sE-selectin were typical for ACS men. Thus, several inflammatory cytokines
and chemoattractants, the same as IL-6, IL-8, IL-18, hsCRP, sCD40-L, MCP-1
and EMAP-II can be key blood biomarkers not only for ACS, but for vulnerable
atherosclerotic plaques of coronary artheries too.
The study was supported by Grant RFBI 09−04–00374.
383 ANNEXIN A5 PREVENTS REACTIVE STENOSIS IN A DOSEDEPENDENT FASHION: POTENTIAL FOR CLINICAL APPLICATION
M.M. Ewing1,2 , K. Pettersson3 , J.C. Karper2 , M.R. de Vries2 , M.L. Sampietro4 ,
J.W. Jukema1 , P.H. Quax2 . 1 Cardiology, 2 Surgery, Leiden University Medical
Center, Leiden, The Netherlands, 3 Athera Biotechnologies AB, Stockholm,
Sweden, 4 Human Genetics, Leiden University Medical Center, Leiden, The
Netherlands
Background: Annexin A5 is an anti-coagulant protein that binds to exposed
phosphatidylserine on activated and apoptotic endothelial cells and platelets.
We recently showed that it inhibits restenosis in a murine CABG model. Here,
we report a potential role for annexin A5 against vascular inflammation.
Methods and Results: To study the effects on inflammatory-driven intimal
thickening in vivo, ApoE−/− mice on a Western-type diet underwent femoral
arterial cuff placement to induce reactive stenosis and were treated with annexin
A5 (0.1, 0.3 and 1.0 mg/kg/d) for 3 or 14days. Annexin A5 as compared
to controls dose-dependently reduced inflammation with fewer adhering and
infiltrating leukocytes and macrophages (3d). It also significantly reduced intimal
thickening by 54.6–71.2%, reduced luminal stenosis and intima/media ratio
(14d). All doses were effective, although 1 mg/kg was the most effective.
Furthermore, we report significant association between the rs4833229 and
rs6830321 SNPs in the annexin A5 gene and increased restenosis risk in
patients from the GENDER (Genetic Determinants of Restenosis) population,
undergoing PCI.
Conclusions: Annexin A5 is anti-inflammatory and prevents vascular
remodeling in dosages that do not affect hemostasis. SNPs in the annexin
A5 gene could serve as markers for restenosis after PCI, together indicating
clinical potential for annexin A5 against restenosis.
384 SILENCING OF SOCS-3 GENE DUE TO HYPERMETHYLATION
INDUCED BY BOTH TNF-a AND IGF-1 IN HUMAN CORONARY
ARTERY SMOOTH MUSCLE CELLS
K. Dhar, D. Pankajakshan, D.K. Agrawal. Center for Clinical & Translational
Science, Creighton University School of Medicine, Omaha, NE, USA
Purpose: Inflammatory cytokine, TNF-a, and mitogen, IGF-1, play a critical role
in the pathogenesis of atherosclerosis and intimal hyperplasia. Suppressor of
cytokine signaling-3 (SOCS-3) negatively regulates cytokine signaling through
the JAK/STAT pathway. Here, we investigated the effect of TNF-a and IGF-1,
individually or in combination, on mRNA and protein expression of SOCS-3 in
human coronary artery smooth muscle cells (HCASMCs).
Methods: HCASMCs were stimulated with TNF-a and IGF-1 for 24 hours and
SOCS-3 expression was examined by qPCR and Western blotting.
Results: qPCR data revealed significantly high SOCS-3 expression in
HCASMCs in the presence of TNF-a or IGF-1 (8−10 fold) alone, but a
significant decrease (5-fold) was seen in presence of both TNF-a and IGF-1.
Similar pattern was found in the protein expression. To evaluate the underlying
mechanisms of this phenomenon, DNA methylation studies were done using
methylation-specific PCR in four different CpG regions of SOCS-3 promoter. The
identified methylation site of the DNA was a signal transducer and activators of
transcription (STAT) binding consensus sequence. We further investigated the
mRNA expression level of mammalian DNA methyltransferase, Dnmt1, Dnmt3a
and Dnmt3b, which specifically methylate cytosine of CpG region in the DNA.
Dnmt1, Dnmt3a and Dmnt3b mRNA levels were elevated when the cells were
stimulated with both TNF-a and IGF-1.
Conclusion: Since both TNF-a and IGF-1 are released during coronary
intervention, hypermethylation of SOCS-3 gene could be an underlying
mechanism of intimal hyperplasia and restenosis. Thus, gene therapy with
SOCS-3 gene could prevent restenosis following balloon angioplasty or
intravascular stenting.
385 CHLAMYDIA PNEUMONIAE INFECTION IMPAIRS ENDOTHELIAL
FUNCTION IN THE ABSENCE OF A HOST IMMUNE RESPONSE
J. Deniset1,2 , E. Dibrov2 , G. Pierce1,2 . 1 Department of Physiology, University
of Manitoba, 2 Institute of Cardiovascular Sciences, St. Boniface Research
Centre, Winnipeg, MB, Canada
Introduction: Chlamydia pneumoniae (Cpn) infection initiates atherosclerosis
in animal models. However, it is unclear whether Cpn directly induces changes
in vascular contractile function and the mechanisms that may be responsible.
Aim: The current study aims to determine whether Cpn infection can initiate
endothelial dysfunction in the absence of a host immune response.
Materials and Methods: Isolated porcine coronary arteries were administered
1 of 3 treatments in a tissue culture environment: live Cpn, heat-inactivated
Cpn (mock infection), no treatment (control). Relaxation responses to bradykinin
(Bk) and sodium nitroprusside (SNP) were measured at days 5 and 10 postinfection (pi).
Results: Cpn infection was confirmed via immunofluorescent detection of
chlamydial inclusion bodies within coronary cross sections. SNP-induced
relaxation responses were unchanged at both day 5 and 10 pi. BK-induced
relaxation responses remained unchanged at day 5 pi, however, at day 10
pi Cpn infected vessels displayed decreased relaxation responses of 60%
and 54% compared to control and mock-infected vessels, respectively. These
changes were accompanied by a significant decrease in endothelial nitric oxide
synthase (eNOS) protein expression in Cpn infected vessels compared to the
other groups. Dihydroethidium staining of coronary cross-sections revealed an
increase in superoxide production in Cpn infected vessel compared to mockinfected and control vessels. Protein expression of p22phox was upregulated
in both Cpn and mock infected vessels compared to control vessels.
Conclusion: Our results demonstrate that Cpn infection alone can contribute
to endothelial dysfunction by reduced nitric oxide production and increased
oxidative stress.
Supported by Canadian Institutes of Health Research.
386 ATORVASTATIN SUPPRESS OXIDIZED LOW DENSITY LIPOPROTEININDUCED DENDRITIC CELL-LIKE DIFFERENTIATION OF RAW264.7
CELLS BY INACTIVATION OF THE P38 MAPK PATHWAY
L. Hu1 , L. Shen1 , S. Jin1 , D. Li1 , H. Xiao1 , P. Nie1 , J. Yi2 , B. He1 . 1 Ren Ji
Hospital affiliated to Medical Shool of Shanghai Jiaotong University, 2 Medical
School of Shanghai Jiaotong University, Shanghai, China
Atherosclerosis is an inflammatory disease in which immune mechanisms
interact with metabolic risk factors to initiate and activate lesions in the arterial
tree. Dendtritic cells (DCs) are potent antigen-presenting cells and have an
important role in the pathogenesis of atherosclerosis. We recently found that
mature macrophages could differentiate into dendritic-like cells under oxidized
low-density lipoprotein (oxLDL) treatment in vitro. In this study, we investigated
the molecular mechanisms and the effect of atorvastatin on it. Western blot
analysis showed that the phosphorylation of p38 MAPK, ERK1/2 and IkB-a
were induced in RAW264.7 cells when treated with oxLDL (10 mg/ml), but
only p38 MAPK pathway was involved in oxidized LDL-induced macrophagesDCs transition. Atorvastatin had an inhibitory action on this transition.
Oxidized LDL-treated RAW264.7 cells could not differentiated into dendriticlike cells when incubated with atorvastatin (10 mmol/ml) at the same time.
Correspondingly, the up-regulated dendritic cell markers, such as CD40, CD86,
CD83, CD1d and MHC II, were significantly reduced by atorvastatin. And the
DCs phenotype including reduced endocytic activity, increased allostimulatory
activity and cytokines production were also inhibited by atorvastatin. The effect
of atorvastatin on oxidized LDL-induced macrophages-DCs transition was partly
via p38 MAPK pathway. The findings of the present work give evidence to the
inhibitory effect of atorvastatin on atherogenesis.
79th EAS Congress
387 NEW THIAZOLIDINE-2,4-DIONES IMPROVE LIPID PROFILE AND
DOW-REGULATE PRO-INFLAMMATORY GENE EXPRESSION IN
LDL RECEPTOR-DEFICIENT MICE
F.A. César1 , M. Rudnicki1 , C.A.I. Saito1 , S.L. Galdino2 , M.C.A. Lima2 ,
I.R. Pitta2 , D. Saes Parra Abdalla1 . 1 Dept. of Clinical and Toxicological
Analyses, University of São Paulo, São Paulo, 2 Therapeutical Innovation
Group, Center of Health Sciences, Federal University of Pernambuco, Recife,
Brazil
Introduction and Objectives: Thiazolidinediones (TZDs), insulin-sensitizing
agents, are synthetic ligands for peroxisome proliferator-activated receptor-g.
They have been demonstrated to possess cardioprotective effects in humans
and anti-atherogenic properties in animal models. However, several side
effects are associated with TZDs treatment making necessary the search
for new compounds. In this study, new thiazolidine-2,4-diones (GQ-177, GQ145, LYSO-7) were tested on the metabolic effects and gene expression in
atherosclerotic lesions of low-density lipoprotein receptor-deficient (LDLr−/−)
mice.
Material and Methods: Male LDLr−/− mice were fed an atherogenic diet
containing 0.5% cholesterol for 4 months, and 4 weeks before euthanasia they
received TZDs (20 mg/kg per day) by gavage. Blood samples were collected for
determination of glucose, total cholesterol, HDL-cholesterol and triglycerides,
and the aortic sinus of the hearts for atherosclerotic gene expression by RTqPCR.
Results and Conclusions: GQ-177 significantly increased plasma levels of
HDL, besides to up regulate the expression of PPARg2, ABCA-1 and CD36
mRNA in the atherosclerotic lesions. LYSO-7 significantly decreased plasma
levels of glucose, total cholesterol and triglycerides, and down-regulated the
expression of VCAM and MCP1 genes. GQ-145 significantly up regulated the
expression of PPARg2, CD36, ABCA1, VCAM, PECAM, MCP1 and IL-6 genes.
The new tiazolidine-2,4-diones (particularly GQ-177 e LYSO-7) presented antiatherogenic properties, indicating that further studies on these compound are
warranted, as they may associate a hypoglycemic action with potential beneficial
pleiotropic effects related to inflammation and atherosclerosis, conditions
observed in diabetes.
Financial support: CNPq/INCT_if (grant to D.S.P.A.) and CAPES (scholarship
to F.A.C.).
388 REDUCED NUMBER OF DENDRITIC CELLS IN ATHEROSCLEROTIC
PLAQUES OF UREMIC PATIENTS
D. Neureiter1 , V. Campean2 , D. Wachter2 , A. Yilmaz3 , K. Amann2 . 1 Institute
of Pathology, University Hospital Salzburg of the Paracelsus Private Medical
University, Salzburg, Austria, 2 Institute of Pathology, 3 Department of
Cardiology, University of Erlangen-Nuremberg, Erlangen, Germany
Aims: Dendritic dells (DC) are known stimulators of T-cell activity playing
an important role for atherosclerosis. It was the aim of the present study
to analyse the number and localization of DCs together with T- and B-cell
infiltrates in calcified and non-calcified atherosclerotic plaques of uremic and
control patients.
Methods: 50 atherosclerotic plaques from uremic (n = 25) and control (n = 25)
patients were categorized according to the Stary classification and were
investigated using immunohistochemistry for S100 (for DC), CD3 (T-cells), CD20
(B-cells), CD68 (macrophages), CD117 (c-Kit) and mast cells antibody (mast
cells). The number of positive cells was analyzed for intima, media, adventitia
and the different plaque subregions.
Results: Within the plaques DCs were predominantly localized at the plaque
media, basis, boundaries and shoulders, but not within the cap. In control
patients the number of S100+ DCs was significantly higher in calcified plaques
than in non-calcified ones; it was also significantly higher than in calcified
plaques of uremic patients. In general, the scores for macrophages, CD3 and
CRP-positive cells tended to be higher in calcified and non-calcified plaques
of uremic patients compared to control patients. S100 expression in intima
and media correlated positively with the Stary grading. The number mast cells
showed a highly expression in the basis.
Conclusions: In parallel with a lower number of circulating DCs the number of
DCs within atherosclerosic plaques is reduced in uremic plaques compared to
non-renal control ones suggesting that DCs are not involved in T-cell activation
and plaque instability in these patients.
389 5,6-SECOSTEROL AFFECTS MACROPHAGE DIVERSITY AND
INDUCES PRO-INFLAMMATORY MACROPHAGE ACTIVATION
B. Buttari1 , E. Profumo1 , R. Monticolo2 , L. Iuliano2 , R. Businaro3 , R. Riganò1 .
1
Malattie Infettive, Parassitarie ed Immunomediate, Istituto Superiore di Sanità,
2
Department of Internal Medicine, 3 Department of Human Anatomy, Sapienza
University of Rome, Rome, Italy
Introduction: Macrophages play a central role in immunity and homeostasis.
The classically activated macrophages (M1) and the alternative ones (M2)
coexist and contribute to the pathogenesis of atherosclerosis through their
accumulation of cholesterol and the production of inflammatory mediators
83
and cytokines. The oxidised cholesterol product, 3b-hydroxy-5-oxo-5,6secocholestan-6-al (5,6-secosterol) has been shown to be present in
atherosclerotic plaques but its effects on macrophage remain to be elucidated.
Aim: To investigate whether 5,6-secosterol affects human macrophage diversity
and polarizes the two different macrophage subsets M1 and M2 toward a pro- or
anti-atherogenic phenotype.
Methods: Human monocytes obtained from healthy blood donors were induced
to differentiate for 6 days in the presence of either GM-CSF, to obtain activated
macrophages (M1), or M-CSF, to obtain alternative macrophages (M2). By the
use of immunochemical and cytofluorimetric analyses, we investigated surface
molecule expression, caspase activation, phagocytosis and pro- and antiinflammatory cytokine production. The effects of 5,6-secosterol on macrophage
migration were evaluated using Boyden chemotaxis chambers.
Results: Our findings indicate that 5,6-secosterol was able to enhance a
pro-inflammatory phenotype in M1 and to increase macrophage functions
such as phagocytosis and migration. Moreover, this compound prevented the
phenotypical and functional activation of M2.
Conclusion: 5,6-secosterol may exacerbate pro-inflammatory macrophage
activation and polarization subverting the balance between M1 and M2
activation profiles in atherosclerotic arteries.
390 CHARACTERIZATION OF T CELLS INFILTRATING PERIVASCULAR
CORONARY ADIPOSE TISSUE IN ATHEROSCLEROSIS
D. Ludew1 , K. Urbanski1 , B. Kapelak2 , T. Mikolajczyk1 , R. Nosalski1 ,
R. Korbut3 , T.J. Guzik1 . 1 Department of Internal and Agricultural Medicine,
Jagiellonian University Collegium Medicum, 2 Department of Cardiovacular
Surgery and Transplantation, 3 Department of Pharmacology, Jagiellonian
University School of Medicine, Krakow, Poland
Atherosclerosis is associated with perivascular inflammation. Nevertheless
characteristics of leukocytes infiltrating peri-coronary adipose tissue is not
known. Accordingly we aimed to study T cells infiltrating pericardial adipose
tissue surrounding coronary arteries (CA; from 52 patients) in comparison
with adipose tissue of internal mammary arteries (IMA), which do not develop
atherosclerosis. T cell infiltration of adipose tissue was studied by flow
cytometry of tissue digests. Endothelial function and superoxide production
were performed in IMA rings.
Results: The content of total leukocytes was 11-fold higher in adipose tissue
digests surrounding coronary arteries than in IMA (6.5±0.3 vs. 0.6±0.02%;
p < 0.05). CD3+ cells (T cells) constituted 48±1.8% of leukocytes in both
CA and IMA. The absolute number of T cells was higher in CA than IMA
adipose tissue (120.3±18.7 vs. 55.6±7.3 cells/mg of tissue). Interestingly,
a significant population of T cells in CA showed neither CD4 nor CD8 and
were double negative (DN) (33.9 vs. 17.4 cells/mg; CA vs IMA) and these
cells were predominantly CCR5+. DN/CCR5+ cells content was significantly
correlated with elevated BP, obesity, high leukocyte infiltration (R = 0.488,
R = 0.356, R = 0.42; p < 0.05; Manova, forward stepwise multiple regression).
Total leukocyte infiltration is correlated with acetylocholine-induced, endothelium
dependent vasorelaxation (R = −0.348, p = 0.00059).
Conclusion: Development of atherosclerosis in coronary arteries is accompanied by perivascular T-cell infiltration, consisting predominantly with double
negative (CD4−/8−) T cells characterized by high expression of RANTES
receptor CCR5.
This work is funded by European Union Structural Grant for the Foundation for
Polish Science/Welcome/2009/02
391 CD137 ON CD4+T LYMPHOCYTES IN PATIENTS WITH ACUTE
CORONARY SYNDROME MIGHT BE A MARKER FOR VASCULAR
SMOOTH MUSCLE CELL DEATH
J.E. Park1 , J.Y. Lee1 , J.Y. Kim1 , B.S. Kwon2 , B.S. Lee1 . 1 Cardiology, Samsung
Seoul Hospital, Sungkyunkwan University, 2 National Cancer Center, Seoul,
Republic of Korea
Background and Aims: As atherosclerosis progresses, medial smooth muscle
cells (SMC) disappear. We have investigated whether T cells from patients with
acute coronary syndrome are involved in smooth muscle cell (SMC) killing.
Methods: Patients were divided into 4 subgroups: normal (NL; 25 cases),
unstable angina (UA; 29 cases), stable angina (SA; 48 cases), and acute
myocardial infarction (AMI; 40 cases). CD137 level in serum was measured
by sandwich ELISA. Primary SMCs were co-cultured with CD4 or CD8
lymphocytes from the patients.
Results: Serum sCD40L, MCP-1, IL8, or IL6 levels were not significantly
different among subgroups. Although CD137 level showed wide variation within
group, 24% of UA patients and 22% of AMI patients showed above 0.2 ng/ml
of CD137 while only 4% of the patients in SA group showed that level.
CD137 serum level had significant correlation to aspirin (p < 0.0094) or CCB
(p < 0.0223) treatment. CD137 and C reactive protein (CRP) showed correlation
in only MI subgroup (r = 0.37, p < 0.0164). SMC cell death appeared in 19.4%
(p < 0.01) and 44.9% (p < 0.01) of cells when CD4+ cells from normal and
UA patients were added and it increased to 41.9% (p < 0.05) and 50.6%
84
(p < 0.01) in cells activated with phorbol myristate acetate (PMA) and Ionomycin,
respectively.
Conclusions: CD137 level was increased in sera from UA and AMI compared
to SA. Both CD8+ and CD4+ cells are critical in SMC death and CD137 in
activated CD4+ cells from UA patients might play an important role for the
SMC death.
392 MYCOPHENOLIC ACID HAS ANTI-INFLAMMATORY CAPACITY
BY INHIBITION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1
PRODUCTION IN VASCULAR SMOOTH MUSCLE CELLS
M. Schuchardt, M. Tölle, J. Prüfer, S. Huber, W. Zidek, M. van der Giet. CC10
Nephrologie, Charité − Campus Benjamin Franklin, Berlin, Germany
Objectives: Mycophenolic acid (MPA) emerged as immunosuppressive drug
with non-immunmodulating effects, too. For initiation and maintenance of
atherogenesis the production of monocyte chemoattractant protein-1 (MCP-1)
is crucial. The aim of this study was to explore anti-inflammatory properties of
MPA on MCP-1 production in vascular smooth muscle cells (VSMCs).
Methods: MCP-1 was measured via real-time PCR and Luminex™ technology.
Superoxide production was detected in DHE-labeled cells via fluorescence
microscopy and quantified in a multimode-reader. H2 O2 production was
measured in H2 DCFDA-labeled cells via flow cytometry. Rac1 activation was
detected via ELISA. NADPH consumption rate was measured photometrically.
Results: MPA significantly decreased the thrombin-induced MCP-1 expression
and secretion in a dose-dependent manner. Next, we investigated the
production of superoxide in DHE-labeled cells treated with thrombin±MPA.
Pretreatment of MPA significantly decreased the thrombin-induced fluorescence
increase. Superoxide is rapidly converted in H2 O2 . MPA also diminished the
H2 O2 production dose-dependently. A predominant source of reactive oxygen
species in the vasculature is the NADPH oxidase. We measured the NADPH
consumption rate in VSMCs. MPA significantly diminished the thrombin-induced
NADPH consumption. To gain further evidence pointing to NADPH oxidase as
target, we measured Rac1 activation. MPA significantly diminished activation of
Rac1.
Conclusions: MPA is able to significantly attenuate this thrombin-induced
MCP-1 production indicating anti-inflammatory effects of MPA. The vascular
NADPH oxidase is involved in the signal transduction pathway. In summary,
MPA might play a relevant role in anti-inflammatory response and therefore
constitutes an attractive candidate for the modulation of inflammatory activation
in atherosclerosis.
393 TRIGLYCERIDE METABOLISM AND INTERLEUKIN-6 LEVELS
ARE ALTERED IN CARRIERS OF THE MINOR ALLELE OF AN
INTERLEUKIN-1B GENE VARIATION (−1473G/C)
J. Delgado-Lista1 , P. Perez-Martinez1 , A. Garcia-Rios1 , J. Solivera2 ,
E.M. Yubero-Serrano1 , F. Fuentes1 , L.D. Parnell3 , J. Shen3 , P. Gomez1 ,
Y. Jimenez-Gomez1 , M.J. Gomez-Luna1 , C. Marin1 , S.E. Belisle4 ,
F. Rodriguez-Cantalejo5 , S.N. Meydani4 , J.M. Ordovas3 , F. Perez-Jimenez1 ,
J. Lopez-Miranda1 . 1 Lipids and Atherosclerosis Unit, IMIBIC/Hospital
Universitario Reina Sofı́a/Universidad de Córdoba and CIBER Fisiopatologı́a
Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, 2 Neurosurgery
Unit, Hospital Universitario Reina, Cordoba, Spain, 3 Nutrition and Genomics,
JM-USDA Human Nutrition Research Center on Aging, Tufts University,
4
Nutritional Immunology Laboratory, JM-USDA Human Nutrition Research
Center on Aging, Tufts University, Boston, MA, USA, 5 Biochemical Laboratory,
Hospital Universitario Reina, Cordoba, Spain
Background and Aims: Interleukin 1b (IL1B), is a major regulator of the
immune response mainly by its main effector, IL-6. Hypertriglyceridemia
(specially after a saturated fatty acids rich meal) induces inflammation in the
postprandial state, but with high interindividual differences. Although it has been
shown that variations in lipid metabolism influence the inflammatory response in
the postprandial state, is not clear whether primary inflammatory changes may
influence postprandial lipemia. We evaluated the influence of a genetic variant
located in the promoter region of IL1B (−1473G/C) on fasting and postprandial
lipids and IL6.
Patients and Methods: 477 people aged >65 years old were genotyped
for IL1B −1473G/C, and we evaluated the fasting lipids depending on their
genotype, and, subsequently, 88 healthy young men were eventually genotyped,
and were fed a saturated fatty acid-rich meal. Serial blood samples were drawn
for 11 hours after the meal, and lipid fractions and IL6 were assayed.
Results: Aged persons who were CC (minor allele homozygotes) showed
higher triglycerides (TG) and cholesterol levels in the fasting situation. In line
with these results, healthy young men carriers of the minor C allele showed
higher postprandial TG, and those carried into large triglyceride rich lipoproteins
(TRL). In addition, they showed higher postprandial IL6 concentrations.
Conclusions: Inflammatory genes may regulate postprandial lipid metabolism,
a key contributor to the atherosclerosis development process, as carriers of the
minor allele of IL1B (−1473G/C) have a disturbed postprandial lipid metabolism.
IL6 (the natural effector of IL1B) was also elevated in this population.
394 MANNOSE BINDING LECTIN AS CARDIOVASCULAR RISK FACTOR:
A COHORT STUDY
B. Klop, A. Alipour, A. Westzaan, E. Birnie, G.J.M. van de Geijn, T. Njo,
J.W. Janssen, N. van der Meulen, J.W.F. Elte, A.P. Rietveld, M. Castro
Cabezas. Sint Franciscus Gasthuis, Rotterdam, The Netherlands
Introduction: Mannose binding lectin (MBL) is linked to atherosclerosis and
both high and low levels have been reported as risk factor. We measured
MBL as part of our standard cardiovascular risk program and followed patients
prospectively.
Methods: MBL deficiency was established 0.8 mg/l. Cardiovascular disease
(CVD) was defined by coronary artery disease (CAD), cerebrovascular disease
(CVA) or peripheral vascular disease (PVD) after initial MBL measurement.
Results: 478 patients were analyzed. The prevalence of MBL deficiency was
44.4%. The median follow-up was 24.0 months (range 2.0–60.0 months). Age,
gender and medical history of CVD were comparable between patients with and
without MBL deficiency. Type 2 diabetes mellitus (T2DM) was less prevalent
in patients with MBL deficiency (17.1% vs. 25.6%; P = 0.016) in contrast to
heterozygous familial hypercholesterolemia (FH) (10.4% vs. 3.4%; P = 0.002).
MBL deficient patients showed an incidence per 1000 person-years of 4.7 for
CAD, 4.7 for CVA, 11.9 for PVD and 21.3 for all CVD events combined. In
patients with MBL sufficiency, incidence density rates were respectively 16.2
for CAD, 9.0 for CVA, 16.2 for PVD and 41.3 for CVD. The relative risk of MBL
deficiency for CAD was 0.29 (95% CI 0.06–1.36), CVA 0.53 (95% CI 0.32–2.72),
PVD 0.73 (95% CI 0.38–2.19) and for any CVD event 0.52 (95% CI 0.24–1.12).
Conclusion: MBL deficiency was unexpectedly prevalent in our cohort. T2DM
was less prevalent in MBL deficient patients, but FH was more prevalent.
Overall, our study suggests a trend towards a protective effect of MBL deficiency
for atherosclerosis.
395 ASSOCIATION OF PERIODONTAL DISEASE WITH ISCHEMIC
STROKE: A CASE-CONTROL STUDY
M. Ogura1 , H. Iwabuchi2 , M. Ishihara3 , H. Sato4 , Y. Sato5 , R. TsukinokiMurakami6 , K. Matsushita7 . 1 Internal Medicine, First Service School, JMSDF,
Etajima, 2 Division of Dentistry and Dental Surgery, 3 Division of Neurosurgery,
Tochigi National Hospital, Utsunomiya, 4 Department of Medical Informatics,
National Defense Medical College, Tokorozawa, 5 Environmental Health
Science Division, National Institute for Health and Welfare Environmental
Studies, Tsukuba, 6 Translational Research Center, Kyoto University, Kyoto,
Japan, 7 Department of Epidemiology, Johns Hopkins Bloomberg School of
Public Health, Baltimore, MD, USA
Aim: Low-grade inflammation is associated with atherosclerotic disease.
Recently, inflammation in oral cavity, e.g. periodontitis attracts attention as a
source of chronic low-grade inflammation. Although some studies have reported
the association between poor oral health and risk of ischemic stroke in Western
populations, little is known about the association in Japanese. The aim of this
study is to investigate whether poor oral health, including periodontitis and tooth
loss, is a risk factor for ischemic stroke in Japanese.
Methods: We performed a case-control study with 37 cases examined within
7 days after symptomatic ischemic stroke and 64 age- and sex-matched
controls. Exclusion criteria included diabetes and current smoking to eliminate
their confounding effects. All subjects received a complete clinical dental
examination. The individual community periodontal index (CPI: WHO 1982) was
used as the main indicator for periodontitis.
Results: Patients had higher prevalence of periodontal disease than controls
(92% vs. 58%). Subjects with periodontitis had a 10.3-fold higher (95%
confidence interval, 2.3 to 29.8) odds of cerebral ischemia than subjects without
periodontitis after adjustment for age, sex, vascular risk factors and life style
factors. Furthermore, the risk of ischemic stroke increased along with severity
of periodontitis (P for trend < 0.001).
Conclusion: Our study suggests that periodontitis is an independent risk factor
for ischemic stroke in non-diabetic and non-smoking Japanese subjects.
396 ROLE OF ROSUVASTATIN AND CLOPIDOGREL ALONE AND
COMBINED IN ENDOTHELIAL PROGENITOR CELLS AND
ENDOTHELIAL MICROPARTICLES
C. Nunes França, L.F.M. Pinheiro, M.C.O. Izar, F.A. Helfenstein Fonseca.
Medicine, Federal University of São Paulo, São Paulo, Brazil
Introduction: Atherosclerosis is an inflammatory disease complicated with
thrombotic events related to endothelial dysfunction or apoptosis. Statins and
antiplatelet drugs are commonly used in coronary artery disease patients and
share common pathway in their metabolism. Endothelial progenitor cells (EPC)
and endothelial microparticles (EMP) are new biomarkers of cardiovascular
disease and are related to the turnover of endothelial cells. These EPC can
be characterized by proteins, such as CD34, KDR and CD133, while EMP
can be identified by flow cytometry by expression of CD51. We examined the
pharmacokinetic interaction of rosuvastatin with clopidogrel in the balance of
EPC and EMP.
Methods: We enrolled stable coronary disease patients (79% males, median
age of 58.66 years). All subjects were under stable statin dose (>3 mo). We
79th EAS Congress
quantified by flow cytometry the baseline levels of EPC and EMP after 1-wk
of statin withdrawal and after 1-wk of a daily dose of 40 mg of rosuvastatin;
after 1-wk of clopidogrel 300 mg (starting dose) followed by 75 mg/day plus
rosuvastatin 40 mg; and after 1-wk of clopidogrel alone (rosuvastatin stopped
for 1-wk).
Results: The mean (EPM) EMP CD51+ plasma levels (microparticles/mL) were:
1498 (489) at baseline; 1397 (543) 1-wk after rosuvastatin; 2975 (790) 1-wk
after rosuvastatin+clopidogrel; and 3737 (931) with clopidogrel alone (p = 0.04).
No significant changes were seen for EPC.
Conclusion: Short period of statin discontinuation was associated with early
increase in endothelial microparticles showing potential harm among subjects
under chronic statin therapy.
397 THE ROLES OF CHOLESTEROL AND INFLAMMATION IN THE
FORMATION AND PROGRESSION OF ATHEROSCLEROSIS
H.S. Seog1 , E.J. Kim1 , H.J. Baek2 , J.O. Na2 , C.U. Choi2 , H.E. Lim2 ,
S.-W. Rha3 , C.G. Parl2 , D.J. Oh3 , B.W. Yeom2 . 1 Cardiovascular Center, 2 Korea
University Guro-Hospital, 3 Kobe University Graduate School of Medicine,
Seoul, Republic of Korea
The inflammation caused by cholesterol deposition into arterial walls develops
atherosclerosis, yet the effects of other types of inflammation on the artery
are not clear in the setting of normal or high serum cholesterol level.
Twenty rabbits were assigned into the following groups: Group I (control),
Group II (carrageenan injection), Group III (a cholesterol diet), and Group
IV (carrageenan injection and a cholesterol diet). Groups I and II received
normal chow and groups III and IV received a 1% cholesterol diet. Groups II
and IV received subcutaneous carrageenan injection to promote inflammation
with TLR4 signaling. The animals were sacrificed after 3 months. The total
cholesterol, LDL-C and HDL-C were markedly increased in groups III and
IV. IL-1, IL-6, TNF-a and C-reactive protein (CRP) were increased in all the
rabbits except the controls. The level of cytokines was correlated with the
level of CRP. Focal medial thickening and calcification with chondrodysplasia
at medial layer were observed in Group II, but there was no atherosclerosis.
Group III showed early atherosclerotic lesions, including intimal hyperplasia and
punctuated calcium deposits. In group IV, there were advanced atherosclerotic
lesions, including acellular lipid cores and medial penetration by foam cells.
Arteries can be affected by inflammation that’s associated or not associated
with cholesterol. While cholesterol and its associated inflammation are essential
for atherogenesis, the inflammation that is independent of cholesterol seems to
be critical for the further progression of atherosclerosis.
398 CIRCULATING SOLUBLE INTERCELLULAR ADHESION
MOLECULE-1 AND TRANSFORMING GROWTH FACTOR-BETA-1
IN PATIENTS WITH RECURRENT ISCHEMIA AFTER CORONARY
ARTERY BYPASS GRAFTING SURGERY
K.V. Korzhenevskaya1 , N.A. Gavrisheva2 , A.V. Panov1 , T.P. Ses2 ,
G.A. Alekseeva2 , M.Z. Alugishvili1 , I.T. Abesadze1 , N.L. Lohovinina1 . 1 Federal
Almasov Heart Blood and Endocrinology Center, 2 Saint-Petersburg Pavlov
State Medical University, Saint-Petersburg, Russia
Objective: To evaluate proinflammatory soluble intercellular adhesion
molecule-1 (sICAM-1) and anti-inflammatory transforming growth factor-beta-1
(TGF-beta-1) blood serum levels in patients underwent urgent coronary artery
bypass grafting surgery (CABG).
Materials and Methods: We observed 130 patients, who had non-ST elevation
acute coronary syndrome (non-ST ACS) at the moment of surgery. The average
age was 58.4±0.8 years. The control group consisted of 28 patients with
stable angina treated conservatively. Immune-enzyme analysis was used to
measure sICAM-1, TGF-beta-1 levels in blood serum samples. Follow-up period
averaged 4 years.
Results: Recurrent myocardial ischemia was developed in 26.9% patients.
The mean angina recurrence time was 13.6±2.0 months. TGF-beta-1 serum
levels were higher in the recurrent angina group than in non-recurrent patients
(100.5±19.8 ng/ml and 66.9±8.2 ng/ml, respectively, p < 0.05). There was no
significant difference between sICAM-1 serum concentrations in the recurrent
and non-recurrent patients (611.3±26.9 and 592.6±17.9 ng/ml, respectively,
p > 0.10). However, postoperative non-recurrent patients had lower TGF-beta-1
(66.9±8.2 ng/ml) and sICAM-1 (592.6±17.9 ng/ml) values than before CABG
(99.4±5.5 ng/ml, p < 0.05; 680.0±40.0 ng/ml, p < 0.05, respectively), but had
similar levels to the control (61.8±14.1 ng/ml, p > 0.10; 580.0±30.0 ng/ml,
p = 0.098, respectively).
Conclusion: The recurrent ischemia in patients after urgent CABG is strongly
associated with high levels of TGF-beta-1. The postoperative both pro- and
anti-inflammatory markers values in the patients with recurrent angina pectoris
are tended to be similar to the non-ST ACS, while in the non-recurrent patients
are tended to the control group level. These data underline the critical role of
systemic chronic inflammation in the progressive atherosclerosis.
85
399 INVOLVEMENT OF TOLL LIKE RECEPTOR-4 IN INFLAMMATORY
RESPONSES OF ENDOTHELIAL CELLS
R. Menghini1 , M. Tesauro1 , V. Rovella1 , A. Marino1 , R. Lauro1 , M. Federici1 ,
C. Cardillo2 . 1 Internal Medicine, Tor Vergata University, 2 Internal Medicine,
Catholic University Medical School, Rome, Italy
Growing evidence supports the role of inflammation as pathophysiological
mechanism in atherosclerosis. Lipopolysaccharide (LPS), a major component
of the outer surface of Gram negative bacteria, represents a potential source
of inflammatory vascular injury leading to the development of atherosclerosis.
Alternative ligands, such as mmLDL (minimal modify LDL) can also activate
TLR4. E5564 (eritoran) is a second generation synthetic lipodisaccharide
designed to antagonize the toxic effects of endotoxins. To investigate the
potential anti-atherosclerotic effects of E5564, we incubated human aortic
endothelial cells for 6h with eritoran (10 nM) in the presence or absence of
LPS (100 ng/ml) or oxPAPC, a component of mmLDL (100 mg/ml), and analyzed
cytokines expression by real time PCR and western blot. Administration of
eritoran significantly reduced the expression of LPS-induced cytokines, such
as TNF-a, MCP-1, IL-6, as well as that of cell adhesion molecules, such as
ICAM and VCAM; concurrently, eritoran increased both the expression and
phosphorylation of eNOS and reduced IL-8 expression by oxPAPC, hence
suggesting that TRL-4 receptor activation is potentially involved in LPS- and
oxPAPC-induced inflammatory responses in HAEC. Additional data indicated
that treatment with eritoran significantly reduces I-úB phosphorylation induced
by LPS, thereby suggesting that modulation of I-úB phosphorylation and
subsequent activation of NF-úB is a mechanism by which TRL-4 receptor
stimulation affects HAEC rersponse to endotoxin. In conclusion, these results
support the notion that TRL-4 receptors are involved in inflammatory and
proatherogenic responses of endothelial cells, thus suggesting that blockade
of these receptors may exert protective effects in the vascular endothelium.
400 ATHEROGENIC IN VIVO LDL OXIDATION,
THROMBOXANE-MEDIATED PLATELET ACTIVATION AND
SIMULTANEOUS OCCURRENCE OF NATURAL IMMUNITY IN
ATHEROSCLEROSIS-PRONE (LDLR−/− AND APOE−/− ) MICE
E. Matsuura1 , L. Shen2 , Y. Matsunami1 , N. Quan1 , K. Kobayashi1 ,
Y. Shoenfeld3 , K. Oguma2 , L.R. Lopez4 . 1 Cell Chemistry, 2 Bacteriology,
Okayama University Graduate School of Medicine, Okayama, Japan,
3
Department of Medicine ‘B’ and Zabludowicz Center for Autoimmune
Diseases, Tel-Aviv University, Tel-Aviv, Israel, 4 Corgenix, Inc, Broomfield, CO,
USA
Introduction: Several murine models are prone to atherosclerosis such as
LDL receptor deficient (LDLR−/− ) and apolipoprotein E deficient (apoE−/− ) mice,
and used to study pro-atherogenic mechanisms. Abnormal lipid metabolism
is commonly associated with thoracic/abdominal aortic plaques. Heterozygous
LDLR+/− /apoE+/− mice did not exhibit significant dyslipidemias nor progression
of atherosclerosis. We investigated the time relationship and pathogenic role
of novel atherogenic biomarkers involved in oxidative stress, platelet activation
and immunity in experimental atherosclerosis-prone mice.
Methods: Six week old male mice (LDLR−/− , apoE−/− ) atherosclerosis-prone
and heterozygous LDLR−/− /apoE−/− mice were fed with normal chow or highcontent fat diet and sacrificed at different age intervals until week 16 to measure
aortic plaque size. Plasma lipids, oxidized LDL (oxLDL), oxLDL/b2-glycoprotein
I (oxLDL/b2GPI) complexes, IgM antibodies against native LDL, oxLDL, or
oxLDL/b2GPI were measured by ELISA. Urine 11-dehydro-thromboxane B2
(11dhTxB2 ) or 8-hydroxy-deoxyguanosine (8OHdG) were also measured by
ELISA.
Results: There was a parallel increase in plaque size, plasma cholesterol,
and urinary 11dhTxB2 in atherosclerosis-prone (LDLR−/− and apoE−/− ) mice.
In contrast to LDLR−/− and apoE−/− strains, an elevation of urinary
11dhTxB2 with no significant plaque generation was observed in heterozygous
LDLR+/− /apoE+/− mice. The atherogenic autoantigen oxLDL/b2GPI complex was
detected only in LDLR−/− mice and seemed to correlate with plaque size.
There was a strong correlation (r2 =0.97) between IgM antibodies to oxLDL
with antibodies to oxLDL/b2GPI in apoE−/− mice indicating that these natural
antibodies recognized oxLDL epitopes.
Conclusions: Progression of atherosclerotic lesions was associated with
increasing dyslipidemia, platelet activation, oxLDL/b2GPI complexes and natural autoimmune-mediated regulatory mechanism(s) in murine atherosclerosisprone models.
86
401 AN APPROACH BASED ON LIPID NANOPARTICULES TO
DETERMINE THE ROLE OF MONOCYTES/MACROPHAGES IN THE
DEVELOPMENT OF AORTIC VALVE ATHEROSCLEROTIC LESIONS
M. Calin1,2 , I. Manduteanu2 , E. Butoi2 , D. Stan2 , E. Dragan2 , A.-M. Gan2 ,
M. Simionescu2 . 1 Institute of Macromolecular Chemistry “Petru Poni”, Iasi,
2
Institute of Cellular Biology and Pathology “ N. Simionescu”, Bucharest,
Romania
Monocyte-derived macrophages have a significant role in the early stages
of vascular and valvular inflammatory process associated with atheromatous
plaque formation. To determine the role of macrophages in lesion formation in
heart valves in hyperlipidemia, systemic depletion of monocytes/macrophages
was obtained by intravenous administration of clodronate-encapsulated
liposomes (Lclod), a treatment that selectively induces significant monocyte
apoptosis. We examined whether the absence of monocytes has a beneficial
or adverse effect on the development of aortic valve lesions in hyperlipemic
hamsters injected twice weekly (for 2 months) with Lclod. Our results show that
during the development of an aortic valve lesion, the presence of macrophages
is beneficial, and their depletion is a two edged sword. Thus, the selective
induction of monocytes/macrophages apoptosis has the beneficial effect of
reducing the valvular cell activation caused by diminished expression of proinflammatory cytokine IL-1b and decreased activity of MMP-9 and MMP-2, but
also a detrimental effect, inducing the expansion of aortic valve atherosclerotic
lesions and a significant increase in lipid and collagen accumulation in the
lesions, both of which may lead to the impairment and degeneration of valvular
motion and function.
This work was supported by the Romanian Academy and a grant from
the Romanian Ministry of Education and Research, National Program
VIASAN (grant no. 330). M. Calin acknowledges the financial support of
European Social Fund “Cristofor I. Simionescu” Postdoctoral Fellowship
Programme (ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme
Human Resources Development 2007–2013.
402 ADVANCED ABDOMINAL AORTIC ANEURYSMS DISPLAY A
SPECIFIC INFLAMMATORY-PROATHEROSCLEROTIC PROFILE
WITH RENAL DYSFUNCTION, RAISED D-DIMER, EXPANDED
CD14++ CD16+ -MONOCYTES HAVING HIGH CD143
C. Barisione1 , G. Ghigliotti1 , D. Palombo2 , D. Palmieri2 , G. Spinella2 , B. Pane2 ,
P. Fabbi1 , P. Altieri1 , P. Spallarossa1 , C. Brunelli1 , S. Garibaldi1 . 1 Dept of
Internal Medicine, University of Genova, Genova, 2 Unit of Vascular and
Endovascular Surgery, San Martino Hospital and University of Genoa, Genoa,
Italy
AAA patients definitely share risk factors with atherosclerosis, but the chronic
inflammatory response seen in AAA that includes extreme medial thinning
associated to VSMC apoptosis, adventitial inflammatory cell infiltration, elastin
fragmentation and degeneration that are rarely seen in atherosclerotic lesions
of other districts, such as carotid and coronary atherosclerosis. In inflammatory
chronic disease, such atherosclerosis and chronic kidney disease (CKD),
circulating monocytes display expanded CD14++CD16+ subset and higher
surface expression of CD143. Plasma D-dimer is increased in patients with
abdominal aortic aneurysm (AAA), and might contribute to the systemic proinflammatory response associated to circulating monocytes. Our work aimed
to determine the frequency of CD14++ CD16+ monocytes in AAA patients, and
to investigate the relationship between this monocyte subset, D-dimer levels,
monocyte CD143 expression, renal function and other laboratory parameters
of inflammation.
Peripheral blood from 78 large AAA patients, and 22 healthy controls (Contr)
was analyzed to determine the percentage of CD14++ CD16+ and the CD143
expression in monocytes by means of flow-cytometry.
Patients with AAA have expanded CD14++ CD16+ monocyte subsets (median
value and IQR) (7.2, 5.7−9.3 vs 5.2, 4.3−6.4, p < 0.05, AAA vs Contr), whose
levels were directly associated to D-dimer and age, and inversely related to
GFR and cholesterol. Moreover, there was a significant positive relationship
between CD14++ CD16+ frequency and CD143 expression.
These results indicate that the presence of a moderate renal dysfunction,
raised D-dimer levels, expanded CD14++ CD16+ monocyte frequency and high
expression of CD143 constitute a specific proatherogenic profile that promotes
systemic inflammatory response in patients with large AAA.
403 THE ROLE OF NICOTINAMIDE N-METHYLTRANSFERASE (NNMT)
IN ANTI-INFLAMMATORY AND ANTI-ATHEROSCLEROTIC ACTION
OF NICOTINIC ACID IN MOUSE MODEL OF ACCELERATED
ATHEROSCLEROSIS
Ł. Mateuszuk1 , M. Sternak1 , M. Gajda2 , M. Szafarz3 , J. Szymura-Oleksiak3 ,
M. Franczyk-Żarów4 , R. Kostogrys4 , S. Chłopicki1 . 1 Chair of Pharmacology,
2
Chair and Department of Histology, 3 Department of Pharmacokinetics and
Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University, Collegium
Medicum, 4 Chair of Human Nutrition, University of Agriculture, Kraków,
Poland
Introduction: Nicotinic acid (NicA) is the most effective HDL-raising drug, but
it has additional anti-inflammatory properties, that seems to be independent
of its effect on lipid profile. It is possible, that many of this actions are
connected with 1-methylnicotinamide (MNA), synthesized by nicotinamide
N-methyltranspherase (NNMT). MNA was considered as an inactive metabolite
of NicA. Recent studies, however, has revealed anti-atherothrombotic and antiinflammatory activities of MNA, linked to COX/PGI2 pathway.
The aim of this study was to compare the antiatherosclerotic and antiinflammatory activity of NicA, MNA (100 mg/kg and 1 g/kg) and perindopril
(2 mg/kg) given for 2 months to ApoE/LDLR −/− mice fed with high-cholesterol
diet.
Materials and Methods: Plaque progression (ORO), macrophage content
(CD68 IHC) and MMP activity (zymography) were examined inside aortic
roots. Endogenous NicA derivatives concentrations (LC/MS), SAA (ELISA)
and total Cholesterol/Triglicerides levels (spectophotometry) were measured in
plasma. NNMT activity (fluorometry) was estimated in liver homogenates from
ApoE/LDLR−/− mice.
Results: NicA, MNA and perindopril reduced the area of atherosclerotic lesions
and plasma concentration of SAA without changing the levels of lipids. MNA
and perindopril also lowered MMP activity in the plaque area and macrophage
accumulation. The activity of NNMT in ApoE/LDLR−/− mice was increased, and
this correlated with higher MNA concentration in plasma.
Conclusions: Exogenous MNA displays anti-atherosclerotic activity comparable with NicA, suggesting MNA-dependent mechanisms in NicA antiatheroclerotic action. High levels of endogenous MNA and increased NNMT
activity during atherosclerosis suggest, that NNMT-MNA pathway may have a
regulatory role in atherosclerosis.
404 EVALUATION OF A DIABETIC RABBIT MODEL FOR IN-STENT
RESTENOSIS
P.W. Radke1 , A. Joost1 , A. Kaiser2 , M. Basler2 , M. Mandapathil2 , C. Weber2 ,
S. Yla-Herttuala3 , W. Ito1 . 1 Medizinische Klinik II, Universität zu Lübeck,
Lübeck, 2 RWTH Aachen, Aachen, Germany, 3 University of Kuopio, Kuopio,
Finland
Aims: Aim of this study was to evaluate
1. the feasibility of a diabetic rabbit model for stent implantation and
2. to characterise the extent of neointimal inflammation and proliferation in this
model.
Methods: New Zeeland White (NZW, 3.0±0.2kg) were fed a cholesterolrich diet. Diabetes mellitus was induced by alloxan injection (70 mg/kg i.v.).
Animals with a fasting blood sugar (BS) 250 mg/dl were classified as
“diabetic” (n = 9). Control animals were not subjected to Alloxan injections
(n = 9). Stent implantation was performed after 4 weeks in the iliac artery
(after endothelial denudation). 8 weeks post intervention, the extent of
neointimal inflammation (macrophage marker RAM-11 immonuhistochemstry)
and neointimal proliferation (morphometric measurements) were quantified.
Results: Induction of diabetes by alloxan injection was associated with a
36% mortality. In diabetic survivors, fasting blood glucose (416±40 mg/dl vs.
120±13 mg/dl, p < 0.01) and cholesterol serum levels (42.1±30.7 mmol/l vs.
30.1±15.5 mmol/l, p < 0.05) were significantly higher as compared to controls.
Peri- and postprocedural mortality was comparable between groups. The
degree of neointimal inflammation was significantly higher in diabetic animals,
however, the neointima thickness (0.88±0.38 mm vs. 1.83±0.68 mm, p < 0.05)
as well as the intima/media ratio (3.94±1.52 mm vs. 8.38±3.15 mm, p < 0.05)
were significantly lower in diabetic animals.
Conclusions: Alloxan application is associated with a significant early, but
not late (or procedural) mortality in rabbits. Diabetic rabbits, however, exibit
hyperglycemia and hypercholersterolemia leading to an exaggerated neointimal
inflammation after stent implantation.
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405 RELATIONSHIPS BETWEEN MARKERS OF APOPTOSIS AND
ANTIBODIES TO OXIDIZED LOW-DENSITY LIPOPROTEINS IN
PROGRESSING ATHEROSCLEROSIS
V. Gurevich1 , L. Vasina2 , N. Petrischev3 , A. Lugovaya4 . 1 Center of
Atherosclerosis and Lipid Disorders, Mechnicov’s State Medical Academy,
Saint Petersburg, 2 Lab of Microcirculation, Amasov’s Center of Heart, Blood
and Endocrinology, Saint-Petersburg, 3 Chair of Pathology, 4 Pavlov’s State
Medical University, Saint Petersburg, Russia
The purpose of this study was to examine the relationships between soluble
apoptosis markers sBcl-2 and sApo-1/Fas and antibodies to oxidized LDL in
acute coronary syndrome.
Methods: Blood samples of 190 patients with clinical and instrumental signs of
acute coronary syndrome were examined. Antibodies to oxidized LDL, sBcl-2
and sApo-1/Fas in the blood plasma were determined by ELISA.
Results: Significantly higher levels of antibodies to oxidized LDL and an
increase of sApo-1/Fas and sBcl-2 in the blood plasma have been shown.
In unstable angina sAro-1/Fas and sBcl-2 levels were 1980.4±112.62 pg/ml
and 29.1±8.16 IU/ml, respectively, in acute myocardial infarction with non-STsegment elevation (NSTEMI) sAro-1/Fas and sBcl-2 levels were 1898.2±132.41
pg/ml and 30.5±9.28 IU/ml, respectively, while in acute myocardial infarction
with ST-segment elevation sAro-1/Fas and sBcl-2 levels was 2951.0±184.41
pg/ml and 61.5±15.23 IU/ml, respectively (p < 0.05). In the control group sAro1/Fas level was 1464.0±209.28 pg/ml, sBcl-2 level was 13.7±3.15 IU/ml. The
positive correlation between the antibodies to oxidized LDL and the content of
sBcl-2 and sApo-1/Fas was demonstrated (r = 0.65, and r = 0.71, respectively,
p < 0.05).
Conclusions: The data obtained confirm the involvement of modified LDL in
the development of Fas-mediated apoptosis of endothelial cells in progressing
atherosclerosis.
406 THE DELETERIOUS INFLUENCE OF TENOFOVIR-BASED
THERAPIES ON THE PROGRESSION OF ATHEROSCLEROSIS
IN HIV-INFECTED PATIENTS
G. Aragones1 , P. Pardo2 , R. Beltrán-Debón1 , A. Rull1 , L. Fernández-Sender2 ,
J. Joven1 , C. Alonso-Villaverde3 . 1 Centre Recerca Biomedica, 2 Servei
de Medicina Interna, Institut Investigació Sanitària Pere Virgili, Hospital
Universitari Sant Joan, Universitat Rovira i Virgili, Reus, 3 Servei de Medicina
Interna, Hospital Son Llàtzer, Palma de Mallorca, Spain
Objective: To investigate the potential differential effects of antiretroviral
therapies on unbalanced chemokine hemostasis and on the progression of
atherosclerosis in HIV patients.
Methods and Design: We conducted a two-year prospective study in a cohort
of 147 HIV-infected patients. We assessed changes in the circulating levels of
inflammatory biomarkers and in the progression of subclinical atherosclerosis.
The expression levels of selected genes (MCP-1, CCR2, CCR5 and CXCR-4) in
circulating leukocytes were also assessed at the end of the follow-up period.
Results: Control subjects showed significantly lower plasma concentrations
of CRP (p < 0.001), tPA (p < 0.001), IL-6 (p = 0.04) and MCP-1 (p = 0.03) than
HIV-infected patients at a baseline time-point. After two years of follow-up,
the observed decreases in plasma inflammatory biomarker levels were only
significant for MCP-1 (p < 0.001), tPA (p = 0.001) and IL-6 (p = 0.01). A decrease
in the plasma MCP-1 concentration was associated with the progression of
atherosclerosis, and this effect was negligible only in patients receiving tenofovir
(TDF) therapy. Multivariate analysis confirmed that treatment with TDF was
positively and significantly associated with a higher likelihood of subclinical
atherosclerosis progression (OR = 7.1; p = 0.002; 95% CI: 2−24). However, the
expression levels of selected genes in blood cells showed associations with the
viral load and plasma total and HDL-cholesterol levels.
Conclusion: Current antiretroviral treatments may partially attenuate the
influence of HIV infection on certain inflammatory pathways, though patients
receiving TDF therapy must be carefully monitored with respect to the presence
and/or progression of atherosclerosis.
407 DUFFY BLOOD ALLOANTIGEN SYSTEM INFLUENCES
INFLAMMATORY CHEMOKINE CONCENTRATION IN SERUM
BUT NOT PLASMA: GEOGRAPHIC DIFFERENTIATION AS AN
ADDITIONAL CONFOUNDING FACTOR
M. Barreda, G. Aragones, A. Rull, R. Beltrán-Debón, J. Camps, J. Joven.
Centre Recerca Biomedica, Institut Investigació Sanitària Pere Virgili, Hospital
Universitari Sant Joan, Universitat Rovira i Virgili, Reus, Spain
Objectives: The Asp42Gly polymorphism (rs12075) in the Duffy antigen/receptor for chemokines (DARC) gene is a major determinant of chemokine
binding in endothelial and red blood cells. We explored whether this genetic
variant represents a confounding factor in the interpretation of monocyte
chemoattractant protein-1 (MCP-1) concentration in circulating blood.
Methods: Chemokines concentrations in serum and plasma were measured in
293 healthy Caucasian participants who are representative of our geographic
area. The rs12075 genotype distribution was also assessed in this population.
87
Results: Plasma MCP-1 concentration did not vary among the rs12075
polymorphism derived genotypes (pg/mL, AA: 178.5±56.4; AG: 190.1±75.3
and GG: 185.5±73.6). However, there were significant increases in serum
MCP-1 related to the presence of the A allele (pg/mL, AA: 365.6±138.8; AG:
329.2±132.6 and GG: 262.2±73.8). We also observed a significant variation
in the genotype frequency in our population with respect to other Caucasian
populations, indicating the presence of an additional genetic confounding
factor.
Conclusions: Differences between serum and plasma MCP-1 concentrations
suggest a clotting effect that is associated with the rs12075 polymorphism in
DARC. These findings limit the value of circulating MCP-1 as a biomarker and
apparently indicate a pathophysiological role for chemokine receptors that do
not activate intracellular signaling pathways.
408 PLASMA CHEMERIN IS ASSOCIATED WITH MARKERS OF
INFLAMMATION AND CORONARY ARTERY DISEASE IN PATIENTS
NOT ON ASPIRIN
M. Herová1,2 , M. Schmid1 , M. Hersberger1,2 . 1 Division of Clinical Chemistry
and Biochemistry, University Children’s Hospital Zurich, 2 Zurich Center for
Integrative Human Physiology (ZIHP), Zurich, Switzerland
Background: Chemerin is a peptide chemoattractant of macrophages and
dendritic cells and was recently identified as an adipokine. Active chemerin
is found in plasma and was shown to regulate inflammation and adipocyte
differentiation. Inflammation, obesity and metabolic syndrome are pronounced
risk factors for coronary artery disease (CAD). Therefore we investigated
whether plasma chemerin levels are associated with inflammatory markers and
atherosclerosis in a case-control study for CAD.
Research design and Method: Total chemerin levels were measured by ELISA
in plasma of angiographically documented CAD patients (n = 249) and healthy
controls (n = 221).
Results: Plasma chemerin levels were highly associated with high sensitive
C-reactive protein, creatinine, BMI and hypertension and negatively associated
with HDL levels. Plasma chemerin levels were similar in controls and CAD
patients. However, chemerin levels were significantly higher in CAD patients
not undergoing aspirin treatment than in CAD patients on aspirin treatment and
in controls.
Conclusion: Chemerin is strongly associated with markers of inflammation and
components of the metabolic syndrome and is associated with CAD in patients
not receiving anti-inflammatory treatment. This suggests that high chemerin
levels are associated with inflammation accompanying CAD.
409 CHANGES OF ENDOGLIN LEVELS IN SERUM AND MICE AORTA
WITH RESPECT TO CHOLESTEROL AND ATORVASTATIN
L. Večeřová, Z. Strasky, J. Rathouska, M. Slanarova, E. Brcakova, P. Nachtigal.
Charles University, Faculty of Pharmacy, Hradec Kralove, Czech Republic
Endoglin (CD 105) also known as a type III TGF-b receptor is able
to modulate TGF-b1 activity suggesting its role in atherogenesis. In this
study, we hypothesized, whether endoglin expression in aorta and its blood
serum levels are affected by cholesterol levels and/or atorvastatin treatment.
ApoE/LDLR double knockout mice were divided into 4 groups (n = 32).
Mice on chow diet, mice fed with 1% cholesterol diet, mice on chow diet
with atorvastatin 50 mg/kg/day and mice fed with 1% cholesterol diet with
atorvastatin 50 mg/kg/day. Cholesterol levels, ELISA analysis of endoglin in
blood serum and Western blot analysis of endoglin in aorta were performed.
Cholesterol feeding resulted in a significant increase of cholesterol and endoglin
levels in blood serum, whereas endoglin expression in aorta decreased, when
compared to mice on chow diet. Atorvastatin treatment in mice on chow diet
resulted in a significant increase of cholesterol levels and endoglin expression
in aorta with no changes of endoglin levels in blood. Atorvastatin treatment
in cholesterol fed mice resulted in a significant decrease of cholesterol and
endoglin levels in blood serum, with concurrent increase of endoglin expression
in aorta, when compared to untreated mice.
In conclusion, changes in endoglin blood serum levels are conversely related
to its expression in aorta, suggesting that endoglin might be interesting blood
marker, which could reflect atherosclerotic process and/or efficiency of statin
treatment.
in Prague number 129208/C and by the grant SVV-2010–261−00.
88
410 THE EFFECT OF PERCUTANEOUS TRANSLUMINAL ANGIOPLASTY
UPON MMP2, MMP9, TIMP1 AND NOX1 RELEASE IN PATIENTS
WITH STABLE ANGINA
C. Revnic1 , C. Ginghina2 , A. Mereuta3 , D. Gradinaru4 , F. Revnic5 . 1 UMF Carol
Davila Bucharest/Faculty of General Medicine, 2 Cardiology, 3 Interventional
Cardiology, C.C.Iliescu Cardiovascular Institute, 4 Pharmachology, UMF
Carol Davila, Bucharest, 5 Biology of Aging, NIGG’ANA ASLAN’, Bucharest,
Romania
Introduction: Vascular wall remodeling is a main factor contributing to
restenosis after angioplasty which is related with migration and proliferation
of vascular smooth muscle cells. The release of matrix degrading
metaloproteinase-2 and -9, facilitates remodelling. Literature data have shown
that nitric oxide decreases the activity of metalloproteinases and this may
attenuate remodeling after baloon injury.
The aim of this study was to see if metalloproteinase 2 and 9 and TIMP 1 and
NO metabolites are released into coronary sinus blood during angioplasty in
coronary patients.
Patient selection: 15 patients with stable angina have been admited in
Cardiovascular Clinique of C.C.Iliescu Cardiovascular Institute, X=58.3±6.2 for
undergrowing percutaneous transluminal coronary angioplasty of an isolated
stenosis of the proximal left anterior descending coronary artery
Method: Blood has been collected at baseline, immediately and after 2 minutes
after each baloon inflation (60 seconds duration). Plasma release of MMP2
and 9 and TIMP1 was assayed by ELISA method using Chem Well 2910
Automated EIA and Chemistry Analiser and liberation of NOx1 metabolites was
measured colorimetricaly using Nitrite/nitrates Sigma kit and the results have
been recorded on Chem Well Analyser as well.
Results: Two consecutive baloon inflations each of 60 seconds, ended up into
a significant increase in MMP 9 (p < 0.05) levels followed by normalisation to
the basal level within 2 minutes Serum levels of NOx1 remained unchanged
(X = 33.92±8.5umol/L, TIMP1 was at lower values during inflations and returned
to normal values after angioplasty.
Conclusions: Rapid release of MMP9 after baloon inflation may contribute to
remodeling and protect the vascular wall from post angioplasty trombosis.
411 A SHORT-TERM HIGH COMPLEX-CARBOHYDRATE DIET REDUCES
THE BASELINE INFLAMMATORY PROFILE
I. Shapira, O. Rogowski, S. Berliner, O. Raz. Tel Aviv Sourasky Medical
Center, Tel Aviv, Israel
Objective: Atherothrombosis is associated with the presence of low grade,
subclinical and smoldering internal inflammation. It is suggested that life style
modification might reduce the inflammatory response and be beneficial in terms
of atherosclerosis progression. The objective of this study was to compare a
high complex-carbohydrate diet with the American Heart Association Step 1
(AHAS1) Diet regarding potential anthropometric changes, as well as eventual
improvements in the lipid and inflammatory profile in two groups of apparently
healthy volunteers.
Methods: A prospective open labeled study. Each participant was evaluated
before and following 8 weeks of a complex-carbohydrate based or
AHAS1diets.
Results: Two groups of sixty one overweight volunteers (BMI > 30.9±5.0 and
30.6±4.7), were given two different types of diets for a time period of eight
weeks each. In the first group the diet was a high complex carbohydrates one
(HCCD), while the second group was on AHAS1. At a baseline, anthropometric
measurements, inflammatory profile, including the Westergren erythrocyte
sedimentation rate, high sensitivity C-reactive protein (hs-CRP), fibrinogen
concentrations and the white blood cell count (WBCC), did not differ significantly
between both groups. We found that both diets significantly reduced weight,
BMI, waist and hip circumferences of the participants, whereas the inflammatory
biomarkers were significantly reduced only in the group that was given the high
complex carbohydrate diet.
Conclusion: A short-term high complex-carbohydrate based diet is effective
in improving the individuals’ baseline inflammatory profile as compared to the
AHAS1diet..
412 EFFECTS OF INTENSIVE LIPID-LOWERING ON PLATELET
ACTIVATION & MARKERS OF SYSTEMIC INFLAMMATION IN
PATIENTS AT ELEVATED CARDIOVASCULAR RISK
E.C. Wicks1 , P. Maciocia2 , S. Bournazos3 , R. Hilling-Smith3 , I. Dransfield3 ,
N. Uren4 . 1 Cardiology, The Heart Hospital, London, 2 Bristol Royal Infirmary,
Bristol, 3 University of Edinburgh, 4 Royal Infirmary of Edinburgh, Edinburgh,
UK
Background: Statins reduce fatal and non-fatal cardiovascular events through
lipid-lowering and pleiotropic effects. However the relationships between statin
dose and platelet activation and other markers of systemic inflammation are not
well established. We therefore sought to investigate these.
Methods: 36 patients (16 men; mean age 62.8years, range 33.2–83.2) with
stable coronary heart disease (CAD) and/or multiple cardiovascular risk factors
were randomised in a single-blinded manner to receive either atorvastatin 80 mg
(n = 16) or 10 mg (n = 20) once daily for 3 months. Baseline characteristics
and usage of anti-platelet medication were recorded. Peripheral blood samples
were collected at baseline and at study end. Adhesion of platelets to CD14hi
monocytes (platelet-monocyte binding, PMB) and to neutrophils (PPMNC)
was measured using multi-parameter flow cytometry. Serum pro-inflammatory
cytokine levels (IL-8, IL-12, TNF-a) were measured using cytometric bead array
techniques.
Results: Baseline characteristics and anti-platelet therapy were similar in both
groups. Mean total cholesterol reduction was 26.5±14.3% with a greater
reduction in the high dose group (32.2±14.9% v 21.9±12.3%, p = 0.03).
Platelet adhesion to neutrophils increased in all patients (22.7% [15.8–21.4]
v 26.9% [21.1–36.3], p = 0.04) irrespective of dose used. No significant
differences in PMB, IL-8, IL-12 or TNF-a were found with either dosage.
Patient data
Parameter
Pre statin therapy
Post statin therapy
p value
Cholesterol (mmol/L)
PPMNC (%)
PMB (%)
IL-8 (pg/mL)
IL-12 (pg/mL)
TNF-a (pg/mL)
6.4 [5.5−6.8]
22.7 [15.8–21.4]
77.7 [61.3–85.7]
12.6 [9.1–17.4]
23.7 [18.8–32.9]
17.4 [12.9–23.9]
4.7 [3.8−5.3]
26.9 [21.1–36.3]
61.9 [79.6–86.1]
12.2 [7.9–15.4]
25.1 [16.2–32.4]
18.8 [16.6–26.4]
<0.0001
0.04
NS
NS
NS
NS
Values are presented as median [IQR] unless otherwise stated.
Conclusions: Lipid-lowering using atorvastatin leads to increased plateletneutrophil binding in patients at increased cardiovascular risk, independent
of dose used. Other markers of inflammation are not altered. These findings
suggest that the anti-inflammatory actions of atorvastatin are complex. It
is possible that atorvastatin activates neutrophils, thus enhancing plateletneutrophil complex formation and clearing activated platelets from the
circulation.
413 ERK SIGNALING IS INVOLVED IN INFLAMMATION AND VASCULAR
REMODELING PROCESS IN PREMATURE CORONARY ARTERY
DISEASE
V. Dhawan1 , N. Mahajan1,2 , O.M. Bhat1 , A. Bahl3 , I. Sharma1 . 1 Experimental
Medicine and Biotechnology, Postgraduate Institute of Medical Education &
Research, Chandigarh, India, 2 Department of Molecular Pharmacology and
Biological Chemistry, Robert H. Lurie Comprehensive Cancer Center, Feinberg
School of Medicine, Northwestern University, Chicago, IL, USA, 3 Cardiology,
Postgraduate Institute of Medical Education & Research, Chandigarh, India
C-reactive protein (CRP) induces pro-inflammatory micro-environment within
an atherosclerotic plaque, creating an imbalance between the MMPs and
their tissue inhibitors (TIMPs), rendering it prone to rupture. Keeping in view
multifactorial aspects of CAD, it becomes imperative to identify molecular
targets for this disease. Circulating levels of hsCRP, MMP-1, 2 & 9 and
TIMP-1 were determined in the serum of 80 subjects with angiographically
proven premature CAD (Group II) along with 80 age and sex-matched
controls (Group I) using ELISA kits. Intracellular levels of IL-6 and TNF-a
were determined by flow cytometry in PBMCs of the study subjects before
and after atorvastatin treatment. Effect of atorvastatin was determined on
transcriptional expression of MMP-1, MMP-2, MMP-9 and TIMP-1 in PBMCs
of the study subjects using RT-PCR. Specific MAPK inhibitors (ERK-PD 98059,
p38-SB 203580 and JNK-SP 600125) were used to elucidate the signaling
pathways involved in PBMCs. Significantly augmented levels of hsCRP, MMP-9,
TNF-a and IL-6 were observed in the patients with premature CAD as
compared to controls. Transcriptional expression of MMPs was also found
to be significantly augmented in these subjects and atorvastatin significantly
attenuated expression of MMPs and pro-inflammatory cytokines, whereas it
augmented TIMP-1 expression. Signaling experiments revealed that MMPs
utilized ERK signaling pathway to transduce signals in PBMCs probably via
CRP as a mediator. Our study points in the direction that monitoring MMP-TIMP
imbalance and pro-inflammatory cytokines may prove vital in better evaluation
of the subjects with premature CAD. Targeted inhibition of the ERK pathway
could serve as therapeutic strategy.
79th EAS Congress
414 OXIDATIVE STRESS MEDIATES APL-INDUCED THROMBOSIS AND
ATHEROSCLEROSIS DEVELOPMENT IN ANTIPHOSPHOLIPID
SYNDROME
P. Ruiz-Limón1 , C. Perez-Sanchez1 , M.A. Aguirre2 , N. Barbarroja1 ,
R.M. Carretero1 , A. Rodriguez-Ariza1 , E. Collantes-Estevez2 , J.M. Villalba3 ,
F. Velasco4 , M. Khamashta5 , M.J. Cuadrado5 , C. Lopez-Pedrera1 . 1 Research
Unit, 2 Rheumatology Service, Reina Sofia Hospital/IMIBIC, 3 Biologı́a Celular,
Fisiologı́a e Inmunologı́a, Universidad de Córdoba, 4 Haematology, Reina Sofia
Hospital/IMIBIC, Córdoba, Spain, 5 Lupus Research Unit, St Thomas Hospital,
London, UK
The aim of this study was to investigate the association of oxidative stress and
mitochondrial membrane potential (MMP) in leucocytes from 20 APS patients
with the development of thrombosis and atherosclerosis. Increased expression
of TF and PARs were found in monocytes from APS patients, with higher
plasma levels of VEGF, tPA, IL8 and CRP. APS monocytes and neutrophils
showed increased peroxides and superoxide levels and more depolarized
mitochondria, while intracellular GSH was decreased. Healthy monocytes
stimulated with APS, but not with control IgG, also displayed increased
peroxides and superoxide levels and decreased MMP and GSH. Monocyte
MMP and peroxides showed significant negative and positive correlations,
respectively, with TF expression. Peroxide levels further showed a significant
positive correlation with plasma levels of other pro-atherosclerotic markers
(tPA and CRP). aPL-IgG positively correlated with peroxides, TF, VEGF and
PAR2, and negatively with MMP and GSH. That data suggest that an excess
of oxidative stress, most likely caused by effect of anticardiolipin antibodies,
may act as a key determinant of atherothrombosis, possibly through a shift
of the coagulation balance towards a procoagulant state. In support for that
hypothesis, carotid intimate-media thickness was associated to increased TF
and PAR2 expression and markers of oxidative stress. Thrombosis development
was further associated with NO, peroxides, MMP, and GSH levels.
Conclusion: Oxidative stress could be considered a pathogenic link between
aPL, thrombosis and atherosclerosis development in APS. Antioxidant treatment
might have efficacy in preventing the clinical manifestations of this autoimmune
disease.
Supported by JA0246/2009, P08CVI04234 and PS09/01809.
415 MONOCYTE CHEMOATTRACTANT PROTEIN-1 AND
PARAOXONASE-1 IN PERIPHERAL ARTERIAL DISEASE: A MODEL
OF INTERACTION BETWEEN DEFENSE AGAINST OXIDATIVE
STRESS AND INFLAMMATION
A. Rull1 , R. Garcı́a2 , L. Fernández-Sender1 , R. Beltrán-Debón1 , G. Aragonès1 ,
J.M. Alegret1 , C. Alonso-Villaverde3 , M. Mackness1 , J. Camps1 , V. MartinParedero2 , J. Joven1 . 1 Centre de Recerca Biomèdica, Institut d’Investigació
Sanitària Pere Virgili (IISPV), Universitat Rovira i Virgili (URV), Reus, 2 Servei
d’Angiologia, Cirurgia Vascular i Endovascular, Hospital Universitari Joan XXIII
de Tarragona, Universitat Rovira i Virgili, Tarragona, 3 Servei de Medicina
Interna, Hospital Son Llàtzer, Mallorca, Spain
Background: Atherosclerosis in symptomatic peripheral arterial disease affects
wide portions of numerous arteries in lower extremities representing active
inflammation in a considerable amount of arterial tissue. This facilitates systemic
detection via inflammation-related variables measurement. We reasoned that
the interaction between defense against oxidative stress and inflammation
may be also easily detected using paraoxonase-1 (PON1) and monocyte
chemoattractant protein-1/chemokine (C-C motif) ligand 2 (CCL2) as putative
biomarkers.
Methods and Results: Plasma CCL2 and serum PON1-related variables
were measured in a cross-sectional study in patients with symptomatic PAD
assessed by their interaction with functional genetic variants. We found that
the combination of plasma CCL2 and PON1 related values, especially PON1
concentration, segregated almost perfectly controls from patients and that the
expression generally co-localized in the atherosclerotic lesion; CCL2 apparently
limited to areas rich in inflammatory cells and PON1 extended to a variety
of cellular types. Since no association with genetic variants was found, such
relationship is probably the result of the disease.
Conclusions: Our data suggest a coordinated role between CCL2 and PON1
that may be detected in blood with simple measurements and may represent
an indicator of the extent of atherosclerosis or endothelial damage.
416 TAQIB AND I405V CETP POLYMORPHISMS DO NOT MODIFY
PLASMA INFLAMMATORY MARKERS: A STUDY IN A BRAZILIAN
POPULATION SAMPLE
E. Soler Parra, F.L. D’Alexandri, N.B. Panzoldo, D.B. Kaplan, E.C. de
Faria. Departamento de Patologia Clı́nica, Núcleo de Medicina e Cirurgia
Experimental, Faculdade de Ciências Médicas, Universidade Estadual de
Campinas, São Paulo, Brazil
Objective: This study determined CETP activity interactions with plasma
inflammatory markers in carriers of 2 well-known polymorphisms of the CETP
gene.
89
Methods: TaqIB and I405V polymorphisms of CETP gene were investigated
in 223 adult volunteers of both sexes and their lipid profiles were determined.
The concentrations of the pro-inflammatory factors CRP (immunoturbidimetric
assay), TNF-a (ELISA) and abLDLox (in house ELISA) were measured and
their associations with the polymorphisms and CETP activity investigated.
Results: Lower concentrations of abLDLox were found in the VV polymorphism
(I405V) and higher levels were found in B1B2 genotype (TaqIB), the
only differences observed between the genotypes. The distribution of the
polymorphisms did not differ between the percentiles and quartiles of these
markers, but an interesting inverse relationship between CETP activity and
sistolic and diastolic blood pressure was found in genotypes B2B2 and IV,
presenting lower CETP activity.
Conclusion: TaqIB and I405V polymorphisms, used in this study as model of
CETP deficiency, did not modify plasma levels of inflammatory markers. Further
studies are ongoing in the laboratory to better understand the CETP association
with blood pressure.
Funding Agencies: Fapesp, CNPq
417 SOLUBLE CELLULAR ADHESION MOLECULES,
MYELOPEROXIDASE, AND NEOPTERIN IN METABOLIC SYNDROME
PATIENTS WITH STABLE AND UNSTABLE ANGINA PECTORIS
P. Tretjakovs1 , A. Jurka2 , I. Bormane2 , I. Mikelsone2 , D. Reihmane2 ,
G. Krievina2 , K. Elksne1 , J. Verbovenko3 , G. Bahs3 , D. Fuchs4 . 1 Riga Stradins
University, 2 University of Latvia, 3 Riga Eastern Clinical University Hospital,
Riga, Latvia, 4 Medical University of Innsbruck, Innsbruck, Austria
Background: The aim of the present study was to evaluate differences
in serum levels of neopterin, soluble cellular adhesion molecules (sCAMs),
and myeloperoxidase (MPO) between coronary artery disease and metabolic
syndrome (CAD-MetS) patients with stable and unstable angina pectoris (SAP,
UAP), and to clarify relationships between neopterin and other biomarkers.
Material and Methods: The study included 60 patients with CAD-MetS
who were classified into two groups, 30 patients with SAP and 30 patients
USP. 20 healthy subjects were selected as controls (C). Serum soluble
vascular cell adhesion molecule-1 (sVCAM-1), intercellular cell adhesion
molecule-1 (sICAM-1), sE-selectin and MPO levels were measured by Luminex
xMAP technology, but serum neopterin concentrations were measured by
radioimmunoassay.
Results: Serum levels of neopterin, MPO, sVCAM-1, sICAM-1, and sE-selectin
were significantly higher in patients with UAP in comparison with the group
of healthy controls (p < 0.05). Patients with SAP also had higher levels of
these biomarkers than healthy controls (p < 0.05), except for sE-selectin. The
biomarkers did not differ between the two patient groups, except for MPO, which
was significantly higher in the USP group (p < 0.05). Neopterin was significantly
correlated only with sVCAM-1 (p < 0.05).
Conclusion: CAD-Met patients with SAP have more apparent elevations of
serum sICAM-1 and sVCAM-1 levels, simultaneously with higher MPO and
neopterin concentrations than healthy subjects, but UAP is also associated with
more substantial changes in MPO and significantly increased sE-selectin levels.
Neopterin has a close correlation only with sVCAM-1.
418 ENDOGLIN-RELATED PATHWAYS ARE NOT RELATED TO DISTINCT
PREDISPOSITION TO ATHEROSCLEROSIS IN C57BL/6J AND
C3H/HEJ MICE
J. Rathouska, Z. Strasky, L. Vecerova, E. Brcakova, M. Slanarova, P. Nachtigal.
Department of Biological and Medical Sciences, Charles University, Faculty of
Pharmacy, Hradec Kralove, Czech Republic
In general, mice, being resistant to atherosclerosis, do not develop
atherosclerotic lesions on a low-fat diet. It was demonstrated that C57BL/6J
mouse strain is the most susceptible to atherosclerosis, while C3H/HeJ is the
most resistant. Endoglin-related pathways, including ALK and Smad proteins,
are suggested to play a protective role in atherogenesis. In this study, we
hypothesized, whether expressions of two endoglin-related pathways, namely
endoglin/ALK-5/Smad2/eNOS and endoglin/ALK-1/Smad1/VEGF, are related to
distinct predisposition to atherosclerosis in C57BL/6J and C3H/HeJ mice.
Female normocholesterolemic mice were divided into two groups (n = 16);
C3H/HeJ and C57BL/6J group. Both groups were fed with chow diet for 12
weeks after the weaning. Biochemical analysis of lipid profile and western blot
analysis of endoglin, ALK-1, ALK-5, phosphorylated Smad-2, phosphorylated
Smad-1, eNOS and VEGF expression in aortas were performed.
Cholesterol levels were significantly higher in C3H/HeJ mice, including HDL,
when compared with C57BL/6J. However, atherogenic index did not differ
between these strains. Western blot analysis showed no significant differences
in endoglin, ALK-1, ALK-5, p-Smad2, eNOS and VEGF expression, however,
there was a significant difference in p-Smad1 expression in aortic sinus of
C3H/HeJ and C57BL/6J mice.
In conclusion, expressions of two endoglin-related pathways, endoglin/ALK5/Smad2/eNOS and endoglin/ALK-1/Smad1/VEGF, do not seem to differ in
90
aortas of both studied mouse strains, suggesting that these pathways are not
related to distinct predisposition to atherosclerosis.
in Prague number 129208/C and grant SVV-2010–261−00.
419 CORRELATIONS BETWEEN CLINICAL, IMMUNOLOGICAL AND
METABOLIC CONTRIBUTORS AND ATHEROSCLEROSIS IN
SYSTEMIC LUPUS ERYTHEMATOSUS
E. Ammirati1,2 , R. Contri2 , E. Bozzolo3 , S. Tramontana4 , K. Garlaschelli4 ,
A. Palini3 , D. Cianflone2,3 , A.L. Catapano4,5 , M.G. Sabbadini2,3 , G.D. Norata4,5 .
1
Heart Care Foundation, Florence, 2 Vita-Salute San Raffaele University,
Milano, 3 San Raffaele Scientific Institute, 4 Centro SISA per lo Studio
dell’Aterosclerosi, Bassini Hospital, 5 Department of Pharmacological Sciences,
Università degli Studi di Milano, Dip. Scienze Farmacologiche, Milan, Italy
Objective: Patients with systemic lupus erythematosus (SLE) are at higher
risk for cardiovascular (CV) events, mainly due to an increased atherosclerotic
burden. Traditional, immunological, disease- and treatment-related factors could
explain such difference, but their single contributions on the pathogenesis of
atherosclerosis is unclear.
Methods: In fifty patients (44±10 years, 86% female) with a median SLE
duration of 14 years, and 50 sex- and age-matched controls 56 subsets of
circulating CD4 T-cells were investigated by 8/color/polycromatic flow cytometry
(CD3/CD4/CD45RO/CD45RA/CCR7/CCR5/CXCR3/HLA-DR) and correlated to
lipid, metabolic, inflammatory profile and carotid intima-media thickness
(CIMT).
Results: Patients with SLE did not differ in terms of CV risk factors and
metabolic profile compared to controls, except for a reduction in highdensity lipoprotein levels (p < 0.001). SLE patients presented significant
changes concerning the immunological profile (increase in C-reactive protein,
lymphopenia and modified levels of circulating HLA-DR+, CCR5+T and effector
memory T-cells). Age-adjusted mean CIMT was significantly higher in patients
compared to controls (p = 0.03). In univariate analysis correlations were
observed between age-adjusted mean CIMT and systolic blood pressure
(p = 0.048), LDL cholesterol (trend p = 0.06) and body-mass index (p = 0.01)
but not with immunological parameters. Among variables associated to the
treatment only antimalarian hydroxycloroquine therapy was associated with
lower CIMT.
Conclusions: As traditional CV risks factors are significantly associated with
accelerated atherosclerosis in SLE patients, management of CV risk factors
should be part of the treatment. As the inflammatory status in SLE is not
a constant but a waxing and waning pressure, that could explain the lack of
correlation between CIMT and immunological features.
420 EZETIMIBE INHIBITS PMA-INDUCED MONOCYTIC
DIFFERENTIATION INTO MACROPHAGE-LIKE PHENOTYPE
THROUGH THE MICRORNA PATHWAY
P. Muñoz-Pacheco, A. Ortega-Hernández, A. Fernández-Cruz, D. GómezGarre. Vascular Biology Laboratory, Lipid Unit, Hospital Clinico San Carlos,
Madrid, Spain
Introduction: Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, effectively reduces plasma cholesterol both in monotherapy or combined
with a statin. However, its effect on atherosclerosis plaque progression
is certainly unknown. MicroRNAs are short non-encoding RNA molecules
dynamically implicated in monocytic differentiation which is considered an
essential process during atherosclerosis development.
Aims: The purpose of this study was to investigate the effect of ezetimibe on
monocyte/macrophage differentiation in vitro.
Methods: The human monocitic THP-1 cell line was differentiated into a
macrophage-like phenotype by incubation with phorbol-12-myristate-13-acetate
(PMA) in the presence or absence of ezetimibe. Adhesion assays were
performed with a crystal violet solution and measured by spectrophotometry.
CD11b, a macrophage surface marker, was analyzed by flow cytometry.
Expression of microRNAs, mir-155, mir-222, mir-424 and mir-503, was
quantified by real-time PCR.
Results: THP-1 differentiation with PMA became cells adherent to the plastic
surface (100±3 vs 423±15%, P < 0.01), induced the expression of adhesion
molecules such as CD11b (100±2 vs 143±13%, P < 0.05) and up-regulated
the expression of microRNAs (mir-155: 2-fold; mir-222: 2.1-fold; mir-424:3-fold;
mir-503: 7-fold, P < 0.05). In the presence of ezetimibe (10-5 M), both PMAinduced THP-1 cell adhesion and cell surface antigen CD11b expression were
reduced by 30%. miRNA expression was almost totally inhibited (mir-155: 87%,
mir-222: 99%, mir-424: 76% and mir-503: 97%).
Conclusion: Ezetimibe inhibits PMA-induced THP-1 cell differentiation into
macrophage-like cells through inhibition of microRNA pathway. Our study
suggests that inhibition of microRNAs might form a novel mechanism of antiatherosclerotic effect of ezetimibe.
421 ATHEROSCLEROSIS RISK FACTORS, INFLAMMATION AND DENTAL
STATE IN PATIENTS WITH CARDIO-CEREBROVASCULAR DISEASE
AND EXCESSIVE OBESITY (BMI OVER 40) FROM ROMANIA
I.A. Gutiu1 , L.I. Gutiu2 , F.S. Radulescu3 . 1 Medical Emergencies Dpt.,
University of Medicine and Pharmacy ‘Carol Davila’, Bucharest, 2 Neurology,
Military Emergencies Hospital, 3 Faculty of Pharmacy, University of Medicine
and Pharmacy ‘Carol Davila’, Bucharest, Romania
Purpose: The role and incidence of atherosclerosis risk factors in patients
with excessive obesity are more little known and may present some specific
features.
Methods: We analyzed a group of subjects with excessive obesity (BMI > 40
Kg/squared meter) concerning the traditional atherosclerosis risk factors (TRF):
dyslipidaemia, arterial hypertension, smoking, etc.; presence of non specific
inflammation syndrome (serum fibrinogen level, C reaction protein, etc.);
number of tooth missing (NTM) indicating an old chronic low inflammation
presence (by parodontosis/parodontitis). From 571 patients with cardiocerebrovascular disease (CCVD), we selected all subjects with BMI over 40
Kg/sm and, for comparison, the subjects with BMI under 25 kg/m2 , all included
in 3 groups: A (137 patients, 24.0% of all) − normal BMI; B (19, 3.3%) −
severe obesity (BMI 40 and <45 kg/m2 ), and C (7, 1.2%) − excessive obesity
(BMI 45 Kg/m2 ).
Results: Some TRF significantly differ especially between normal and
excessive obese groups: age (63.7±12.4 versus 51.4±7.9 years, P < 0.001),
serum triglycerides (124.7±72.1vs 185.3±94.7 mg%, P < 0.001), systolic
arterial pressure (153.3±18.6 vs 150.3±18.1 mmHg, P < 0.003), NTM
(15.1±8.3 vs 8.2±3.9, P < 0.002). Incidence of metabolic syndrome presence
was in these 3 groups: 35% (A), 93% (B), 100% (C) (P < 0.001). Other variables
do not significantly differ in these 3 groups. Multivariate analysis ordered:
triglycerides, systolic arterial pressure, age and NTM (all at p < 0.001).
Conclusions: In CCVD and excessive obesity are present some TRF, mainly
indicating dyslipidaemia. Only tooth loss number indicated an old low level
inflammation presence in atherogenesis process. Comparing with normal
weight patients, excessive obesity group is younger but incidence of metabolic
syndrome is greater.
422 ASSESSMENT OF INSULIN RESISTANCE, DYSLIPIDEMIA,
OXIDATIVE STRESS AND INFLAMMATORY RESPONSE IN MALE
PATIENTS WITH ANGIOGRAPHICALLY PROVEN CORONARY
ARTERY DISEASE
B. Goswami1 , D. Tayal2 , S. Tyagi3 , V. Mallika3 . 1 Biochemistry, Maulana Azad
Medical College and Associated Hospitals, 2 Lady Hardinge Medical College
and Associated Hospitals, 3 Maulana Azad Medical College and Associated
Hospitals, Delhi, India
Background: Extensive research has proven the role of multiple etiologies such
as dyslipidemia, inflammation, endothelial dysfunction and insulin resistance
in the pathogenesis of Coronary artery disease. The following study was
undertaken to assess the roles of insulin resistance, inflammation, oxidative
stress and dyslipidemia; which are important risk factors for CAD in the
atherosclerosis-prone North Indian male population.
Materials and Methods: The present study was conducted in 100 patients of
myocardial infarction (MI) diagnosed on electrocardiographic and biochemical
criteria, who subsequently underwent coronary angiography and 100 age
matched healthy controls. The parameters that were evaluated include lipid
profile, hsCRP, apolipoprotein B, Nitric Oxide, insulin levels and HOMA-IR.
Results: Significantly higher serum levels of cholesterol, triglycerides, LDL and
apolipoprotein B was observed in the patients as compared to the controls.
On further classification, the dyslipidemia was marked in the patients with
triple vessel disease as compared to single and double vessel disease. Similar
pattern was observed for insulin resistance, NO and CRP. Upon plotting the
ROC curves, hsCRP emerged as the strongest predictor for CAD.
Conclusions: The present study illustrates the roles of insulin resistance,
inflammation, oxidative stress and dyslipidemia in the pathogenesis of
atherosclerosis in the CAD prone north Indian population. It also highlights
the superiority of hs CRP in risk stratification of patients with angiographically
proven coronary artery disease.
423 ASSOCIATION BETWEEN INFLAMMATION AND
ATHEROSCLEROSIS IN METABOLIC SYNDROME PATIENTS: THE
SAME IN ALL ARTERIAL BEDS?
P. Rein1,2,3 , C.H. Saely1,2,3 , S. Beer1,2,3 , A. Vonbank1,2,3 , C. Boehnel1,3 ,
V. Drexel1,3 , V. Kiene1 , H. Drexel1,2,3,4 . 1 VIVIT Institute, 2 Internal Medicine,
Academic Teaching Hospital Feldkirch, Feldkirch, Austria, 3 Private University
of the Principality of Liechtenstein, Triesen, Liechtenstein, 4 Drexel University
College of Medicine, Philadelphia, PA, USA
Background: While inflammatory markers are firmly established as predictors
of clinical atherothrombotic events, data on their association with directly
visualized atherosclerosis are controversial. We aimed at investigating the
79th EAS Congress
association of C-reactive protein and of leukocytes with atherosclerosis in
different arterial beds.
Methods: We enrolled 405 consecutive Caucasian patients with the Metabolic
Syndrome (MetS) who were referred to coronary angiography for the evaluation
of stable coronary artery disease (CAD) and who did not have a history of
peripheral arterial disease (PAD). Significant CAD was diagnosed in patients
with significant coronary artery lumen narrowing 50% (n = 232); subjects
without such lesions served as controls (n = 173). Additionally, we enrolled 200
MetS patients who underwent routine duplex sonography for the evaluation of
suspected or established PAD and in whom PAD was verified sonographically.
Results: The inflammatory markers CRP and leukocytes did not differ
significantly between patients with stable CAD and controls (0.47±0.71 vs.
0.43±0.48 mg/dl; p = 0.472; 6.9±1.7 vs. 6.9±1.8 G/L; p = 0.856, respectively).
In contrast, CRP as well as leukocytes were significantly higher in PAD patients
compared to both, patients with stable CAD (0.94±1.88 vs. 0.47±0.71 mg/dl;
p = 0.001 and 7.6±2.3 vs. 6.9±1.7 G/L; p = 0.003, respectively) and controls
(0.94±1.88 vs. 0.43±0.48 mg/dl; p = 0.008; 7.6±2.3 vs. 6.9±1.8 G/L; p = 0.005,
respectively).
Conclusion: In conclusion, CRP and leukocytes do not differ between MetS
patients with or without stable CAD but are significantly elevated in MetS
patients with PAD. This is well in line with the extremely high cardiovascular
event risk of PAD patients.
424 STATE CELLULAR LEVEL OF IMMUNE RESPONSE IN PATIENTS
WITH CHD, DEPENDING ON THE AVAILABILITY OF SIMPLE OR
COMPLEX CORONARY STENOSIS
O. Lomakovskyi. Institute of Cardiology, Kyiv, Ukraine
The goal was to link the cellular level of immune response in patients with CHD
and morphological type of stenosis of coronary arteries.
Materials and Methods: Depending on the morphological state of
atherosclerotic plaque on the angiographic classification of Ambrose J.A.
(1985), patients with a lesion of the coronary arteries (n = 79) were divided
into three groups.
Results: In CHD patients with simple stenosis of the arteries of the heart
(stable plaque) compared with the control group was observed more Th
(43.8±1.9 vs. 36.2±0.5%; p < 0.05), a large quantity of immunoregulation Index
Th/Ts (1.91±0.11 vs. 1.36±0.14 arbitrary units; p < 0.05) and lower phagocytic
activity of neutrophils (33.2±3.0 vs. 45.4±3.3%; p < 0.05). In patients with
complex coronary artery stenosis (unstable plaque) compared with patients with
simple stenosis revealed painful amount of general T-lymphocyte (68.4±1.7
vs. 63.4±1.5%; p < 0.05), large phagocytic activity of neutrophils (44.1±2.8 vs.
33.2±3.0%; p < 0.05). In patients with acute thrombosis of the heart arteries
(plaque rupture) compared with patients with complex stenosis with numerous
B-lymphocytes (16.0±1.1 vs. 10.5±1.4%; p < 0.05) and reduced their ability to
apoptosis (10.5±1.4 vs. 16.0±1.1%; p < 0.05).
Conclusion: State cellular level of immune response is associated with
morphological type of stenosis of coronary arteries and can affect the
destabilization of atherosclerotic plaques of patients with coronary artery
disease.
425 CARDIOVASCULAR RISK FACTORS AND INFLAMMATORY
MARKERS IN SYSTEMIC LUPUS ERYTHEMATOSUS: GENDER
DIFFERENCES
T.A. Panafidina, T.V. Popkova, N.G. Klukvina, D.S. Novikova, E.N. Alexandrova,
Z.S. Alekberova, E.L. Nasonov. Research Institute of Rheumatology of RAMS,
Moscow, Russia
Background: Patients with Systemic lupus erythematosus (SLE) have an
increased risk for cardiovascular disease (CVD) and develop atherosclerosis
(AS) likely associated with a combination of factors, including disease and its
therapy related factors and classic coronary artery disease risk factors.
Objectives: To determine gender differences in cardiovascular risk factors AS
and inflammatory markers in SLE patients (pts).
Methods: We studied 227 SLE pts: 156 women (mean age 35.4±0.8 years)
and 71 men (36.1±1.3 years, p > 0.05), fulfilling ACR criteria for SLE. We
considered traditional cardiovascular risk factors and SLE-related factors (age
at onset, duration of SLE, clinical features, disease activity and treatment
with steroids). We measured serum levels of soluble tumor necrosis factor-a
(sTNF-a), CD40 ligand (sCD40L) and C-reactive protein (CRP) by the highly
sensitive particle-enhanced immunonephelometric assay.
Results: CVD was found in 13% (20/156) of women and 31% (22/71) of
men (p = 0.001); subclinical AS (carotid plaque) − 11% vs 32% (p = 0.0001),
respectively. Reliable differences were found in the intima-media wall thickness
(IMT) of common carotid arteries: 0.80 (0.70–0.92 mm) for women and 0.90
(0.76–1.25 mm) for men (p < 0.001). We observed statistical differences in SLE
females vs males in: smoking − 21% vs 61% (p < 0.001), cumulative steroid
doses − 39.1±3.4 g vs 80.1±6.6 g (p < 0.001), concentration of sCD40L −
7.3±0.4 vs 10.1±0.7 ng/ml (p < 0.001) and sTNF-a − 4.2±0.3 vs 5.2±1.1
ng/ml (p < 0.05).
91
Conclusions: CVD and AS were determined frequently at men than women
and were associated with smoking, cumulative steroid doses and sTNF-a,
sCD40L.
426 ASSOCIATION OF TNF-ALPHA AND ANTICARDIOLIPIN
ANTIBODIES IN TYPE II DIABETES MELLITUS
M. Becarevic1,2 , J. Seferovic2 , S. Ignjatovic2,3 , N. Majkic-Singh2,3 . 1 University
of Novi Sad, Medical Faculty, Novi Sad, 2 Clinical Center of Serbia, 3 University
of Belgrade, Pharmaceutical Faculty, Belgrade, Serbia
Objective: Patients with type II diabetes mellitus have a greater prevalence of
accelerated atherosclerosis. It was estimated that traditional risk factors can
account for 30% of excess cardiovascular risk factors in diabetec patients, and
that inflammation is a relevant in all stages of atherosclerosis. Therefore, we
investigated the TNF-alpha and anticardiolipin (aCL) antibodies concentrations
in patients with type II diabetes mellitus.
Methods: Study included 78 patients with type II diabetes mellitus (53 male).
Mean age was 53.86±9.24; mean BMI was 27.75±4.49, and mean HbA1c
was 7.09±0.96. Measurement of TNF-alpha and anticardiolipin antibodies
concentrations was done by ELISA.
Results: Mean concentrations of aCL antibodies of the IgG isotype were
6.35±5.22 GPLU/mL and for the IgM isotype 4.93±3.07 MPLU/mL. Although
mean concentrations of aCL antibodies were not detected in high titers,
weak, but statistically significant correlation was found between concentrations
of TNF-alpha and IgG class (r = 0.303, p = 0.007) and IgM class (r = 0.386,
p = 0.000) of anticardiolipin antibodies.
Conclusion: Although in vitro it was shown that stimulation of monocytes with
monoclonal aCL antibodies resulted in increased secretion and expression
of TNF-alpha, a larger prospective study is needed to further explore this
association of TNF-alpha and anticardiolipin antibodies in type II diabetes
mellitus.
427 DECREASED TITERS OF ANTI-OXLDL ANTIBODIES
AFTER ACUTE CORONARY SYNDROME IN PATIENTS WITH
METABOLIC SYNDROME ARE ASSOCIATED WITH SEVERITY
OF ATHEROSCLEROSIS
H.A. Fonseca1 , C.M. Monteiro1 , L.F. Pinheiro1 , F.A. Fonseca1 , S.A. Brandao1 ,
S.C. Fischer1 , A.O. Santos1 , M.A. Gidlund2 , A.M. Monteiro2 , A.M. Figueiredo
Neto2 , M. Izar1 . 1 Medicine, Federal University of São Paulo, 2 University of
São Paulo, São Paulo, Brazil
Objectives: Inflammation and adaptive immunity have been recognized to
modulate atherosclerosis and plaque instability. We investigated the role of
autoantibodies against oxLDL in subjects with metabolic syndrome (MetS) after
an acute coronary syndrome (ACS).
Methods: Patients of both genders (n = 116) were included in the first 1−3 days
after hospital discharge due to ACS. Anti-oxLDL antibodies (Abs) of IgG class
were assayed by ELISA (OD). Carotid intima media thickness and the severity
of coronary disease (Gensini score) were examined.
Results: Six weeks after the first evaluation, there was improvement on lipid
profile, reduction on TBARS, high-sensitivity C-reactive protein (hs-CRP) and
anti-oxLDL Abs titers (P < 0.005). The decline of anti-oxLDL Abs was observed
in males (P = 0.012), in those under 65 y (P = 0.032), and in subjects with
Gensini score above the median (P = 0.040).
Conclusions: In patients with MetS after an ACS circulating anti-oxLDL Abs
continue to decrease in the short term follow-up. The consumption of Abs
antioxLDL was associated with the severity of atherosclerosis and can be a
marker of the immune response.
428 PERIODONTAL TREATMENT INDUCES REDUCTION RISK
MARKERS FOR ATHEROSCLEROSIS
A. Moreira Monteiro1 , M. Jardini2 , S. Alves3 , A. Figueiredo Neto3 , M. Gidlund4 .
1
Institute of Biomedical Sciences, São Paulo, 2 School of Dentistry of São José
dos Campos, São Paulo State University, Sao Jose dos Campos, 3 Institute
of Physics, 4 Institute of Biomedical Sciences, University of São Paulo, São
Paulo, Brazil
Objectives: The purpose of this study was to report the benefits of periodontal
treatment in patients with chronic periodontitis in reducing the well-established
risk markers for atherosclerosis.
Material and Methods: Forty patients with chronic periodontitis between 31 to
60 years of age were referred and submitted periodontal treatment. Probing
depths and bleeding on probing were evaluated clinically before and 3, 6
and 12 months after periodontal treatment. Blood samples were collected and
total and differential white blood cells counts, total cholesterol, triacyglycerol,
high-density lipoprotein, low-density lipoprotein, levels of cytokines, C-reactive
protein, antibodies against oxidized low-density lipoprotein and non-linear index
of refraction were measured and compared over time.
Results: After periodontal treatment the pathogenic gingival pockets and
bleeding on probing decreased significantly. One year after the ending
of treatment, triacyglycerol concentrations were significantly decreased.
92
Interleukin-6, -8 and C-reactive protein levels were significantly lower after
periodontal treatment. Treatment had no effect on plasma levels total
cholesterol, HDL and LDL. The value of non-linear index of refraction of
low density lipoprotein and the IgG antibodies against oxidized low-density
lipoprotein were significantly higher and lower, respectively 12 months after
periodontal treatment. Leukocyte and neutrophils counts were lower after 3
months of periodontal treatment.
Conclusion: In conclusion, this study indicates that standard treatment for
periodontal disease induces systemic changes in several biochemical markers
that reflect the risk for atherosclerosis.
429 HLA-G AND NPC1L1 GENE POLYMORPHISMS IN CAD PATIENTS
WITH DIFFERENT LEVELS OF LDL
C. Boiocchi, E. Maggioli, S. Bozzini, M. Cuccia, C. Falcone. University of
Pavia, Pavia, Italy
Objective: We investigated the allele and genotype frequencies of 14bp Ins/Del
polymorphism of HLA-G gene (HLA class III 6p21.3) in 664 CAD patients and
of 2093 A/G polymorphism of NPC1L1 gene (7p14) in 194 patients but in
the context of differing cholesterol levels. We also investigated 215 controls,
opportunely matched. There is evidence to suggest that tissue expression of
HLA-G tolerogenic molecule can be influenced by genetic polymorphisms of
3’UTR. Recent studies have shown a correlation between NPC1L1 mutation
and plasma cholesterol levels.
Materials and Methods: Polymorphisms were detected by PCR-RLFP and
Real Time PCR. Patients were subdivided in two groups: patients with level of
LDL <150 mg/dL and patients with LDL >150 mg/dL.
Results: Frequency of the Ins/Ins HLA-G genotype was significantly higher in
patients with CAD than controls. After confounding variables were controlled for,
results showed that the Ins/Ins was significantly and independently associated
with CAD (P = 0.03). NPC1L1 polymorphism: a significant increase of GG
and AG frequencies in CAD patients vs controls (P = 0.001; P = 0.004) was
found. Significant increase of GG genotype in patients with LDL <150 mg/dL
vs controls (P = 0.002) and with LDL >150 mg/dL vs controls (P = 0.0144) were
demonstrated.
Conclusion: Our data show a significant association between the HLA-G
Ins/Ins genotype and CAD adding a novel risk factor to genetic contribution
to this inflammatory disease. Our results on NPC1L1 polymorphism show the
hypothesis that the GG genotype may be involved in the predisposition to
hypercolesterolemia. Epistatic interaction of two gene will be presented.
430 CRYPTOCOCCUS NEOFORMANS CAUSES LIPID PEROXIDATION;
THEREFORE IT IS A POTENTIAL INDUCER OF ATHEROGENESIS
C. Jarrstrand Hall1 , U. Dizcfalusy1 , K. Sandstedt2 , L.R. Bouhafs3 . 1 Laboratory
Medicine, Karolinska Institute, 2 Department of Laboratory Medicine, Karolinska
Institutet, 3 Department of Laboratory Medicine, Karolinska University Hospital
Huddinge, Stockholm, Sweden
Abstract. Certain viral and bacterial species play a role in the development
of atherosclerosis. Our hypothesis was that yeasts, such as Cryptococcus
neoformans, may also be a cause of lipid peroxidation (LPO), which can lead to
atherosclerosis. Rabbits were inoculated with heat-killed C. neoformans several
times during the first 6 wks out of a 10-wk observation period. The following
tests were done at regular intervals: (1) blood cell count, (2) the nitroblue
tetrazolium (NBT) test on isolated neutrophils to determine their super oxide
anion production and (3) LPO of plasma. The histopathology of the lungs
was also evaluated. In the inoculated rabbits we found an increase in the
number of neutrophils in blood with an elevated NBT reduction, an increase
in lipid peroxidation of plasma and bronchopneumonia with various types of
inflammatory cells. Our findings suggest that the ability of C. neoformans to
induce LPO in human asymptomatic carriers should be studied. Moreover, the
cryptococcal-rabbit model we use rapidly induces LPO and may be of value in
the assessment of therapy for atherosclerosis.
431 LEVELS OF ADHESION MOLECULES (S-ICAM-1 AND S-VCAM-1)
IN DIFFERENT DEGREE OF ISCHEMIC CHRONIC HEART FAILURE:
CORRELATION WITH HS-CRP
S. Radovanovic1 , D. Popovic-Lisulov1 , N. Ninkovic1 , A. Djokovic1 , S. Hinic1 ,
M. Krotin1 , T. Simic2 , A. Savic-Radojevic2 . 1 University Hospital Bezanijska
Kosa, 2 Institute of Medical and Clinical biochemistry, Faculty of Medicine,
University of Belgrade, Belgrade, Serbia
Background: Several lines of basic research indicate that inflammation in the
vessel wall plays a critical role in the initiation and progression of atherosclerosis
and in the conversion of stable plaques to unstable lesions.
Aim of this study was to investigate whether subjects with chronic heart failure
(CHF) had higher plasma levels of soluble cell adhesion molecules (s-VCAM-1
and s-ICAM-1) than controls and whether they correlate with other markers of
inflammation.
Methods: 120 consecutive CHF patients, with angiographically demonstrated
CAD, divided into four groups according to New York Heart Association
classification (NYHA) and 69 healthy subjects as a control group were included
in this study. Circulating levels of ICAM-1 and VCAM-1 were assayed using a
specific sandwich ELISA kit. Tumor necrosis factor a (TNFa) and High sensitive
C reactive protein (hs-CRP) as inflammation indexes, were evaluated.
Results: In comparison to healthy sex- and age-matched controls, CHF
patients had significantly higher levels of s-ICAM-1 (367.8±138.1 ng/ml versus
281.3±188.4 ng/ml, p = 0.001). Significant difference was also found in the
levels of s-VCAM-1 (1171.3±386.1 ng/ml versus 860.2±340.9 ng/ml, p = 0.001)
in these groups. VCAM-1 was significantly correlated with hs-CRP levels,
(r = 0.189; p = 0.04) but no significant relationship was noted between VCAM-1
and ICAM-1 and TNFa.
Conclusion: Plasma adhesion molecule levels are increased in patients with
heart failure and VCAM-1 and ICAM-1 correlated with the severity of ischemic
heart failure. Circulating levels of VCAM-1 also correlated with one of the most
important inflammatory marker, hs-CRP. The clinical significance of our findings
warrants further investigation.
432 THE IMPORTANCE OF DE NOVO LIPOGENESIS OF LIPID LOADING
IN HUMAN MACROPHAGES
L. Mattsson Hultén, B. Liu, M. Levin, E. Lu, L. Håversen, C. Ullström,
S.-O. Olofsson, L. Li. Sahlgrenska Center for Cardiovascular and Metabolic
Research, Wallenberg Laboratory, University of Gothenburg, Gothenburg,
Sweden
Atherosclerotic lesions are characterized by lipid-loaded macrophages (foam
cells) and hypoxic regions. Although it is well established that foam cells
are produced by uptake of cholesterol from oxidized LDL, we previously
showed that hypoxia also promotes foam cell formation even in the absence
of exogenous lipids. The hypoxia-induced lipid accumulation results from
increased triglyceride biosynthesis but the exact mechanism is unknown.
However, we hypothesize that glucose uptake may be important because the
major glucose transporter in macrophages, glucose transporter 3 (Glut3), is
highly expressed in macrophages in hypoxia and advanced atherosclerotic
plaques. Our aim was therefore to investigate the importance of glucose and
Glut3 in promoting de novo lipid synthesis in human macrophages.
In the absence of serum, increased glucose levels promoted the accumulation of
Oil Red O-stained lipid droplets in human monocyte-derived macrophages. Lipid
analysis showed that increased glucose in the medium enhanced the levels of
triglycerides and phospholipids but not cholesterol in macrophages. Knockdown
of Glut3 with siRNA reduced glucose uptake and decreased Oil Red O-stained
lipid droplets by 50%. Most importantly, we showed that triglycerides extracted
from hypoxic foam cells were synthesized from glucose.
In conclusion, we have shown that glucose uptake through Glut3 is important
for lipid synthesis in macrophages, and may be important for the foam cell
formation in the hypoxic regions of atherosclerotic lesions.
433 HYPOXIC CELLS ARE ABUNDANT IN MURINE ATHEROSCLEROTIC
LESIONS
N. Junker1 , E.D. Bartels2 , T.X. Pedersen1 , C. Christoffersen2 , L.B. Nielsen2 .
1
Department of Biomedical Sciences, University of Copenhagen, 2 Department
of Clinical Biochemistry, Copenhagen University Hospital Rigshospitalet,
Copenhagen, Denmark
Human and rabbit atherosclerotic lesions contain hypoxic macrophages. The
hypoxia has been proposed to develop mainly as a consequence of increased
diffusion distance between the blood and the deep areas of the lesions. Hypoxia
likely has profound effects on cell biology and therefore could affect the growth
of lesions and perhaps necrotic core formation. We have examined whether
the mouse could be used a as model to study the impact of lesion hypoxia
in atherogenesis. When aortic sections from apoE and LDL-R KO mice were
stained with antibodies against Hypoxia Inducible Factor 1a (HIF1a) we saw
abundant HIF1a accumulation in the luminal part of the lesion. The presence
of hypoxia was confirmed by injecting pimonidazole (PIMO, which selectively
accumulates in hypoxic cells) into apoE KO mice. PIMO staining confirmed the
presence of hypoxia in the HIF1a staining areas. Co-staining of lipids (with Nile
Red) and HIF1a showed co-localization of foam cells and HIF1a. When primary
bone-marrow derived macrophages and foam cells were exposed to hypoxia the
expression of MCP-1 and ABCG1 decreased markedly.
These data show that hypoxic cells are present in mouse models of
atherosclerosis. We hypothesize that hypoxia in murine atherosclerotic
plaques arise because of increased oxygen consumption in metabolic active
macrophages/foam cells rather than because of increased diffusion distance.
Since expression of genes involved in inflammation and lipid transport are
affected in hypoxic macrophages and foam cells it is likely that cellular hypoxia
affects plaque progression.
79th EAS Congress
434 HIGH MAST CELL NUMBERS IN HUMAN CAROTID
ATHEROSCLEROTIC PLAQUES HAVE PREDICTIVE VALUE FOR
FUTURE CARDIOVASCULAR EVENTS
S. Willems1 , A. Vink2 , I. Hoefer1 , P. Quax3 , G. Pasterkamp1 . 1 Experimental
Cardiology Laboratory, 2 Department of Pathology, University Medical Centre
Utrecht, Utrecht, 3 Department of Cardiology, Leiden University Medical Center,
Leiden, The Netherlands
Mast cells (MCs) are inflammatory cells known to contribute to the pathogenesis
of atherosclerotic disease. Experimental studies have revealed that MCs might
be involved in the progression of atherosclerosis by inducing intraplaque
neovascularization.
Objective: This study was designed to assess total MC numbers and relate
the score to neovascularization in human atherosclerotic plaques. In addition
we related the presence of plaque MCs with the occurrence of adverse
cardiovascular events during follow-up.
Methods and Results: In the current study atherosclerotic plaques of 253
patients suffering from carotid artery stenosis, were stained for the presence
of MCs and microvessels (CD34). Furthermore, during a follow-up of 3 years,
cardiovascular related endpoints were assessed in all patients.
A cut-off point of 100 MCs per plaque was used to divide the cohort into a
group with low (n = 122) and high (n = 131) total MC numbers. Plaques with
high MC numbers revealed an unstable phenotype, a large lipid core, high
macrophage numbers, and low smooth muscle cell numbers. High MC numbers
were associated with high microvessel density 6.0 [4.3−9.0] versus 10.7 [6.5–
14.2] (P = 0.000). Patients with high plaque MC numbers showed significantly
more cardiovascular events during follow up (33 versus 56 events (P = 0.008)).
Macrophage and neutrophil presence did not show this association with the
occurrence of cardiovascular events.
Conclusions: This study shows for the first time, that intraplaque MC numbers
are predictive for future cardiovascular events. These data corresponds with the
strong association found between high MC numbers and the histopathologic
features of rupture-prone atherosclerotic lesions.
435 HIGH ENDOTHELIAL SHEAR STRESS INDUCES RUPTURE-PRONE
PLAQUES FORMATION DUE TO ANGIOGENESIS
G. Wang1 , J. Qiu1 , J. Hu1 , H. Liu1 , L. Ye1 , Y. Zheng1 , Y. Teng1 , D. Zhang2 .
1
Key Laboratory of Biorheological Science and Technology (Chongqing
University), Ministry of Education; Chongqing Engineering Laboratory
in Vascular Implants; Bioengineering College of Chongqing University,
Chongqing, China, 2 Chongqing Emergency Medical Center, Chongqing,
China
Background: Recent studies provided evidence for a predominant upstream
with high shear stress location of vulnerable plaque. However, the reason
that high shear stress in atherosclerotic vulnerable plaque occurs is currently
unknown.
Methods: In hypercholesterolemic rabbits, perivascular silastic collars were
placed around the left carotid arteries of rabbits (n = 6) to form high
shear stress on the upstream of the stenosis. The ratio of intraplaque
hemorrhage and necrosis and the percentage of collagen and lipoprotein
were calculated. Microvessel density (MVD) in the atherosclerotic plaque
was measured by immunohistochemistry for endothelial cells (CD31). The
microvessel morphology was performed by transmission electron microscopy.
Results: Atherosclerotic vulnerable plaque mainly localized at the regions
with high shear stress and high percentage of intraplaque hemorrhages and
necrosis, and contained less collagen and more lipids. The intraplaque MVD
in upstream plaques was significantly higher than that in the downstream
plaques (20±3 n/mm2 vs. 4±1 n/mm2 , p < 0.01), which correlated with lesion
morphology. Intraplaque microvascular endothelial cells on the high shear stress
region tend to apoptosis with membrane blebs, more leukocyte infiltration, less
mural cell coverage, and less collagen around the endothelial cells.
Conclusion: Our findings indicated that rupture-prone plaques mainly localize
at the upstream of stenosis with high shear stress. High shear stress
increases the number of new capillary and induces the incomplete structure of
microvascular endothelium. So the new born microvessels may be responsible
for intraplaque hemorrhage and inflammation, and finally lead to formation of
vulnerable plaques.
436
18
FDG-PET IMAGING AND GENE EXPRESSION OF MARKERS
OF NEOANGIOGENESIS, MICRO VESSEL DENSITY AND
VULNERABILITY IN ATHEROSCLEROTIC HUMAN CAROTID
PLAQUES
S.F. Pedersen1 , M. Græbe2 , A.M.F. Hag1 , L. Højgaard3 , H. Sillesen2 , A. Kjær1 .
1
Cluster for Molecular Imaging, University of Copenhagen, Copenhagen N,
2
Department of Vascular Surgery, Rigshospitalet, University of Copenhagen,
Copenhagen Ø, 3 Department of Clinical Physiology, Nuclear Medicine & PET,
Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
Neoangiogenesis and inflammation is a pivotal feature of the vulnerable
atherosclerotic lesion. The imaging modality 2-[18 F] fluoro-2-deoxy-D-glucose
93
(18 FDG) positron emission tomography (PET), 18 FDG-PET is an emerging
technique for identification of vulnerable plaques. However, it is unknown how
18
FDG uptake relates to neoangiogenesis and micro vessel density (MVD).
We performed gene expression analysis using quantitative PCR on
atherosclerotic carotid artery plaques obtained from symptomatic patients
undergoing carotid endarterectomy. The following genes were measured for
neoangiogenesis: VEGF, integrin aV and integrin b3 subunits, for MVD: CD34
and finally for plaque vulnerability: CD68, MMP-9 and cathepsin K.
Gene expression analysis was compared to 18 FDG-uptake of the carotid
arteries of which two measures were calculated: the mean and maximum
standardized uptake value (SUVmean and SUVmax). We found that VEGF
and integrin aV b3 gene expression did not correlate with 18 FDG-uptake,
whereas CD34 gene expression exhibited an inverse correlation with both
SUVmean (R = −0.381, P < 0.0001) and SUVmax (R = −0.319, P = 0.0046) in
univariate analysis. Additionally we established that markers of vulnerability
were correlated with 18 FDG-uptake (SUVmean and SUVmax) represented by
CD68, MMP-9 and cathepsin K gene expression.
In conclusion MVD (CD34) and markers of vulnerability (CD68, MMP-9 and
cathepsin K) correlate with 18 FDG-uptake, whereas neoangiogenesis (VEGF,
integrin aV and integrin b3 ) do not. The absence of correlation between
markers of neoangiogenesis and 18 FDG-uptake suggests a temporal separation
between the process of neoangiogenesis and inflammatory activity.
437 THE INFLUENCE OF MYOCARDIAL REVASCULARIZATION
ON ANGIOGENIC GROWTH FACTORS IN CORONARY ARTERY
DISEASE PATIENTS
I.V. Sergienko1 , E.V. Merculov2 , A.E. Semenova1 , V.P. Masenko3 .
1
Atherosclerosis, 2 Endovascular Treatment, 3 Immunology, Russian Cardiology
Research Complex, Moscow, Russia
Purpose: To estimate a status of angiogenic humoral regulation in CAD
patients. To assess the influence of myocardial revascularization on angiogenic
growth factors in CAD patients.
Methods: 228 patients with CAD and 30 healthy volunteers were included.
29 patients underwent CABG and 199 − PCI. Serum levels of vascular
endothelial growth factor (VEGF), transforming growth factor beta (TGF-b1)
and endostatin were detected three times − before, in 6 days and in 6 months
after revascularization.
Results: In 6 days after myocardial revascularization growth factors hadn’t
changed significantly. But in 6 month VEGF levels decreased from 293.9
(161.3–461.2) to 273.6 (154.4–399.9) pg/ml (p = 0.04), TGF-b1 levels
increased from 3.9 (3.1−4.8) to 4.7 (3.5−5.0) ng/ml (p = 0.01) and endostatin
levels increased from 151.8 (131.6–170.6) to 165.7 (142.3–204.6) ng/ml
(p = 0.001). The separate analysis of growth factor’s dynamics depending on
revascularization method showed similar changes. There were a decrease of
VEGF levels from 287.7 (159.4–459.3) to 215.3 (131.4–352.5) pg/ml (p = 0.01),
an increase of TGF-b1 levels from 3.9 (3.1−5.1), to 4.6 (3.5−5.0) ng/ml (p = 0.02)
and an increase of endostatin levels from 151.4 (130.5–170.5) to 167.1 (142.5–
204.8) ng/ml (p < 0.001) in 6 month after PCI. And we also found a decrease of
VEGF levels from 299.5 (170.3–497.1) to 223.2 (148.8–394.4) pg/ml (p > 0.05)
and an increase of TGF-b1 levels from 3.5 (3.0−4.7) to 4.8 (3.6−5.0) ng/ml
(p < 0.05) without any significant changes of endostatin levels in 6 months after
CABG.
Conclusions: Myocardial revascularization in CAD patients modifies angiogenic growth factor’s levels, making it closer to healthy volunteers’ levels.
438 IS HYPOXIA THE COMMON TRIGGER FOR INFLAMMATION AND
APOPTOSIS IN HUMAN CAROTID ATHEROSCLEROTIC PLAQUES?
A. Bitto, G. De Caridi, F. Spinelli, F. Squadrito. Clinical and Experimental
Medicine and Pharmacology, University of Messina, Messina, Italy
Introduction: Apoptosis and inflammation are important features of atherosclerotic plaques. Hypoxia inducible factor (HIF-1a) is known to participate in
atherosclerosis and to stimulate apoptosis signal-regulating kinase 1 (ASK-1),
one of the mitogen-activated protein kinases, which is activated by various
extracellular stimuli and involved in a variety of cellular function.
Objective: We investigated whether a common signal molecule can trigger
these two apparently separate pathways.
Patients and Methods: We tested carotid artery specimens from 50 subjects
who underwent angioplasty and five age-matched controls for either Western
blot or histologic analysis. The hypoxic status was investigated by means of
HIF-1a expression in carotid specimens.
Results: HIF-1a was significantly upregulated in carotid specimens with respect
to controls (P < 0.05), ASK-1 was detected in plaques of any composition from
lipidic to calcific, and this expression increased with the stage of the plaque
and with the expression of inflammatory (p-ERK, RANK-L, OPG) and apoptotic
molecules (caspase 9, p-p-38, and p-JNK).
Conclusion: Our data suggest that hypoxia is the key regulating factor
that triggers inflammation as well as apoptosis in the human atherosclerotic
plaque.
94
439 POLYPHENOLS FROM RED PROPOLIS SUPPRESSES
ANGIOGENESIS BY INDUCING VON HIPPEL-LINDAU-DEPENDENT
HIF-1 ALPHA DEGRADATION
J.B. Daleprene1 , M. Rudnicki2 , T.P. Ong1 , M. Ikegaki3 , H. Menrad4 , T. Geis4 ,
T. Schmid4 , N. Dehne4 , B. Bruene4 , D. Saes Parra Abdalla2 . 1 Dept. of Food
Science, 2 Dept. of Clinical and Toxicological Analyses, University of São Paulo,
São Paulo, 3 Dept. of Pharmacy, Federal University of Alfenas, Alfenas, Brazil,
4
Institute of Biochemistry, Johann Wolfgang Goethe-University, Frankfurt,
Germany
Natural products have shown great potential for the prevention and/or therapy
of various diseases. Recently, polyphenol-enriched fractions from red propolis
(PRP) have been proposed to interfere with angiogenesis. We aimed at
characterizing molecular mechanisms of PRP towards this action. PRP
reduced sprouting of endothelial cells (EC) in an aortic ring assay, attenuated
formation of new blood vessels in the chorio-allantoic chicken embryo assay
(CAM), decreased differentiation of embryonic stem cells into CD31 positive
cells in a spheroid culture and lowered migration of endothelial cells. PRP
inhibited hypoxia- or DMOG-induced mRNA and protein expression of vascular
endothelial growth factor (VEGF), which is important to promote angiogenesis.
Reduced VEGF amount was attributed to a decrease in hypoxia-inducible
factor 1a (HIF-1a) protein accumulation in response to PRP under hypoxia.
Specifically, PRP reduced HIF-1a protein half-life, but not its mRNA amount,
from ~58 to ~38 min under hypoxic conditions. The reduced protein halflife was due to increased von Hippel-Lindau (pVHL)-dependent proteasomal
degradation of HIF-1a, which was correlated with PRP-evoked downregulation
of Cdc42 but a subsequent pVHL increase. The ability of PRP to attenuate HIF1a accumulation and HIF-1 signaling provides an explanation to understand its
anti-angiogenic ability and to further address its therapeutic value.
Financial support: FAPESP (grant to D.S.P.A. and scholarship to J.B.D.), DAAD
(scholarship to J.B.D), DFG (grant to B.B.)
440 INTRA-PLAQUE VESSEL DENSITY, INFLAMMATORY CELLS AND
EXISTENCE OF THROMBIN-CLEAVED FORM OF OSTEOPONTIN
ARE THE RISK FACTORS FOR SYMPTOMATIC STROKE
M. Kurata1 , T. Okura2 , Y. Kumon3 , H. Watanabe3 , M. Tagawa3 , J. Higaki2 ,
M. Nose1 . 1 Department of Pathology, Division of Pathogenomics, 2 Department
of Integrated Medicine and Informatics, 3 Department of Neurosurgery, Ehime
University Graduate School of Medicine, Toon-city, Japan
Background and Aim: Intra-plaque hemorrhage is one cause of plaque
vulnerability. Osteopontin (OPN) which secreted in the atherosclerotic lesion
is well known an inflammatory cytokine. However, the functional role of
thrombin-cleaved form of osteopontin (trOPN) is not clear. In this study, we
clarified the relationship between intra-plaque hemorrhage and clinical and
histological phenotypes; location and density of intra-plaque micro-vessels and
the counts of inflammatory cells. We also showed the existence of trOPN in
the atherosclerotic plaque. To clarify the trOPN function, we stimulated mouse
macrophages by OPN and trOPN and analyzed transcriptional outcome.
Method: The participants were 40 patients including 20 symptomatic patients
who were operated by carotid endo-atherectomy. The micro-vessels and trOPN
were estimated by immuno-histochemsitry. RAW264.7 cell line was treated
with recombinant OPN and trOPN and analyzed of inflammatory cytokines by
realtime PCR.
Results: The patients who had symptom of stroke within 6months before
operation, the presence of intra plaque hemorrhage were higher than no
symptomatic patients. There were significant positive relationship between
vascular density and intra-plaque hemorrhage. The higher density of microvessels, more inflammatory cells infiltrated. Further, trOPN was co-localized
with infllamatory cells which located beside the micro-vessels. In vitro study,
trOPN induced macrophages to express of IL1b and TNFa in a concentration
dependent manner as well as OPN.
Conclusion: These results suggest the vascular density, infiltration of
inflammatory cells, and trOPN are the risk factors of plaque hemorrhage. TrOPN
has an significant role in the pathogenesis of atherosclerosis.
441 THE ROLE OF PRENATAL CHRONIC HYPOXIA ON MYOCARDIAL
ISCHEMIA/REPERFUSION INJURY IN ADULT RABBITS OFFSPRING
Z. Huang, Z. Wang, J. Yang. Department of Cardiology, Second Affiliated
Hospital of Fujian Medical University, Quanzhou, China
Objective: To evaluate the role of prenatal chronic hypoxia on myocardial
ischemia/reperfusion injury in adult rabbits offspring and explore the relevant
mechanism.
Methods: The pregnant New-Zealand rabbits were divided randomly into
normoxic (n = 8) and hypoxic (12% O2 from days 10 to 28 of gestation, n = 8)
groups. One male offspring of each maternal rabbit was randomly selected
to study. The offspring rabbits were subjected to heat stress (42ºC for 15
min) at 6 months of age. After 24 h, left anterior decending branches were
excised and subjected to ischemia for 30 min and reperfusion for 120 min.
Cardiac histopathological observation was performed by light microscope. The
expression of heat shock protein 70 (HSP70 ) in myocardium was detected
by immunohistochemistry. Myocardial enzyme activity, apoptotic index and
caspase-3 activity in myocardium were examined as well.
Results: Ischemia-reperfusion after heat stress pretreatment increased
myocardial enzyme activity, apoptotic index and caspase-3 activity in
prenatal chronic hypoxia rabbits (4720.31±744.39 IU/L, 1849.13±416.58 IU/L,
40.43±5.03%, 12.43±1.77 unit, respectively) when compared with control
(3388.95±532.43 IU/L, 1435.13±92.08 IU/L, 34.40±4.66%, 10.58±1.42 unit,
respectively). Heat stress pretreatment induced HSP70 significant expression
in left ventricular myocardium was not observed in prenatal chronic hypoxia
rabbits but in normoxic control rabbits.
Conclusions: Prenatal chronic hypoxia inhibits HSP70 synthesis in the heart
of adult offspring in response to body heat stress, which might insult
cardioprotection against ischemia-reperfusion injury.
442 FAR INFRA-RED THERAPY PROMOTES ISCHEMIA-INDUCED
ANGIOGENESIS IN DIABETIC MICE AND RESTORES HIGH
GLUCOSE-SUPPRESSED ENDOTHELIAL PROGENITOR CELL
FUNCTIONS
S.-J. Lin1 , P.-H. Huang2 , J.-W. Chen2 . 1 Department of Medical Research and
Education, Taipei Veterans General Hospital, 2 Division of Cardiology, Taipei
Veterans General Hospital, Taipei, Taiwan R.O.C.
Objective: Emerging evidence indicate that far infra-red (FIR) therapy exerts
beneficial effects in the cardiovascular system, but limited data are available
on potential mediating mechanisms. Here, we tested the hypothesis that
FIR radiation can restore collateral flow to ischemic tissue in diabetic mice
and improve high glucose-suppressed endothelial progenitor cells (EPCs)
functions.
Methods and Results: Hindlimb ischemia was induced in streptozotocine
(STZ)-induced (type 1) diabetic mice, which were divided into control and
FIR therapy groups. The latter mice were placed in a far-infrared dry sauna
at 34ºC for 30 min once daily for 5 weeks. Laser Doppler perfusion imaging
demonstrated that the ischemic limb/normal side blood perfusion ratio in the
thermal therapy group was significantly increased beyond that in controls, and
significantly greater capillary density was seen in FIR therapy group. These
beneficial effects were markedly abolished by an eNOS inhibitor (L-NAME).
Bone marrow-derived EPCs differentiated into endothelial cells defined as
GFP+ /CD31+ double positive cells were significantly increased in ischemic
tissue around the vessels in diabetic mice received FIR radiation as compared
with those in control group. In in-vitro studies, EPCs were incubated with high
glucose (25 mM) and treatment of cultured EPCs with FIR radiation for 30 minutes markedly augmented high glucose-impaired EPC functions, and inhibited
high glucose-induced EPC senescence and reduced H2 O2 production.
Conclusions: The administration of FIR therapy promoted collateral flow
recovery and new vessels formation in STZ-induced diabetic mice, and these
beneficial effects may derive from enhancement of EPC functions and homing
process.
443 THE TRANSCRIPTION FACTOR HNF-4a: A KEY ACTOR OF THE
INTESTINAL UPTAKE OF FATTY ACIDS IN MOUSE
V. Frochot1 , M. Alqub1 , A.-L. Cattin1 , V. Carrière1 , A. Houiller1 , L. Barbot2 ,
S. Saint-Just1 , A. Ribeiro-Pillet1 , P. Cardot1 , J. Chambaz1 , M. Rousset1 ,
J.-M. Lacorte3 . 1 INSERM U872, 2 APHP Pitié Salpêtrière, 3 Centre de
Recherche des Cordeliers, INSERM U872, Paris, France
Background and Aims: An excessive postprandial accumulation of intestinederived triglyceride-rich lipoproteins being a risk factor of cardiovascular
diseases, it is essential to characterize the mechanisms controlling the intestinal
transfer of dietary lipids. Our aim was to investigate the role of the transcription
factor HNF-4a in this process.
Methods: Transgenic mice allowing a specific and inducible intestinal
invalidation of Hnf-4a gene were used.
Results: One hour after a lipid bolus, in the presence of the lipase inhibitor
tyloxapol, lower amounts of triglycerides were found both in plasma and
intestinal epithelium of the invalidated Hnf-4aIntD mice (0.64±0.02 mmol L-1 and
0.21±0.04 mmol/g of proteins respectively) as compared to the Hnf-4aloxP/loxP
control mice (1.11±0.03 mmol L-1 and 0.83±0.09 mmol/g of proteins). These
discrepancies were due to a net decrease of the uptake of fatty acid in Hnf4aIntD mice, as assessed by the amount of radioactivity recovered from triolein
or oleic acid in intestine (2.2±0.25 cpm/ml) and plasma (4.05±0.65 cpm/ml)
of Hnf-4aIntD mice, as compared to Hnf-4aloxP/loxP mice (14.83±2.7 cpm/ml and
14.3±1.2 cpm/ml respectively). This decreased fatty acid uptake was associated
with lower levels of the fatty acid transport protein 4 mRNA (2.5 fold) and protein
(3.5 fold) and with a lower acyl-CoA synthetase activity in Hnf-4aIntD mice as
compared to the control mice (1125±60 against 1566±40 nmoles/min/mg of
proteins).
Conclusion: The transcription factor HNF-4a is a key actor of the intestinal
transfer of dietary lipids, which controls this process as early as in the initial
step of fatty acid uptake by enterocytes.
79th EAS Congress
444 ROLES OF LXRa AND LXRb IN THE REGULATION OF INTESINAL
CHOLESTEROL ABSORPTION: DISTINCTIVE EFFECTS ON BILE
ACID METABOLISM IN MICE
X. Hu1 , K. Steffensen2 , Z.-Y. Jiang3 , P. Parini1 , J.-Å. Gustafsson2 , M. Gåfvels1 ,
G. Eggertsen1 . 1 Department of Laboratory Medicine, 2 Department of
Biosciences and Nutrition, Karolinska Institutet − Huddinge, Stockholm,
Sweden, 3 Department of Surgery, Ruijin Hospital, Shanghai Jiaotong
University School of Medicine, Shanghai, China
Introduction: Activation of the liver X receptors (LXRs) has been shown to
modulate cholesterol absorption in the small intestine, although the individual
effects of LXRa and LXRb have not been fully understood. Our study aimed to
investigate how cholesterol absorption is modulated by the two isoforms in the
small intestine.
Methods: Wild type (WT), LXRa−/− or LXRb−/− mice were subjected either to
standard diet or 0.2% cholesterol-enriched diet.
Results: No differences of cholesterol absorption were observed in all
genotypes of mice under normal diet. However, LXRb−/− mice showed a
significant lower cholesterol absorption rate than WT mice when fed 0.2%
cholesterol diet. This profile was altered when GW3965 was added to
the 0.2% cholesterol diet, while the absorption rate increased significantly
only in LXRa−/− mice. Analysis of gallbladder bile demonstrated that 0.2%
cholesterol diet significantly lower the ratios of CA/b-muricholic acid (b-MCA)
in LXRb−/− and WT mice compared to LXRa−/−, even when GW3965 was
added. Additionly, hepatic Cyp8b1 mRNA was reduced in WT fed with
0.2% cholesterol ± GW3965, which agreed with the CA% in the bile. Similar
patterns were seen in LXRb−/− mice, but not in LXRa−/−. The Cyp7a1 mRNA
in LXRa−/−was repressed by the 0.2% cholesterol treatment, and remained low
when adding GW3965. Fecal analysis showed a strong reduction of bile acid
excretion in LXRa−/− mice subjected to 0.2% cholesterol ± GW3965.
Conclusion: Our data indicate that LXRa and LXRb affect the synthesis of
individual bile acids in different ways upon activation, leading to alterations in
the intestinal cholesterol absorption.
445 PPARA GENE LEVEL DIFFERENTLY AFFECTS LIPID METABOLISM
AND INFLAMMATION IN APOLIPOPROTEIN E2 KNOCK-IN MICE
F. Lalloyer1 , K. Wouters1 , E. Vallez1 , J. Vanhoutte1 , M. Baron1 , R. ShiriSverdlov2 , M. Hofker3 , B. Staels1 , A. Tailleux1 . 1 Inserm U1011-Institut
Pasteur de Lille-Université Lille Nord de France, Lille, France, 2 Department
of Molecular Genetics, Nutrition and Toxicology Research (NUTRIM) and
Cardiovascular Research (CARIM) Institutes of Maastricht, University of
Maastricht, The Netherlands, Maastricht, 3 Department of Medical Biology,
University Medical Center Groningen, University of Groningen, Groningen,
The Netherlands
Objective: PPARa is a ligand-activated transcription factor which controls lipid
metabolism and inflammation. PPARa is activated by fibrates, hypolipidemic
drugs used in the treatment of dyslipidemia. Previous studies assessing the
influence of PPARa agonists on atherosclerosis in mice yielded conflicting
results and the implication of PPARa therein has not been assessed. The
human apoE2 knock-in (apoE2-KI) mouse is a model of mixed dyslipidemia,
atherosclerosis and non-alcoholic steatohepatitis (NASH). The aim of this study
was to analyze the consequences of quantitative variations of PPARa gene
levels and its response to the synthetic PPARa agonist fenofibrate, on NASH
and atherosclerosis in apoE2-KI mice.
Methods and Results: Wildtype (+/+), heterozygous (+/-) and homozygous
(−/−) PPARa-deficient mice in the apoE2-KI background were submitted to a
western diet supplemented or not with fenofibrate. Western diet-fed PPARa−/−
apoE2-KI mice displayed an aggravation of steatosis and hepatic inflammation
compared to PPARa+/+ and PPARa+/− apoE2-KI mice, indicating a role
of PPARa in liver protection. Moreover, PPARa expression was required
for the fenofibrate-induced protection against NASH. Interestingly, fenofibrate
treatment induced a similar response on hepatic lipid metabolism in PPARa+/+
and PPARa+/− apoE2-KI mice, whereas, for a maximal anti-inflammatory
response, both alleles of the PPARa gene were required.
Surprisingly, atherosclerosis development was not significantly different
between PPARa+/+, PPARa+/- and PPARa−/− apoE2-KI mice. However,
PPARa gene level determined both the anti-atherosclerotic and vascular antiinflammatory responses to fenofibrate in a dose-dependent manner.
Conclusions: These results demonstrate a quantitatively different role of
PPARa in the modulation of liver metabolism, inflammation and atherogenesis.
446 ATHEROSCLEROTIC LESION REMODELLING IN
REV-ERBa-DEFICIENT MICE
E. Bauge, C. Comte, C. Duhem, J. Vanhoutte, B. Staels, H. Duez. Inserm
U1011, Univ Lille Nord de France, Institut Pasteur de Lille, Lille, France
Rationale: Rev-erba is a nuclear receptor which has been shown to influence
numerous physiological processes such as circadian rhythm, lipid, glucose, bile
acid metabolism and adipogenesis. Rev-erba is expressed in different cell types
of the vascular wall where it regulates the expression of genes involved in
95
the inflammatory and thrombotic response. Finally, Rev-erba in vitro dampens
oscillation of PAI-1, an important inhibitor of fibrinolysis cascade which may
influence the development of atherothrombosis. Howewer, implication of Reverba in atherosclerotic processes has never been investigated.
Objective: Here, we investigated the effect of Rev-erba deficiency on
atherosclerotic lesion formation in apoE-deficient mice.
Methods and Results: We demonstrate that Western diet-fed Reverba-deficient mice on the apoE−/− background display a significant increase
in total aortic atherosclerotic lesion area as compared to apoE−/− Rev-erba+/+
mice fed the same diet. Plaque immunostaining examinations showed that
apoE−/− Reverba−/−mice display atherosclerotic lesion characterized by a
decreased content in macrophages and increased collagen deposition.
Conclusion: These results demonstrate that Rev-erba plays an important role
in atherosclerosis and demonstrate for the first time a role of a clock machinery
component in atherosclerosis development. This suggests that Rev-erba may
play a role in circadian disorder related cardiovascular disease.
447 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS
REGULATE NADPH OXIDASE EXPRESSION AND FUNCTION IN
HUMAN AORTIC SMOOTH MUSCLE CELLS
A. Manea1,2 , S.A. Manea2 , I.M. Fenyo2 , M. Raicu2 . 1 Petru Poni Institute
of Macromolecular Chemistry, Iasi, 2 Molecular Biology and Pharmacology,
Nicolae Simionescu Institute of Cellular Biology and Pathology, Bucharest,
Romania
Objective: Elevated NADPH oxidase (Nox) activity promotes oxidative injury
of the vascular cells. Peroxisome proliferator-activated receptors (PPARs) play
key roles in the regulation of energy homeostasis, fatty acid metabolism and
inflammation. Evidence exists that PPARs control vascular redox state. Hitherto,
the molecular mechanisms are scantily elucidated. In this study, we investigated
the role of PPARs in the regulation of Nox in human aortic smooth muscle cells
(SMCs).
Methods and Results: Cultured SMCs were exposed to various PPAR
agonists or 4-hydroxynonenal (4-HNE) up to 24h. Dihydroethidium and
dichlorofluorescein assays, real-time PCR, and Western-blot analysis, revealed
that PPAR agonists and 4-HNE induce a dose-dependent up-regulation of Nox
activity and Nox1/4/5 expression. Knock-down of PPARa/d/g partially diminished
the 4-HNE-dependent induction of Nox. In silico analysis indicated the presence
of typical retinoid X receptor (RXR)a elements within human Nox1/4/5 and
p22phox promoters. The promoter activities of the Nox1/4/5 and p22phox genes
were significantly enhanced in SMCs overexpressing hPPARa/d/g or hRXRa;
a finding that indicates the presence of functionally PPRE elements. The
transcriptional activities of each component were significantly up-regulated by
PPAR agonists.
Conclusions: These findings support a new concept whereby Nox-derived
ROS elicit anti-inflammatory activities via PPARs. Thus, autogeneration of
endogenous PPAR ligands (4-HNE) may represent an important mechanism
of Nox regulation in the vasculature.
Work supported by an EFSD New Horizons grant. Adrian Manea acknowledges
448 PLASMA PHOSPHOLIPID TRANSFER PROTEIN (PLTP) IS A
PROCOAGULANT FACTOR IN VIVO
C. Desrumaux1,2 , V. Deckert1,2 , S. Lemaire-Ewing1,2,3 , C. Mossiat2,4 ,
A. Athias1,2 , D. Vandroux2,3 , L. Dumont2,3 , S. Monier1,2 , J.-P. Pais de
Barros1,2 , A. Klein1,2 , E. De Maistre2,3 , D. Blache1,2 , A. Beley2,3 , C. Marie2,4 ,
P. Garnier2,4 , L. Lagrost1,2,3 . 1 INSERM UMR866, 2 Université de Bourgogne,
3
CHU, Hôpital du Bocage, 4 INSERM U887, Dijon, France
Objective: Plasma phospholipid transfer protein (PLTP) is a determinant of
lipoprotein metabolism and atherogenesis. Although earlier in vitro studies
suggested a putative role for PLTP in the modulation of blood coagulation,
the pathophysiological relevance of this effect is currently unknown in vivo. To
address this question, blood coagulation was compared in the present study
in wild-type (WT) and PLTP-deficient (PLTP−/−) mice under both basal and
oxidative stress conditions.
Methids and Results: Under basal conditions, PLTP deficiency was associated
with a significant, 45% increase in tail bleeding time (P < 0.05) despite
a reduction in vascular alpha-tocopherol content and an impairment of
endothelial function. When acute oxidative stress was generated in vivo in the
brain vasculature, the sequestration of alpha-tocopherol in blood of PLTP−/−
mice was associated with a significant reduction in steady state levels of
oxidized lipid derivatives (−22%, P < 0.01), decreased blood vessel occlusion
(−52%, P < 0.05), and decreased volume of ischemic lesions (−39%, P < 0.05)
compared to those measured in WT mice.
Conclusion: In addition to its recognized hyperlipidemic, proinflammatory and
proatherogenic properties, PLTP increases blood coagulation and worsens
the extent of ischemic lesions in response to acute oxidative stress. Thus,
96
PLTP arises here as a cardiovascular risk factor for the late thrombotic events
occurring in the acute phase of atherosclerosis.
449 MICRORNAS 340* AND 624* ARE UPREGULATED IN PLATELETS
IN PATIENTS WITH CORONARY ARTERY DISEASE
B.M. Sondermeijer1 , A. Bakker1 , A. Halliani2 , S. Maiwald1 , S. Sivapalaratnam1 ,
T.A. Mulders1,3 , M.W.J. de Ronde1 , A.J. Tijsen2 , J.A. Marquart4 ,
P.D. Moerland5 , M.D. Trip1 , J.C.M. Meijers4 , E.E. Creemers2 , S.J. PintoSietsma1,5 . 1 Vascular Medicine, 2 Heart Failure Research Center, University of
Amsterdam, Amsterdam, 3 University of Maastricht, Maastricht, 4 Experimental
Vascular Medicine, 5 Clinical Epidemiology, Biostatistics and Bioinformatics,
University of Amsterdam, Amsterdam, The Netherlands
Coronary artery disease (CAD) is a cause of mortality, underscoring the need for
innovative diagnostic strategies. Platelets play a role not only in acute thrombotic
disease, but also in the process of atherosclerotic plaque formation. This is
exemplified by the beneficial use of anti-platelet therapy.
Objective: We aim to identify novel biomarkers for diagnostic strategies.
MiRNAs are emerging as attractive biomarkers since they exhibit tissue- and
cell-specific expression. We hypothesized that platelet specific miRNA
expression patterns differ between patients with stable premature CAD and
controls and this pattern may be used as a biomarker.
Methods: We isolated RNA from platelets of 12 male patients with premature
CAD and 12 age- and sex-matched healthy controls and performed miRNA
array expression profiling (Illumina beadchips). We validated our findings in two
independent cohorts by RT-PCR.
Results: Microarray profiling identified 214 of the 893 mature human miRNAs
to be differentially expressed (p < 0.05;). When corrected for background noise
and corrected for multiple testing, miR340*, miR451, miR454*, miR545:9.1,
miR615−5p, and miR624* and miR1280* were identified with an expression
level that was at least 1.5-fold different in patients as compared to controls.
Validation in two independent cohorts was done. The validation demonstrated
that miR340* and miR624* had significantly higher expression levels in patients
as compared to controls (P < 0.05).
Conclusion: Platelets of subjects with stable premature CAD have higher
miRNAs expression profiles compared to controls.
450 GENETIC VARIATIONS, MRNA EXPRESSION AND CIRCULATING
LEVELS OF MATRIX METALLOPROTEINASE-9 IN PATIENTS WITH
T. Baur Opstad1,2 , A.Å. Pettersen1,2 , S. Åkra1 , T. Weiss1,2 , H. Arnesen1,2,3 ,
I. Seljeflot1,2,3 , Center for Clinical Heart Research. 1 Department of Cardiology,
Center for Clinical Heart Research, Oslo University Hospital, Ullvål, 2 Center
for Heart Failure Research, Oslo University Hospital, 3 Faculty of Medicine,
University of Oslo, Oslo, Norway
Objective: To investigate the influence of the promoter −1562 C/T and the exon
6 R279Q A/G polymorphisms on the MMP-9 gene on MMP-9 gene expression
in circulation leukocytes and circulating levels of the protein in patients with
angiographically verified stable coronary artery disease (CAD).
Methods: The frequencies of the polymorphisms were investigated in 1001
patients and in 204 healthy controls (mean age 62 and 55 years, respectively).
Allelic discrimination and MMP-9 gene expression (isolated from PAXGenetubes) were determined by the Applied Biosystems 7900HT Real-time PCR
system. Serum concentrations of MMP-9 and its inhibitor TIMP-1 were
measured by commercial ELISA kits (R&D Systems) and MMP-9 activity was
measured by ELISA and zymography.
Results: The variant alleles of both polymorphisms (−1562 -T and R279Q-G)
associated significantly with lower and higher mRNA levels, respectively
(p < 0.01, both). The T-allele was significantly associated with higher
concentrations of MMP-9 whereas the G-allele associated with lower MMP-9
activity (p 0.01, both). MMP-9 mRNA levels in circulating leukocytes and
serum levels of TIMP-1 were higher in patients with previous myocardial
infarction (44%), the latter also elevated in patients with type 2 diabetes (20%)
(p < 0.05, for all). No difference in allele frequencies between CAD patients and
controls were observed, however, in patients, the G-allele of R279Q associated
strongly with hypertension (56%) (p < 0.01).
Conclusion: In patients with CAD, variations in the MMP-9 gene strongly
influence gene expression and circulating levels of MMP-9, which may have
clinical implications, especially in patients with hypertension.
451 NADPH OXIDASE-RELATED OXIDATIVE STRESS ENHANCES
PLATELET THROMBOXANE PRODUCTION IN ASPIRIN-TREATED
PATIENTS UNDERGOING ELECTIVE PERCUTANEOUS CORONARY
INTERVENTION
P. Pignatelli1,2 , S. Basili3 , G. Tanzilli3 , S. Di Santo3 , R. Carnevale3 , D. Pastori3 ,
M. Schillizzi3 , C. Calabrese3 , E. Mangeri3 , P. Ferroni4 , F. Violi3 . 1 Divisione
I Clinica Medica, Sapienza University of Rome, 2 Policlinico Umberto I,
3
Sapienza University of Rome, 4 Department of Laboratory Medicine and
Advanced Biotechnologies, IRCCS San Raffaele Pisana, Rome, Italy
Background: Percutaneous Coronary Intervention (PCI), a typical ischemiareperfusion procedure, is associated with a burst of reactive oxygen species
(ROS). Experimental models of ischemia-reperfusion indicate that ROS may
activate platelet function through a mechanism involving arachidonic acid
metabolism and increased formation of thromboxane (Tx) A2 .
Aim: We analyzed serum TxB2 , before, 60 and 120 min after PCI procedure in
aspirin-treated patients (n = 56) randomly allocated to receive either placebo or
vitamin C iv infusion (1 g). In addition, circulating levels of NADPH oxidase
isoform 2 (NOX2), the catalytic core of NADPH oxidase (one of the most
important cellular producer of ROS) were measured to investigate the enzymatic
pathway implicated. Finally, an in vitro study aimed at substantiating the role of
NOX2 in ROS formation and Tx biosynthesis activation was also performed.
Results: Serum TxB2 and 8OH-dG, plasma 8-iso-PGF2a and sNOX2
significantly increased after 60’ and 120’ from PCI in placebo-treated patients.
Vitamin C infusion resulted in a significant decrease of 8OH-dG, 8-iso-PGF2a
and sNOX2 after 60’ and 120’ from PCI, whereas TxB2 was unmodified from
baseline. In vitro experiments showed that, compared to platelets kept at room
air, platelets that underwent anoxia-re-oxygenation had higher platelet NOX2
activation and isoprostanes and TxB2 generation, which were counteracted by
inhibitors of NADPH oxidase and by Vitamin C.
Conclusion: We provide evidence that patients undergoing elective PCI have
an over-production of platelet Tx despite concomitant aspirin treatment. The
inhibitory effects exerted by Vitamin C infusion suggests a major role for
oxidative stress in PCI-induced platelet activation.
452 TIMING AND DOSE OF STATIN THERAPY DEFINE ITS IMPACT
ON INFLAMMATORY AND ENDOTHELIAL RESPONSES DURING
MYOCARDIAL INFARCTION
L. Pego1 , S.N. Santos1 , E.C. Faria2 , D.S.P. Abdalla3 , A.A. Sousa Soares1 ,
A.V.T. Japiassú1 , J.C. Quinaglia e Silva4 , J.A.F. Ramires5 , O.R. Coelho6 ,
A.C. Sposito6 , on behalf of the Brasilia Heart Study Group. 1 University
of Brasilia Medical School (UnB), Brasilia, 2 Faculty of Medical Sciences,
State University of Campinas (Unicamp), Campinas, 3 Faculty of Pharmacy,
University of São Paulo, São Paulo, 4 Hospital de Base do Distrito Federal,
Brasilia, 5 Heart Institute (InCor), University of São Paulo Medicine School,
São Paulo, 6 Cardiology Division, Faculty of Medical Sciences, State University
of Campinas (Unicamp), Campinas, Brazil
Clinical trials of statins during myocardial infarction (MI) have differed in their
therapeutic regimes and generated conflicting results. This study evaluated the
role of the timing and potency of statin therapy on its potential mechanisms of
benefit during MI.
ST-elevation MI patients (n = 125) were allocated into 5 groups: no statin,
simvastatin 20, 40 or 80 mg/day starting at admission, or 80 mg/day 48-h after
admission. After 7 days all patients switched their treatment to simvastatin
20 mg/day for additional 3 weeks and then underwent flow-mediated dilation
(FMD) in the brachial artery.
As of the 2nd day, C-reactive protein (CRP) differed between non-statin
users (1.2±4.1 mg/L), and patients treated by 20 mg (8.5±4.0 mg/L), 40 mg
(3.8±2.5 mg/L) and 80 mg/day (1.4±1.5 mg/L) and the daily differences
remained significant until the 7th day (p < 0.0001). The higher the statin dose the
lower the elevation of IL-2, TNF-a, the greater the reduction of 8-isoprostane
and LDL(−) and the greater the increase in nitrate/nitrite levels (NOx) during
the first 5 days (p < 0.001). Later initiation of statin was less effective than its
early introduction in relation to attenuation of CRP, IL-2, TNF-a, 8-isoprostane
and LDL(−) as well as in NOx increase (p < 0.0001). At the 30th day, there was
no longer difference in lipid profile or CRP between groups, the FMD, however,
was proportional to the initial statin dose and was higher for those who started
the treatment early (p = 0.001).
Conclusion: This study demonstrates that the timing and potency of statin
treatment during MI are key elements for their main mechanisms of benefit.
453 PITAVASTATIN PROVIDES CONSISTENT LONG-TERM
HDL-CHOLESTEROL IMPROVEMENTS IN A RANGE OF PATIENTS
WITH DYSLIPIDAEMIA: RESULTS FROM A PHASE III STUDY
PROGRAMME
N. Hounslow. Kowa Research Europe, Wokingham, UK
Aims: To determine whether pitavastatin provides long-term increases in highdensity lipoprotein cholesterol (HDL-C) in patients at high risk of coronary heart
disease (CHD).
79th EAS Congress
Methods: Evaluation of changes in HDL-C in patients recruited to a 52-week
open-label extension study of pitavastatin (NK-104–307), to two blinded activecontrolled 44-week extension studies (NK-104–309 and NK-104–310) or to a
60-week open-label extension study (65 years of age; NK-104–308). The
daily dose of pitavastatin was 4 mg or 2 mg (NK-104–308). Patients had mixed
(combined) dyslipidaemia or primary hypercholesterolaemia and a range of
CHD risk factors, including type 2 diabetes and hypertension, and entered the
extension studies from 12-week blinded core studies. HDL-C was measured in
a central research laboratory.
Results: Pitavastatin 2−4 mg consistently provided clinically relevant elevations
in HDL-C that increased during long-term treatment across all studies (Table).
Table: Mean changes in HDL-C with pitavastatin
Study
NK-104–307
(n = 1346; 4 mg)
NK-104–309
(n = 120; 4 mg)
NK-104–310
(n = 141; 4 mg)
NK-104–308
(n = 376; 2 mg)
a
Core study
Extension study
Baselinea
(mg/dL)
Endpoint
(mg/dL)
Change
from
baseline
Endpoint
(mg/dL)
Change from
core study
baseline
50.5
52.9
+5.6%
57.0
+14.3%
46.9
51.0
+9.3%
53.4
+14.1%
42.6
45.8
+8.4%
47.5
+12.7%
58.8
59.8
+2.2%
66.3
+9.6%
Following a dietary and washout period of 6−8 weeks.
Conclusions: Long-term studies demonstrate that pitavastatin has consistent
beneficial effects on HDL-C levels in a broad range of patients at risk of CHD.
These results are consistent with those previously reported in 88 Japanese
patients treated with pitavastatin 2 mg for 52 weeks (mean HDL-C increase,
8.2%).
454 INCIDENCE OF CORONARY EVENTS AND CASE-FATALITY RATES
IN RELATION TO CONCENTRATIONS OF BLOOD LEUKOCYTES
S. Adamsson Eryd1 , J.G. Smith2 , O. Melander1 , B. Hedblad1 , G. Engström1 .
1
Department of Clinical Sciences, Lund University, Malmö, 2 Department of
Cardiology, Lund University, Lund, Sweden
Background: Elevated concentrations of leukocytes have been associated with
worse prognosis in subjects with clinically manifest CHD. This population-based
cohort study explores whether lymphocyte and neutrophil concentrations are
associated with incidence of acute coronary events (CE), and also whether
lymphocyte and neutrophil concentrations are associated with fatal outcome in
subjects who subsequently suffered a first coronary event.
Methods: Neutrophil and lymphocyte counts were measured in 27419 subjects
from the general population (mean age 58 years) without a history of coronary
events, heart failure or atrial fibrillation. Incidence of hospitalizations due to CE
was studied in relation to leukocyte counts during a mean follow-up of 13.6
years.
Results: Neutrophil counts were significantly associated with incidence of CE.
After adjustments for confounding factors, the adjusted hazard ratios (HR,
95% CI) were 1.00 (reference), 1.07 (0.94–1.23), 1.09 (0.95–1.25) and 1.39
(1.22–1.59), respectively, for subjects with neutrophils in the 1st , 2nd , 3rd ,
and 4th (highest) quartiles (p for trend < 0.001). Lymphocyte counts were also
significantly associated with CE. The relationship did however not remain after
adjusting for potential confounding factors. Of the 1965 subject who had a
CE, 471 subjects died on the first day of the CE, in- or outside hospital. The
proportions who died the first day were 19%, 21%, 25% and 28%, respectively
in the 1st , 2nd , 3rd , and 4th quartiles (p for trend < 0.001).
Conclusion: Neutrophil counts are associated with incidence of CE. Increased
neutrophil levels are also associated with increased case-fatality rates after a
first coronary event.
455 THE CLASS II PHOSPHOINOSITIDE 3-KINASE ISOFORM b
REGULATES PLATELET FUNCTION AND ARTERIAL THROMBOSIS
IN MICE
G. Tibolla1,2 , S.S. Barbieri1 , S. Gianellini1 , L. Arnaboldi1 , A. Corsini1 ,
E. Tremoli1,3 , M. Falasca4 , G.D. Norata1,2 , A.L. Catapano1,2 . 1 Department
of Pharmacological Sciences, University of Milan, Milan, 2 SISA Center for
the Study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, 3 I.R.C.C.S.
Centro Cardiologico Monzino, Milan, Italy, 4 Barts and The London School of
Medicine and Dentistry, Queen Mary University of London, London, UK
Phosphoinositide 3-kinases (PI3Ks) are crucial components of many signaling
pathways involved in platelet activation. A key role is played by the class I
enzymes and especially by the p110b isoform that regulates aIIb b3 integrin
97
activation. By contrast little is known about the role of the class II PI3Ks, though
the activation of the class II PI3K isoform b (PI3K C2b) via activated aIIb b3
integrin suggests the involvement of these enzymes in the regulation of platelet
functions. Using a PI3K C2b knockout mouse model we showed that PI3K
C2b activity is required for normal collagen and thrombin induced aggregation.
Interestingly absence of PI3K C2b does not affect inside-out signaling, indeed
the a-granules release and the activation of aIIb b3 integrin are similar in PI3K
C2b deficient and wild type platelets. On the other hand analysis of thromboxane
A2 (TxA2) synthesis, show that agonists induced TxA2 production is significantly
impaired in platelets from PI3K C2b knockout mice; the unaltered abundance
and distribution within the different membrane phospholipids of arachidonic acid
(AA) and the similar aggregation in response to AA and U46619, a synthetic
TxA2/PGH2 receptor agonist, suggest that PI3K C2b regulates phospholipase
A2 activation and AA mobilization, possibly downstream activated aIIb b3 integrin.
In vivo studies show that PI3K C2b knockout mice have a normal bleeding time
but thrombus formation after FeCl3 injury of the carotid artery is significantly
delayed in these mice. Taken together our data indicate the PI3K C2b enzyme
as an interesting new target for antithrombotic drugs development.
456 THE IMPACT OF LOW DENSITY LIPOPROTEIN RECEPTOR (LDLR)
GENE MUTATION ON CAROTID INTIMA MEDIA THICKNESS, IN
PATIENTS WITH FAMILIAL HYPERCHOLESTEROLEMIA
V. Metaxa, C. Pitsavos, I. Skoumas, A. Miliou, E. Oikonomou, K. Aznaouridis,
K. Masoura, C. Stefanadis. 1st Cardiology Clinic, Hippokrateio Hospital,
University of Athens, Athens, Greece
Background: Familial hypercholesterolemia (FH) is caused by mutations of the
LDLR gene.
Aim: To evaluate the impact of the most common LDLR gene mutations in
Greece, on carotid intima media thickness (c-IMT), in patients with FH.
Methods: We enrolled 115 patients, aged 29.63±17.15 years (49 men),
clinically diagnosed as FH, according to the Dutch Lipid Clinic Criteria. Their
lipid profile was assessed before any treatment was provided and molecular
analysis was performed by direct sequencing of the LDLR gene. C-IMT and
plaque were assessed by B-mode ultrasonography.
Results: An LDLR gene mutation was found in 57% of the patients. In 42
patients (35.6%) we found Genoa-Palermo (1646 G>A), in 18 patients (15.3%)
Africaner 2 (1285 G>A), in 4 patients (3.4%) Greece 2 (858C>A) whereas in
3 patients (2.5%) Sicily (1775G>A). A single noucleotide polymorphism (SNP)
1773T/C, was found in 72.9% of the patients. Totally, 41 patients had both, an
LDLR gene mutation and the SNP. We compared carriers of Africaner 2 and
Genoa-Palermo. Student’s t test analysis revealed that patients with GenoaPalermo had statistically significantly increased c-IMT compared to those with
Africaner 2 (1.00±0.11 mm vs. 0.78±0.15, p < 0.001). No differences were
found among other risk factors such as age, sex, total cholesterol, LDL,
HDL, APOA1, APOB, LPa and BMI. Multi-linear regression analysis revealed
that patients with Genoa-Palermo present 0.2 mm greater c-IMT compared to
Africaner 2 patients, after adjustment for age, sex, TC, LDL, HDL, APOA1,
APOB, LPa, BMI, arterial hypertension, smoking habits and fasting glucose
(b = 554, p = 0.001).
Conclusions: According to our findings, Genoa-Palermo seems to have greater
impact on c-IMT, compared to Africaner 2.
457 THE INFLUENCE OF TF AND TFPI POLYMORPHISMS ON
THROMBIN GENERATION IN PATIENTS WITH STABLE CORONARY
ARTERY DISEASE
T. Baur Opstad1,2 , A.Å. Pettersen1,2 , V. Bratseth1 , H. Arnesen1,2,3 ,
I. Seljeflot1,2,3 , Center for Clinical Heart Research. 1 Department of Cardiology,
Center for Clinical Heart Research, Oslo University Hospital, Ullvål, 2 Center
for Heart Failure Research, Oslo University Hospital, 3 Faculty of Medicine,
University of Oslo, Oslo, Norway
Objectives: To investigate whether the TF (−1812 C/T, −603 A/G, 5466 A/G)
and TFPI (−399 C/T, −287 T/C, −33 T/C) polymorphisms influence thrombin
generation (TG) in patients with stable coronary artery disease (CAD).
Additionally, associations between levels of TF and TFPI and the different
measures of TG were investigated.
Methods: Patients (mean age 62 years, 22% women) with angiographically
verified CAD (n = 1001) were included. In vivo TG was measured by prothrombin
fragment 1+2 (F1+2) and the Calibrated Automated Thrombogram (CAT) assay
was used for the potential to generate thrombin ex vivo. Allelic discrimination
was determined by the Applied Biosystems 7900HT Real-time PCR system.
Plasma levels of F1+2, TF, and free and total TFPI antigen were measured by
commercial ELISA kits.
Results: The TF 5466 and TFPI-399 polymorphisms associated with higher
F1+2 (TG in vivo), the latter also with a prolonged lag-time of the TG ex vivo
(p < 0.05, all). The TF-1812 TT and the TF-603 GG genotypes associated with
lower peak thrombin (p < 0.05, both) and a slower procoagulant phase (Vmax)
(p = 0.05, both). The TFPI-33 TC genotype prolonged lag-time (p < 0.05) and
98
time to peak (p = 0.06). Strong correlations between TFPI levels, F1+2 and
parameters of the CAT assay were observed.
Conclusion: The influence of TFPI polymorphisms both on in vivo and ex vivo
thrombin generation shown in our population of stable CAD patients might be
a consequence of genotype-induced changes in the circulating TFPI levels [1].
The influence of the TF polymorphisms was not obvious.
Reference(s)
[1] Opstad TB et al, Thromb J 2010;8:7.
458 THE EFFECT OF STATIN TYPE ON THE ACTIVITY OF ANTIPLATELET
THERAPY: POST-HOC ANALYSIS OF CILON-T TRIAL
J.W. Suh1 , M.-J. Cha2 , S.-P. Lee2 , H.-S. Kim2 , CILON-T Investigators. 1 Seoul
National University Bundang Hospital, Seongnam, 2 Internal Medicine, Seoul
National University Hospital, Seoul, Republic of Korea
Background and Objectives: Clopidogrel, cilostazol and atorvastatin have
CYP3A in common as a metabolic pathway and there were controversies about
potential drug interaction among them. Rosuvastatin was postulated to have
less interaction due to its different metabolic pathway. We aimed to investigate
the effect of statin type on the efficacy of antiplatelet regimen and lipid profiles
in this analysis.
Subjects and Methods: Total 915 consecutive patients who underwent
coronary intervention with DES were randomized to receive dual (DAT) or triple
antiplatelet therapy (TAT) for six months. Stratification was performed according
to the statin type (rosuvastatin vs. atorvastatin). We included patients who
took equivalent dose of statin (atorvastatin, 20 mg/day; rosuvastatin, 10 mg/day)
during the study period in this post-hoc analysis. Patients were categorized
into four groups: group A-DAT (n = 217), group A-TAT (n = 220), group R-DAT
(n = 211) and group R-TAT (n = 215). We compared P2Y12 reaction unit (PRU)
assessed with VerifyNow™ and lipid profile among four groups at discharge
and at six months after the index procedure.
Results: Statin types did not have effect on the efficacy of antiplatelet efficacy
of DAT. Rosuvastatin achieved lower LDL level than atorvastatin at six months
in both antiplatelet regimens. The TAT group showed higher HDL and lower TG
level at six months, irrespective of the statin type.
Conclusion: The antiplatelet activity of DAT was not influenced by the statin
type. Rosuvastatin (10 mg/day) showed more powerful LDL lowering action than
atorvastatin (20 mg/day) in Asian patients. The addition of cilostazol seemed to
have beneficial effect on HDL and TG profiles.
459 HOMOCYSTEINE LOWERING AND GLUTATHIONE INCREASING
EFFECTS OF N-ACETYLCYSTEINE IN ACUTE CORONARY
SYNDROME
I. Squellerio1 , G. Marenzi1 , F. Veglia1 , G. Falcucci1 , L. Boccotti2 , S. Eligini1 ,
E. Tremoli1,3 , V. Cavalca1,2 . 1 Centro Cardiologico Monzino − IRCCS,
2
Università degli Studi di Milano, Dip. Scienze Cardiovascolari, 3 Università
degli Studi di Milano, Dip. Scienze Farmacologiche, Milan, Italy
Introduction: Elevated plasma levels of homocysteine (Hcy) and low
concentrations of reduced glutathione (GSH) have been described in coronary
artery disease (CAD). GSH is an important endogenous molecule with both
antioxidant and detoxifying properties. N-acetylcysteine (NAC), a mucolitic drug
used to prevent contrast-nephropathy after coronary angiography, is a precursor
for GSH synthesis. Recent studies have evidenced a Hcy-lowering activity of
NAC in healthy subjects and in renal insufficiency bearing patients.
Aim: In the present study we investigated the ability of NAC administration to
lower Hcy and increase GSH in CAD patients.
Methods: NAC, Hcy, GSH and disulfide form of glutathione (GSSG), as index
of oxidative status, were measured in plasma and blood of patients with acute
coronary syndrome (n = 20) before and 2, 6 and 24 hrs after an intravenous
bolus (600 mg) of NAC.
Results: Mean Hcy concentrations significantly decreased up to 6 hours after
bolus (−40.1%, p < 0.0001), returning to basal levels at 24 hrs, while the
profile of NAC concentrations showed an opposite behaviour. Whole blood
GSH significantly increased after 24 hrs (+18.4%, p = 0.015) with a concomitant
GSSG decrease (−23.8%, p = 0.03).
Conclusions: These preliminary results suggest a beneficial effect of NAC
administration in CAD patients. Further studies are needed to verify whether
NAC may represent a therapeutic tool able to control a cardiovascular risk
factor (Hcy) and to counteract the oxidative stress status associated with the
progression of coronary artery disease.
460 THE SEX-RELATED DIFFERENCES IN ASSOCIATIONS BETWEEN
ACTIVITY OF FIBRINOLYSIS AND SOME CARDIOVASCULAR
DISEASES IN MOSCOW RESIDENTS AGED 55+
V.A. Metelskaya1 , L.A. Ratnikova1 , S.A. Shalnova2 , M.A. Shkolnikova3 ,
A.D. Deev4 . 1 Department of Biomarkers of Atherosclerosis Risk, National
Research Centre for Preventive Medicine, 2 Department of Scientific Research
Planning, Russian Cardilogy Research Centre, 3 Federal Center for Cardiac
Arrhythmias, 4 Department of Biostatistics, National Research Centre for
Preventive Medicine, Moscow, Russia
Background: This analysis was conducted within the framework of Stress
And Health in Russia (SAHR) study − a prospective population survey among
Muscovites aged 55+, and aimed to reveal sex-related associations between
fibrinolytic activity and risk of CHD and diabetes (DM-2).
Material and Methods: All sample (n = 1863; 52% women) was splitted in three
groups according to fibrinolytic activity measured as Euglobulin Clot Lysis Time
(ECLT): normofibrinolysis, ECLT 180–260 min, hypofibrinolysis, ECLT > 260
min, and hyperfibrinolysis, ECLT < 180 min.
Results: At study of relation between fibrinolytic activity and some of clinical
states by logistic regression for men with hypofibrinolysis risk of arterial
hypertension (AH) was by 49% higher than for normofibrinolysis, p = 0.032;
of DM - − by 94%, p = 0.005; and myocardial infarction − by 53%, p = 0.05.
Increased activity of fibrinolysis in men was associated with lower risk of
AH by 48%, p = 0.001; and of DM-2 by 69%, p = 0.009 than in those with
normofibrinolysis. Women with hyperfibrinolysis have lower risk of AH by 57%,
p = 0.0004; of CHD by 39%, p = 0.037; and of angina pectoris by 35%, p = 0.067.
No differences in risk of abovementioned diseases were found in women with
hypofibrinolysis as compared with normofibrinolysis.
Conclusion: Delayed fibrinolysis was associated with higher risk of CHD and
DM-2 only in men, while active fibrinolysis was related to lower CHD risk both
in men and women, and to lower risk of DM-2 in men.
The SAHR Study was supported by research grant No R 01 AG 026786 from
the National Institute of Aging (USA)
461 INFLUENCE OF DAILY ALCOHOL CONSUMPTION ON SERUM
ADIPONECTIN LEVELS IN MEN
S. Makita, A. Abiko, M. Nagai, M. Nakamura. Division of Cardiology,
Department of Internal Medicine, Iwate Medical University, Morioka, Japan
Background: Risk of cardiovascular diseases among moderate drinkers has
been known to be reduced compared with nondrinkers and heavy drinkers. We
found that, in male general population cohort (n = 8,055, mean follow-up of 5.5
years), mild-to-moderate alcohol consumption may have a preventive effect on
future myocardial infarction. This beneficial effect tended to be retained even
in obese subjects. However, factors that can fully account for this U-shaped
protective effect were obscure.
Aim: The present cross-sectional study investigated the relationships between
alcohol consumption and serum adiponectin levels (ADN) or high-density
lipoprotein cholesterol (HDL-C).
Method: In male participants of health check-up programs (n = 527, age range
40−80 years, mean 64.0 years), total ADN was measured. Status of alcohol
intake was categorized into three groups (A1; never or occasional, A2; <50 g/day
and 3 days/week, A3; 50 g/day and 3 days/week) by questionnaire.
Figure: Adiponectin and HDL-C levels in (A1) non-/occasional, (A2) moderate
and (A3) heavy drinkers with and without metabolic syndrome.
Results: In subjects without metabolic syndrome (non-MetS), there were no
significant differences in ADN among three groups, despite linear increase of
79th EAS Congress
HDL-C. In subjects with metabolic syndrome (MetS), a high-risk population,
while ADN was lower compared with non-MetS, only A2 (moderate drinker)
retained adequate ADN, and the linear increase of HDL-C was blunted in A3
(heavy drinker).
Conclusion: These results suggest that U-shaped relationship between alcohol
consumption and risk of cardiovascular events is explained in part by change
of adiponectin level rather than HDL-C.
462 ERECTILE DYSFUNCTION AS A SUBROGATE MARKER OF
A. Hernández-Mijares1 , A. Jover2 , E. Solá1 , C. Bañuls2 , K. Garcı́a-Malpartida2 ,
M. Rocha2 , V.M. Vı́ctor2 . 1 Endocrinology Unit, Hospital Universitario Dr. Peset,
2
Hospital Doctor Peset Research Foundation, Valencia, Spain
Background and Objectives: Erectile dysfunction (ED) is highly prevalent
among patients with cardiovascular risk factors (CVRF). As it usually precedes
cardiovascular disease, it is considered a subrogate marker of subclinical
cardiovascular disease.
The aim of this study was to evaluate the presence of ED among type 2 diabetic
(DM2) patients without clinical macroangiopathy, and to assess the association
between ED and silent myocardial ischaemia (SMI) and other CVRF.
Patients and Methods: 154 male patients with DM2 and without clinical
evidence of cardiovascular disease were included. The presence of ED, SMI
and other CVRF was evaluated.
Results: Prevalence of ED was 68.2%. Patients with ED were older (57.7±7.5
vs. 52.1±7.9 years, p < 0.001) and showed a longer duration of DM2 (9.0 vs.
3.0 years, p < 0.001), a poor metabolic control (HbA1c 7.2% vs. 6.3%) and
higher systolic blood pressure (127.3 vs. 121.2 mmHg, p = 0.017) than patients
without ED. No differences were found in lipid profile, body mass index or smoke
habit.
Independent factors for ED were age (RR 1.1, p = 0.003) and duration of
diabetes (RR 1.1, p = 0.006). SMI was detected in 13.6% of patients (18.1%
in patients with ED vs. 4.1% in patients without) and 90.5% of patients with
SMI had ED.
Conclusions: ED is highly prevalent in DM2, and is associated with the
presence of SMI and higher systolic blood pressure. This fact points out the
usefulness of ED as a marker of poor vascular prognosis in diabetic patients.
463 CHANGING ROLE OF THE “CARDIOVASCULAR RISK FACTOR
PROFILE” IN ACUTE MYOCARDIAL INFARCTION
R. Potluri. Imperial College London, Imperial College London, London, UK
Introduction: Myocardial Infarction (MI) is the main cause of death in Europe
and accounts for over 3 million deaths per year. Cardiovascular risk factors such
as age, smoking, hypertension, high cholesterol, diabetes mellitus, and strong
family history are used to predict MI. Clinical practice has focused on diagnosis
and control of these factors. We hypothesied that increased awareness and
government led primary care initiatives targeting cardiovascular risk factors has
led to changing spectrum of patients presenting with acute MI.
Methods: Anonymous information on acute MI patients attending a large
multi-ethnic general hospital in Birmingham, UK in the period 2000–2007 was
obtained. We looked at the change in the trends of patients presenting with
acute MI according to demographics and co-morbidities.
Results: Out of 12464 acute MI patients.71.1% Caucasian; 5.1% of South
Asian; 0.7% Afro-Caribbean; In-hospital mortality of 11.5%. Divinding the 8-year
period into 4 groups of two years, the prevalence of acute MI has halved in
2006−07 compared to 2000−01. The decrease was uniform amongst all ethnic
groups except Afro-Caribbeans. Moreover, cardiovascular risk factor diagnosis
has improved across all ethnic groups. Interestingly, 33% of these MI patients
in the 2006−07 group compared to 17% in the 2000−01 group did not have any
cardiovascular risk factor apart from age.
Conclusion: Whilst clinical practice focusing on risk factor prevention seems
to have resulted in halving the cases of MI, the risk factor spectrum of patients
has changed and increasing proportions do not have traditional cardiovascular
risk factors.
464 VARYING PROPORTIONS OF PERIPHERAL VASCULAR
DISEASE INTERVENTIONS COMPARED WITH CORONARY
VASCULAR DISEASE IN ENGLAND.IMPLICATIONS FOR THE
AETIOPATHOLOGY OF ATHEROSCLEROSIS?
N. Ahmad, C. Chan. Vascular Surgery, Wirral University NHS Foundation
Trust, Wirral, UK
Introduction: Lower limb peripheral vascular disease has the same underlying
pathology and treatment modalities as coronary heart disease. There are no
UK data describing the surgical interventions for atherosclerosis in the different
arterial territories of ethnic groups.
Aim: To describe the ratio of intervention for peripheral vascular disease (PVD)
in relation to coronary heart disease (CHD) in different ethnic groups aged
50−84 living in England.
99
Method: The combined rates of endovascular and elective surgical
revascularisation for PVD and CHD were calculated using Hospital Episode
Statistics (2003–2009) and the 2006 census estimate. The proportion of PVD
interventions to CHD were calculated for the White British, South Asian and
Black populations.
Results: There were a total of 165 703 combined PVD treatments (133 632
endovascular 32 071 surgical) and 134 312 (29 465 endovascular 104 847
surgical) combined CHD interventions over the 6 year period (ratio of 1.2).
White British females had nearly double the proportion of PVD intervention
than their male counterparts (m=1.3 f=2.3). The proportion of PVD interventions
was much lower in South Asians (m=0.3 f=0.2) compared with Blacks (m=0.9
f=1.2).
Conclusion: The burden of atherosclerosis in different ethnic groups is resulting
in an unequal distribution of intervention by arterial territory. The implication
of these findings on the aetiopathology of atherosclerosis warrants further
investigation.
465 ASSOCIATION OF THROMBOMODULIN GENE VARIANTS IN INDIAN
PATIENTS WITH CORONARY ARTERY DISEASE (CAD)
T. Ashavaid1 , S. Shah2 , C. Ponde3 , R. Rajani3 , R. Mankeshwar4 .
1
Biochemistry, 2 Research Laboratories, 3 Cardiology, 4 Research Department,
P. D. Hinduja National Hospital and Medical Research Centre, Mumbai,
India
Introduction: Coronary artery disease (CAD) is a major cause of mortality
worldwide and is also a growing public health burden in India. The clinical
complication of CAD is the rupture of atherosclerotic plaque followed by
thrombosis, which leads to myocardial infarction. Thrombomodulin (TM), a
membrane glycoprotein is an effective natural anticoagulant present on the
endothelium. TM maintains a thromboresistant state by effectively regulating
thrombin production which if unregulated can participate in progression of
atherothrombosis. Further the TM genetic variants affect the thrombomodulin
function and the thrombomodulin plasma levels.
Objective: To study the association of Thrombomodulin gene variants in Indian
patients with CAD.
Material and Method: Blood samples were collected from 133 CAD cases and
133 age and gender matched controls. Genotyping of TM gene was carried out
by PCR-SSCP technique. Results were validated by DNA sequencing. Plasma
levels of TM were estimated by enzyme immunoassay.
Results: SSCP analysis revealed the identification of 6 genetic variants in the
samples screened. In younger subjects (49 yrs), the Ala455Val polymorphism
was significantly (p-0.006) associated with CAD, increasing the risk of CAD
by 3-fold. Furthermore in presence of smoking and alcohol consumption, the
risk increases by 4.4-fold & 3.7-fold respectively. TM levels were found to be
marginally higher in controls than cases.
Conclusions: TM genetic variant Ala455Val predicts the risk of CAD in subjects
49 yrs thus it can be used as a potential candidate marker of CAD by
cardiologist for the early screening of young asymptomatic Indian subjects.
466 IDENTIFICATION OF HAEMATOLOGICAL MARKERS ASSOCIATED
WITH POOR OUTCOME IN PATIENTS WITH ACUTE CORONARY
SYNDROME
O. Merono, C. Garcia, M. Cladellas, L. Recasens, V. Bazan, N. Ribas,
G. Rivas, A. Sainz, E. Valles, J. Bruguera. Hospital del Mar, Barcelona,
Spain
Introduction: Anemia is an independent predictor of poor outcome in the
setting of an acute coronary syndrome (ACS). We aimed to identify other
haematological markers of unfavorable prognosis in patients presenting with
ACS.
Methods: We prospectively included 174 ACS patients admitted during 2009
in our Coronary Care Unit. Hemoglobin (Hb) levels and other haematological
parameters were assessed. We further compared the clinical outcome of
patients presenting with anemia (Hb <13 or 12 g/dL in men or women,
respectively) at admission as opposed to those who developed anemia during
hospitalization.
Results: A >5 mg/dl value of C-reactive Protein (CRP) was highly predictive
of developing anemia during hospitalization (67% of cases without anemia and
CRP >5 mg/dl developed it, p < 0.05). If anemia was identified at admission, a
higher incidence of bleeding (21% vs. 4%) and in-hospital mortality (11.9%
vs. 1%) was noted as compared to those patients who persisted without
anemia throughout hospitalization; p < 0.05. During a median follow-up period
of 3.15 months (0.26–6.72), anemia at admission was the only haematological
parameter of higher cardiovascular morbidity and total mortality (RR 3; CI 95%:
1.1−8.0, p < 0.05). Developing anemia during hospitalization or >5 mg/dl value
of CRP were associated with a non-significant trend toward presenting higher
cardiovascular morbidity and total mortality (p = 0.13 and 0.14, respectively).
Conclusions: Anemia at admission remains as the strongest haematological
predictor of in- and out-hospital morbidity and mortality after an ACS. A >5 mg/dl
value for CRP predicts the development anemia during hospitalization.
100
467 REVERSIBLE MYOCARDIAL DYSFUNCTION AND LEFT
VENTRICULAR DILATATION IN ACUTE MYOCARDIAL INFARCTION
A. Alavy Lutfullaevich1 , S. Kenjaev Rashidovich1 , M. Kenjaev Latipovich2 .
1
Republican Research Center of Therapy and Medical Rehabilitation, 2 Urgent
Cardiology, Republican Research Center of Emergency Medicine, Tashkent,
Uzbekistan
Mean Hb in Female gender (12.28) was lower than Hb of males (12.91) (t = 2,
P = 0.04). Heart failure has a negative correlation with Hb, t = −4.59, P < 0.001,
but the other factors such as CHD and DM had no effect in Hb.
Patient group
Length of hospital stay
Age
Haemoglobin
Objective: To evaluate whether the presence of reversible myocardial
dysfunction detected by low dose dobutamine stress-echocardiography (DSE),
limits the risk of the left ventricular (LV) dilatation in patients with acute
myocardial infarction (AMI).
Material and Methods: 97 patients were studied by DSE at 3−4 days after AMI.
Echocardiography was repeated after three months. Patients were divided into
two groups depending on the presence (n = 43) or absence (n = 54) reversible
myocardial dysfunction (RMD).
Results: Baseline characteristics were comparable in both groups, except for
localization of MI. LV end-diastolic volume index (EDVI) was stable in patients
with RMD, but the end-systolic volume index (ESVI) decreased significantly
(p = 0.006). Patients without RMD had a significant increase EDVI (p < 0.001)
and ESVI (p = 0.007). Subgroup analysis of patients with non-Q and Q
myocardial infarction (Q MI) have shown that ventricular dilatation occurred
only in patients with Q MI without RMD. This led to an increase value of ESVI
after 3 months in this group compared with patients Q MI plus viability (p < 0.05).
Multivariate regression analysis identified myocardial viability as an independent
predictor of LV dilatation. The changing of wall motion score index during low
dose DSE and the number of pathological Q waves on ECG are also predictors
of LV dilatation.
Conclusions: The presence of viability early after AMI is associated with
preservation of LV size, whereas the absence of viability is an important
predictor of LV dilatation, especially in Q MI.
STEMI
Non-STEMI
Total
9.63±13
6.47±6.29
7.68±1.8
66.33±14.45
70.21±12.46
68.72±13.05
13.29±1.7
12.33±2
12.7±2
468 THE DECREASE OF MEAN PLATELET VOLUME AFTER
EXTRACORPOREAL LDL-CHOLESTEROL ELIMINATION IN
PATIENTS WITH SEVERE FAMILIAL HYPERCHOLESTEROLEMIA
M. Blaha1 , M. Lanska1 , V. Blaha2 , I. Fatorova1 . 1 2nd Dpt. of Medicine,
Hematology, 2 Dpt. of Metabolism and Gerontology, Charles University, Medical
Faculty and Faculty Hospital, Hradec Kralove, Czech Republic
Introduction: Mean platelet volume (MPV) is arousing increasing interest as
a new independent cardiovascular risk factor. Large platelets are likely to be
more reactive. If MPV would drop after LDL-lowering therapy, decreased MPV
could be one of the markers of successful therapy. That is why we investigated
MPV after extracorporeal LDL-cholesterol elimination (EE).
Methods and Patients: MPV was investigated in patients with severe familial
hypercholesterolemia (FH) long-term treated (3−12 years) by LDL-apheresis
(immunoapheresis) or cascade filtration. Plasma was obtained by centrifugation.
Adsorbers Lipopak 400 were used for immunoapheresis and filters Evaflux 4A
were used for cascade filtration. 95 pair samples were measured (before and
after the procedures) in a group of 12 patients − each patient 8 times in 4
years.
Results: Mean MPV before the procedures was 10.891 fl, CI 10.25–11.53.
MPV after the procedures was 10.478 fl, CI 09.84–11.11. The difference is
statistically significant (p = 0.036). MPV did not correlate with age, sex, platelet
count, duration of therapy.
Discussion: MPV is easily available and is often disregarded, and sometimes
may suggest the need for a careful assessment in patients with FH: this
was reported on the association of the MPV with the risk of coronary and
cerebrovascular diseases and atherosclerosis activity.
Conclusion: MPV could be one of the markers of therapeutic efficacy of
patients with FH treated by EE that are simply and inexpensively accessible.
Research supported by the Ministry of Health CZ MZO 00179906, NS/9743−4
and MSM 0021620820.
469 PREVALENCE AND PROGNOSTIC FACTORS OF ANAEMIA IN
PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
I. Kanonidis, G. Rompolas, K. Martiadou, C. Kagiadaki, G. Dadoush,
G. Sakantamis. 2nd Department of Cardiology. Hippokration Hospital, Aristotle
University of Thessaloniki, Thessaloniki, Greece
Purpose: Prevalence of anaemia in patients with AMI and possible prognostic
factors affecting the incidence of anaemia such as age, heart failure (HF),
coronary heart disease (CHD), Diabetes mellitus (DM), gender.
Methods: n = 282 patients were enrolled in our study, n = 108 with STEMI and
n = 174 Non-STEMI. Anaemia was considered when Hb levels < 12 g/dL.
Results: See the table. Mean hemoglobin was significantly different between
STEMI and non-STEMI (t = 2.805, df = 139, P < 0.05) with a preference of
anaemia in non-STEMI pts. There was a negative relation between Hb and
the duration of hospitalization in patients with STEMI, r = −0.434, p < 0.01.
Age was negative correlated with Hb, r = −0.366, p < 0.001 while there was no
difference between STEMI and non-STEMI groups in age variable, p = NS.
Values are mean±SD.
Conclusions: Prevalence of anemia seems to be in approximately one third
of patients with AMI with a preference of anaemia to non-STEMI, elderly and
patients with history of HF. Prevention of anaemia should be a major point for
all cardiologists.
470 DYSLIPIDAEMIA AND ITS COMPONENTS: PREVALENCE IN
HEALTH CARE PROFESSIONALS AND IN THE REST OF THE
WORKING POPULATION
P. Valdivielso-Felices1 , E. Calvo-Bonacho2 , C. Catalina-Romero2 ,
M.-Á. Sánchez-Chaparro1 , J.-C. Sainz-Gutierrez2 , I. Romero-Mateos3 ,
L. Palacio-Olmos3 , M. Gálvez-Godoy3 , J.-A. Gelpi-Méndez3 , L.-M. RuilopeUrioste4 . 1 Internal Medicine, University of Málaga, Málaga, 2 Health Projects,
Ibermutuamur, 3 Ibermutuamur Prevention Society, 4 Hypertension Unit,
Hospital 12 de Octubre, Madrid, Spain
Objective: Compare dyslipidemia prevalence between health care professionals (HCP) and the rest of the workforce.
Methods: Sample of 930404 workers (3688 HCP: 2200 nurses/1448 physicians) who attended a medical check-up between 2004–2008. Dyslipidaemia
was considered if (in mg/dl): Total-Chol >200 or HDL < 40 Male/ < 50 Female
or LDL 160 or Triglycerids 200. A bivariate analysis was performed and
segmented by gender and age (<40 vs. 40 years) to take into account the
potential confounding effect of these variables.
Results: There was no statistically significant difference in favour of HCP in
any of the parameters under study (a =0.05). The prevalence of dyslipidaemia
(54.3% vs. 47.4%, 0.02) and altered levels of LDL (13.4% vs. 9.9%, 0.04) was
higher among male physicians <40 years than in the reference group of the
remaining workforce. In nurses there was a slightly better situation, especially
among younger staff. Male nurses under age 40 had a lower prevalence of
dyslipidemia (39.8% vs. 47.4%, P= 0.01) and altered levels of HDL (10.3 vs.
20.9%, P < 0.001), while among female nurses under age 40, is significantly
lower than the rest of the working population (dyslipidaemia: 38.4% vs. 43.9%;
altered levels of Total-Chol: 4.4% vs. 5.6%; HDL: 17% vs. 21%; and LDL: 2.8%
vs. 4.3% P < 0.001).
Conclusions: With few exceptions, we found little difference in favour of HCP vs
the rest of the working population in the prevalence of dyslipidaemia. Our data
indicate the importance of assessing cardiovascular risk factors and designing
specific programs for this group.
471 PARAMETERS OF PLATELETS ACTIVATION AND METABOLISM
OF CARDIOMYOCYTES IN ACUTE MYOCARDIAL INFARCTION
A. Polek1 , W. Sobiczewski2 , J. Matowicka-Karna3 . 1 Medical Laboratories
BRUSS Group ALAB, Gdynia, 2 Department of Cardiology, Medical University
of Gdansk, Gdansk, 3 Department of Clinical Laboratory Diagnostics, Medical
University of Bialystok, Bialystok, Poland
Aims: We determined the relationship between platelets activation and changes
in the metabolism of cardiomyocytes in acute myocardial infarction.
Methods: We studied 70 patients with acute myocardial (AMI) and 80 healthy
volunteers (K), compared parameters of platelets activate: PLT, MPV, P-LCR,
sP-selectine and parameters of metabolism cardiomyocytes: released ATP, ecto
adenylate kinase (eKA).
Results: WBC (10.2±4.05; p < 0.001) increased, RBC (4.2±0.6; p < 0.05)
and PLT (219.0±54.5; p < 0.001) decreased in AMI versus K. MPV
(11.2±1.3; p < 0.001) increased in AMI versus K. 52% AMI patients had
high percentage of large platelets, MPV (15.0–35.0 fl), and 48% MPV had
greater than 35.0 fl. The concentration of ATP released from cardiomyocytes
and sP-selectin increased due to increasing activity of ecto adenylate
kinase: eKA 0.28, ATP (0.734±0.684): N = 17; eKA 0.28, sP-selectin
(73.6±35.82): N = 8; eKA > 0.43, ATP (1.614±0.887): N = 12; eKA > 0.43, sPselectin (112.7±32.60): N = 7. Increase of TnI level was associated with
decreased ATP released: TnI (31.8±32.6); ATP 0.49; N = 16, and low level
TnI, corresponded to increased released ATP: TnI (13.2±21.5); ATP > 0.49;
N = 40; p < 0.019. High level TnI (14.6±21.3) was accompanied by high values
MPV > 11.2; N = 19.
Conclusion: The activated platelet in AMI accompanied by increased MPV and
P-LCR and short-term decrease PLT, but there was no increase sP-selectin.
High TnI level was accompanied by high value MPV > 11 fL. The increase
79th EAS Congress
ecto adenylate kinase, ATP released and sP-selectin were characteristic of
increased metabolic activity in cardiomiocytes. The high level TnI accompanied
by low concentrations of released ATP, which confirms the degree of myocardial
damage.
472 ENDOTHELIAL DERIVATIVES AS THE POSSIBLE PREDICTORS
OF THE CARDIOVASCULAR RISK IN RHEUMATOID ARTHRITIS
PATIENTS (RA) WITH HYPERCHOLESTEROLEMIA
R. Rupinski, E. Walewska, A. Filipowicz-Sosnowska, P. Gluszko. Department
of Rheumatology, Institute of Rheumatology, Warsaw, Poland
Introduction: The HeartScore (SCORE) assessment tool is used to predict
relative risk of serious atherosclerotic cardiovascular (CV) events. RA patients
are at even higher risk of CV diseases.
Objective: To assess correlation of the SCORE estimated CV risk with serum
levels of endothelial adhesion molecules and other vascular derivatives in RA
patients with cholesterol concentration >200 mg/dl.
Methods: Clinical and metabolic (BMI, atherogenicity index − AI) profile was
evaluated in the group of 72 RA patients (93% female, age 42−74 years).
Serum concentrations of intercellular adhesion molecule-1 (ICAM-1), vascular
cell adhesion molecule-1 (VCAM-1), vascular endothelial growth factor (VEGF)
and E-selectin were measured using ELISA.
Results: The mean values (±SD) in the study group were: age − 58.6 years
(7.6), disease duration − 9.2 years (8.6), CRP − 10.5 mg/l (7.5), BMI − 26.9
(4.3), AI − 4.16 (1.31), ICAM-1–4887 pg/ml (515), VCAM-1–326 pg/ml (77),
VEGF − 134 pg/ml (133) and E-selectin − 6965 pg/ml (1965). The mean
SCORE value (±SD) for the whole RA group was 4.64 (3.84) and 32%
patients exceeded the 5% threshold for high CV risk. The correlations between
VEGF and SCORE values (r = 0.25, p < 0.037), E-selectin and the cholesterol
component of SCORE (r = −0.34, p = 0.021) were found. Other correlations
including CRP, BMI and AI were statistically insignificant.
Conclusions: We found association between elevated VEGF levels and higher
risk of CV events in RA. Interestingly, E-selectin negatively correlated with the
cholesterol component of SCORE.
473 BACK TO THE FUTURE IN TREATMENT OF REFRACTORY
NO-REFLOW AFTER PRIMARY PERCUTANEOUS CORONARY
INTERVENTION: INTRACORONARY EPINEPHRINE
T. Aksu, A. Colak, M. Durukan, U. Guray, H. Kısacık. Turkiye Yuksek Ihtisas
Education and Investigation Hospital, Ankara, Turkey
Background: The goal of this study was to evaluate the safety and efficacy of
intracoronary epinephrine in reversing refractory no-reflow during percutaneous
coronary intervention (PCI).
Methods: We retrospectively evaluated 12 consecutive patients who received
intracoronary epinephrine to reverse refractory no-reflow during primary PCI.
The effects of intracoronary epinephrine on qualitative TIMI flow grade and
quantitative TIMI frame count, cardiac rhythm, and systolic blood pressure were
assessed.
Results: A mean of 333±123 mcg of intracoronary epinephrine was used.
No-reflow was successfully reversed with complete restoration of TIMI 3
flow in 9 of 12 patients (75%). TIMI flow grade improved from 1.33±0.49
prior to epinephrine to 2.66±0.65 after treatment (p < 0.001). There was
improvement in coronary flow of at least one TIMI flow grade in 11 (93%)
patients, two flow grades in 5 (42%) cases. TIMI frame count decreased from
56±10 at the time of no-reflow to 19±11 (p < 0.001). Epinephrine therapy
was well tolerated without serious adverse hemodynamic or chronotropic
effects. Intracoronary epinephrine resulted in significant but tolerable increase
in heart rate (68±13 to 95±16 beats/min; p < 0.001) and systolic blood
pressure (94±18 to 140±20; p < 0.001). Hypotension associated with no-reflow
developed in 5 (42%) patients. During the procedure, intra-aortic balloon pump
counterpulsation was required in two (17%) patients, transvenous pacing in 2
(17%) cases, and both intra-aortic balloon counterpulsation and transvenous
pacing in one (8%) patients. One patient 3.4% died despite therapeutic
measures.
Conclusion: Intracoronary epinephrine may become an effective alternative in
patients suffering refractory no-reflow following primary PCI.
474 EFFECT OF QUERCETIN ON THE DYNAMICS OF THE LEVEL OF
NT-PROBNP IN PATIENTS WITH ACUTE CORONARY SYNDROME
A. Alavy Lutfullaevich1 , S. Varisxanova2 , S. Kenjaev1,2 . 1 Urgent Cardiology,
Republican Research Center of Emergency Medicine, 2 Republican Research
Center of Therapy and Medical Rehabilitation, Tashkent, Uzbekistan
Aim: To study of the concentration of NT-pro BNP in acute coronary syndrome
(ACS+ST) with ST elevation after intravenous infusion of quercetin.
Methods: The study included 68 patients with ACS+ST (age 57.62±4.64 y). All
patients underwent reperfusion therapy. The 1st group (n = 31) patients received
basic therapy. The 2nd group (n = 37) patients who also received the antioxidant
drug quercetin (Corvitin, Ukraine) by intravenous infusion at a dose of 1500–
1000 gr/day immediately after admission in hospital, after 2 and 12 hours for
101
2−3 days. All patients at baseline, at the 3rd and 5-th day was determined
concentration NT-proBNP. Reliability of intergroup differences was calculated
using Student’s t test.
Results: Patients in 1st group showed a significant increase in the
concentration of NT-proBNP to the 3rd day of observation at 52.78% with
subsequent decrease in 32.29% of the baseline, but in the 2 nd group the
peak concentration of NT-pro BNP to the 3-th day was absent, and there was
a progressive decline of 23.52% and 60.21%, respectively, to the 3rd and 5th
days (difference in the dynamics of concentration of NT-pro BNP throughout the
observation period − p < 0.001). As a result, and the third at 5-day concentration
of NT-pro BNP in patients taking Corvitin was significantly lower than patients
receiving only basic therapy (p < 0.001 for 3rd and 5th days of observation).
Conclusion: Inclusion of quercetin in the treatment of ACS+ST contributes to
the gradual decrease in the concentration of NT-proBNP and the disappearance
of the peak on the 3rd day.
475 THE IMPACT OF PERCUTANEOUS CORONARY INTERVENTION (PCI)
WITH STENT IMPLANTATION UPON MMP9 AND INFLAMATORY
MARKERS IN PATIENTS WITH ACUTE CORONARY SYNDROMES
F. Revnic1 , C.R. Revnic2 , C. Ginghina3 , A. Mereuta4 , D. Gradinaru5 , G. Prada6 ,
S. Prada7 , C. Pena8 , C. Borsa9 , C. Ionescu9 . 1 Biology of Aging, NIGG’ANA
ASLAN’, 2 Cardiology, UMF Carol Davila, 3 Cardiology, 4 Interventional
Cardiology, C.C.Iliescu Cardiovascular Institute, 5 Pharmachology,
6
Gerontology and Geriatrics, UMF Carol Davila, 7 Pharmacy, 8 Imunology,
9
Biochemistry, NIGG’ANA ASLAN’, Bucharest, Romania
The aim of this study was to evaluate if serum levels of matrix
metalloproteinase-9 (MMP-9), IL6, IL8, hsCRP and NOx are associated with
development of ISR after stent implantation. We performed PCI with a single
stent in 80 patients (50 with BMS and 30 with DES (58±8 years old)who
had acute coronary syndrome. Serum levels from of MMP-9, IL6, and hsCRP
activity were measured by specific ELISA and NOx levels using a Nitrite/Nitrate
assay before stent implantation and at 24 hours. Patients with ISR at followup showed significantly higher MMP-9 activity levels at baseline (34Â±15 vs.
23Â±10 ng/mL; p < 0.01) and 24 hours after PCI (53Â±28 vs. 26Â±12 ng/mL).
IL-6 0.46±0.03 ng/L versus 0.40±0.07 ng/Patients in the highest quartile
before and after PCI showed a 6.5 (1.7−26) and 7.7 (18.2.1−35)-fold risk
for the development of restenosis, respectively. Plasma hsCRP levels at 24h
(10.12±3.55 vs 6.43±1.63, P = 0.001) after stent implantation were significantly
higher in the BMS group than in the DES. The level of NOx at base line was
46.94±8.5umol/L and 103.98umol/l after 24 h PCI and stent implantation The
occurrence of ISR was significantly associated with MMP-9 activity and NOx
before and after placement of BMS. As MMP-9, Nox, hsCRP and IL6 may play
a role in the pathogenesis of ISR, determination of hsCRP and MMP-9, Nox
and IL6 serum levels might be helpful in the identification of patients with high
risk for development of ISR after DES implantation.
476 ACUTE CORONARY SYNDROME AND LOW-HDL CHOLESTEROL.
RELATIONSHIP WITH LIPOPROTEIN (A)
R. Toro1 , P. Gomez1 , C. Rodriguez1 , I. Tinoco1 , D. Biedma1 , M. Virseda1 ,
A. Mangas2 . 1 Cadiz University, 2 Medicine, Cadiz University, Cadiz, Spain
Several studies have shown that a high serum level of lipoprotein (a), lp(a), may
be an independent risk factor for atherotrombotic disease including myocardial
infarction. Metabolic syndrome (MS), a clustering of cardiovascular risk factors,
including low HDL-C concentration, is linked with an increased risk of developing
cardiovascular disease and diabetes mellitus.
Aim: Was to know the prevalence of MS and low-HDL-c syndrome in patients
with acute coronary syndrome (ACS) and relationship with serum lp(a).
Material and Methods: We examined 65 ACS patients (50 males) with 64.5 y-o.
Blood analysis and lipid profile were done before the sixth day of the hospital
stay. Low HDL-c was defined as a serum concentration <1.04 mmol/l in men
and <1.3 mmol/l in women.
Results: Low HDL-c was observed in 42 (64.6%) patients. 44 (67.7%). MS
was diagnosed using NCEP ATP III criteria. In 25 patients (19 males, 38%) low
HDL-c syndrome is associated with MS. Lp(a) revealed significant elevation in
ACS. We did not found significance differences neither in patients with MS nor
low HDL-c syndrome.
Conclusions: Spanish patients with ACS had a very high prevalence of low
HDL-c syndrome and MS. Lp(a) is an emerging risk factor potentially useful
predicting future cardiac events. We found lp (a) serum concentration elevated
in ACS. Increased lp(a) were not associated with SM nor low HDL-c in ACS.
477 IP-10 AND ACUTE MYOCARDIAL INFARCTION
A.E. Nadimi. Rafsanjan University of Medical Science, Rafsanjan, Iran
Background: Cardiovascular disease are the most common cause of death
in the world and also in Iran. IP-10 “angiostatic chemokine” is an effective
chemokine for decreasing inflammation and injury. This study evaluated and
102
compared the serum level of this chemokine in healthy and in patients with
acute myocardial infarction regarding to age and sex.
Material and Method: Our study was descriptive. Two groups of 63 patients
and healthy participants were evaluated. Patients group was admitted with
acute myocardial infarction. Blood sampling was taken in the first 24 hours of
admission. In patients group with myocardial infarction another sampling was
taken in the seventh day after admission. Serum level of IP-10 and other data
were analyzed with SPSS, T test, chi square and ANOVA test.
Results: In each group 50 participants were male (79.4%) and 13 participants
were female (20.6%). The youngest was 35 years old and the oldest had 84
years old. In patients group, 51 person had first event (80.95%), 37 of them had
no history of ischemic heart disease. At discharge time 40 (63.5%) patients
had Q wave myocardial infarction. Overall there was significant difference
between serum concentrations of IP-10 in control and patient group in the
first sample and also between control and patient’s second sample. In male
participants the difference between control and second sample of patient group
was significant.
Conclusion: Our study showed that IP-10 concentration increased during first
week after myocardial infarction and suggest that IP-10 as an anti-inflammatory
and angiostatic chemokine may be effective in the course of acute myocardial
infarction.
478 COLLAGEN MATRIX OF ATHEROSCLEROTIC PLAQUE
AND LIPID SPECTRUM IN PATIENTS WITH MYOCARDIAL
INFARCTION TREATED WITH ROZUVASTATIN DEPENDING ON
REVASCULARIZATION METHOD
N.A. Koziolova1 , E. Malgina2 . 1 Perm State Medical Academy, 2 Perm Medical
Sanitary Unit #4, Perm, Russia
Objective: To evaluate dynamics of parameters of collagen matrix in
atherosclerotic plaque depending on changes in lipids in STEMI patients
who underwent prehospital thrombolytic therapy (PTT) with tenecteplase or
urgent percutaneous coronary intervention (PCI) during multi-drug therapy with
rozuvastatin.
Materials and Methods: 40 STEMI patients were examined. Average age was
58.28±5.49 years. The patients were divided into 2 groups of 20 people each.
The first group consisted of the patients who underwent PTT. The second group
consisted of the patients who underwent urgent PCI. Duration of multi-drug
therapy including rozuvastatin in dose of 10 mg per day lasted for 1 month. Total
cholesterol (TC), high and low density lipoproteins (HDL, LDL), triglycerides
(TG), serum level of type 1 human tissue inhibitor of matrix metalloproteinases
(hTIMP-1) were evaluated in patients before and after therapy by rozuvastatin.
Results: During multi-drug therapy including rozuvastatin TC, LDL, TG
decreased significantly in both groups. In both groups HDL did not change
significantly. Between the groups no significant differences in terms of lipid
spectrum parameters were revealed. hTIMP-1 level increased significantly in the
first group by 54.7+12.1% (p < 0.001) and in the second group by 48.9+9.4%
(p < 0.001). Correlation analysis showed there were no correlations between
decrease of TC, LDL, TG and hTIMP-1 increase.
Conclusions: During therapy by rozuvastatin of the STEMI patients
encollagenolisis slowed down in atherosclerotic plaque was detected due
to hTIMP-1 increase. The effect of rozuvastatin might be considered as
pleiotropic effect regardless of both its lipid corrective ability and method of
revascularization.
479 GLYCAEMIC CONTROL AND SECONDARY PREVENTION
TREATMENT IN POSTINFARCTION PATIENTS REDUCE THE
INTENSITY OF CARDIOVASCULAR RISK FACTORS CORRELATION
E. Vataman, S. Filimon, D. Lisii, O. Priscu, A. Grivenco. Heart Failure, Institute
of Cardiology, Chisinau (Kishinau), Moldova
Aim: The major target in effective treatment of type 2 diabetes mellitus is to
reduce the micro- and macrovascular complications and as a long-term benefit −
reducing of cardiovascular morbidity and mortality.
Materials and Methods: We studied the correlation between platelet
aggregation, lipids, coagulation and endothelial dysfunction parameters in
54 patients with type 2 diabetes mellitus at one year after Q-myocardial
infarction (34 men and 20 women, mean age 58.88±0.92 years) long term
using secondary prevention treatment and glucose-lowering medications and
maintaining good glycaemic control.
Results: The average values exceeded the normal intima-media thickness (1.11±0.01 mm), total cholesterol (6.56±0.09 mmol/l), triglycerides
(2.05±0.11 mmol/l) and LDL-cholesterol level (4.16±0.10 mmol/l) at our
patients. Light transmittance aggregometry (Born’s method) demonstrated an
important but low level of correlation between the degree of ADP induced
platelet aggregation and fibrinogen level (r = 0.28, p < 0.05), prothrombin index
(r = 0.37, p < 0.05) and blood triglyceride level (r = 0.4, p < 0.05). The duration
of platelet aggregation registered an inverse proportional relationship to the
D-dimers blood level (r = −0.35, p < 0.05). There were direct correlations
between resting brachial artery diameter and platelet aggregation time
sustained after reactive hyperemia testing (r = 0.32 and r = 0.32, p < 0.05).
Conclusions:
1. Patients with diabetes mellitus support multiple atherosclerotic disease and
high level of endogenous risk factors.
2. Observed correlations signify maintaining of constant prothrombotic risk in
postinfarction patients with diabetes mellitus despite of adjusted medical
treatment.
480 CLINICAL AND ECONOMIC IMPACT OF ANTITHROMBOTIC
MANAGEMENT PATTERNS IN ACUTE CORONARY SYNDROMES:
RATIONALE AND DESIGN OF THE EPICOR STUDY
H. Bueno1 , F. Van de Werf2 , N. Danchin3 , M. Tafalla4 , C. Bernaud5 ,
L. Annemans6 . 1 Hospital General Universitario Gregorio Marañón, Madrid,
Spain, 2 University Hospitals Leuven, Leuven, Belgium, 3 Hôpital Européen
Georges Pompidou, & René Descartes University, Paris, France, 4 AstraZeneca
Farmacéutica Spain, Madrid, Spain, 5 AstraZeneca Medical Europe, Zaventem,
6
I-CHER Interuniversity Centre for Health Economics Research UGent, Gent,
Belgium
Aim: To evaluate in a real-life setting of patients hospitalized for ACS the
differences in short- and long-term clinical and economic outcomes according
to different antithrombotic management patterns, and to compare them between
sites and countries.
Methods: EPICOR (long-tErm follow-uP of antithrombotic management
patterns In acute CORonary syndrome patients) is a prospective, observational,
longitudinal cohort study (NCT01171404) that will enrol ~10,600 consecutive
patients 18 years surviving a hospitalization for ACS in ~700 centres from
21 countries in Europe and Latin America, who will be followed up for 2 years
after the index event. The study will include different level hospitals, providing a
representative reflection of real-life management of ACS with pharmacological
therapies and invasive strategies. In-hospital information will be provided by
local staff, while a thorough centralized follow-up will be performed at a country
level through telephone calls using standardized questionnaires, with later
validation of outcomes by physician interviews. The primary endpoint is the
rate of clinical outcomes (ischaemic and haemorrhagic) associated with the
most frequent antithrombotic management patterns used for ACS treatment.
Other variables include quality of life and health resources consumption.
Results: Enrolment started in September 2010 and should finish by February
2011. In-hospital results should be available for the end of 2011. Long-term
results are expected from 2012.
Conclusions: The EPICOR study will generate valuable high quality data on
the different patterns of ACS management used in different types of hospital
and countries, as well as their impact on clinical and economic outcomes in a
real-life setting.
481 HIGH SERUM TESTOSTERONE IS ASSOCIATED WITH REDUCED
RISK OF CARDIOVASCULAR EVENTS IN ELDERLY MEN
C. Ohlsson1 , E. Barrett-Connor2 , S. Bhasin3 , E. Orwoll4 , F. Labrie5 ,
M.K. Karlsson6 , Ö. Ljunggren7 , L. Vandenput1 , D. Mellström1 , Å. Tivesten8 .
1
University of Gothenburg, Gothenburg, Sweden, 2 University of California
San Diego, La Jolla, CA, 3 Boston Medical Center, Boston, MA, 4 Oregon
Health & Science University, Portland, OR, USA, 5 Laval University,
Quebec, QC, Canada, 6 Lund University, Malmö, 7 University of Uppsala,
Uppsala, 8 Wallenberg Laboratory for Cardiovascular Research, University of
Gothenburg, Göteborg, Sweden
Background: Low serum testosterone is associated with increased fat mass, an
adverse metabolic risk profile, and atherosclerosis. However, despite increasing
interest in testosterone treatment of men with low testosterone levels, few
prospective studies have demonstrated a protective link between endogenous
testosterone and cardiovascular outcomes. Polymorphisms in the sex hormonebinding globulin (SHBG) gene are associated with risk of type 2 diabetes, but
few studies have addressed SHBG as a predictor of cardiovascular events. We
tested the hypothesis that serum testosterone and SHBG levels predict incident
cardiovascular events in elderly men.
Methods and Results: We used gas chromatography-mass spectrometry to
analyze baseline levels of testosterone in the prospective population-based
MrOS Sweden study (2416 men, 69−81 years). SHBG was analyzed by
immunoradiometric assay. Cardiovascular outcome data were obtained from
central registers. During a median 5.1 year follow-up, 485 cardiovascular
events occurred. Both serum testosterone and SHBG levels were inversely
associated with the risk of cardiovascular events (trend over quartiles P = 0.009
and 0.012, respectively). In analyses that included both testosterone and
SHBG, testosterone but not SHBG predicted cardiovascular risk. Men in
the highest quartile of testosterone had lower risk of cardiovascular events
compared with men in the three lower quartiles (hazard ratio [HR] 0.70, 95%
confidence interval [CI] 0.56–0.88). This association remained after adjustment
for traditional cardiovascular risk factors and after excluding subjects with
prevalent cardiovascular disease (HR 0.71, 95% CI 0.53–0.95).
Conclusions: High serum testosterone is associated with reduced risk of
cardiovascular events in community-dwelling elderly Swedish men.
79th EAS Congress
482 C-REACTIVE PROTEIN (CRP) AND LIPID LEVELS TO PREDICT
THE RISK OF CORONARY HEART DISEASE IN STATIN-TREATED
STABLE CORONARY PATIENTS
B.J. Arsenault1 , S.M. Boekholdt1 , P. Deedwania2 , D.A. DeMicco3 , W.H. Bao3 ,
G.M. Preston3 , D.D. Waters4 , J.C. LaRosa5 , P. Barter6 , J.J.P. Kastelein1 ,
the Treating to New Targets (TNT) Investigators. 1 Department of Vascular
Medicine, University of Amsterdam, Amsterdam, The Netherlands, 2 Cardiology
Division, School of Medicine, University of California San Francisco, Fresno,
CA, 3 Pfizer Inc., New York, NY, 4 San Francisco General Hospital, San
Francisco, CA, 5 State University of New York Downstate Medical Center,
Brooklyn, NY, USA, 6 The Heart Research Institute, The University of Sydney,
Sydney, NSW, Australia
Objective: To investigate whether CRP levels add to the predictive value of
LDL-C and the apolipoprotein B/apolipoprotein A-I (apoB/apoA-I) ratio in statintreated patients with stable CHD.
Methods: We performed a nested case-control study in the TNT study
population, a randomized trial that compared the efficacy of high (80 mg) vs. low
(10 mg) dose atorvastatin for the secondary prevention of CVD. Fasting levels
of standard lipids and of CRP were obtained after an 8-week run-in period on
atorvastatin 10 mg in 493 patients who experienced CVD during the 4.9-year
study follow-up and in 1013 treatment-matched controls.
Results: Compared to patients with CRP levels <2 mg/L, those with CRP levels
3 mg/L had an age-, sex- and treatment-adjusted relative risk (RR) for future
CVD of 1.16 (95% CI, 0.96–1.40). Compared to patients with LDL-C <70 mg/dL,
those with LDL-C 100 mg/dL had a RR of 1.40 (0.89–2.21). Compared to
patients with an apoB/apoA-I ratio <0.8, those with an apoB/apoA-I ratio 1.0
had a RR of 1.98 (1.54–2.55). Compared to patients with CRP <2 mg/L and
apoB/apoA-I ratio <0.8, those with CRP 2 mg/L and apoB/apoA-I ratio <0.8
had a RR of 1.00 (0.77–1.30), while those with CRP <2 mg/L and apoB/apoA-I
ratio 0.8 had a RR of 1.63 (1.28–2.07) and those with CRP 2 mg/L and
apoB/apoA-I ratio 0.8 had a RR of 1.80 (1.41–2.31).
Conclusion: In statin-treated patients with stable CHD the apoB/apoA-I ratio
is a strong predictor of incident CVD, irrespective of plasma CRP levels.
483 PREVALENCE OF ELEVATED C-REACTIVE PROTEIN LEVELS
IN A PRIMARY CVD PREVENTION POPULATION IN EUROPE:
THE EURIKA STUDY
J. Halcox1 , J.R. Banegas2 , J. Dallongeville3 , G.D. Backer4 , E. Guallar5 ,
E.L. Massó-González6 , J. Perk7 , F. Rodriguez Artalejo2 , P.G. Steg8 ,
F. Tubach9 , C. Borghi10 . 1 Wales Heart Research Institute, Cardiff University,
Cardiff, UK , 2 Universidad Autonoma de Madrid, Madrid, Spain, 3 Institut
Pasteur de Lille, Lille, France, 4 Department of Public Health. University of
Gent, Gent, Belgium, 5 Johns Hopkins Bloomberg School of Public Health,
Baltimore, MD, USA, 6 Medical Department, Epidemiology Unit, AstraZeneca,
Madrid, Spain, 7 School of Health and Caring Sciences, Linnaeus University,
Kalmar, Sweden, 8 NSERM U-698, Recherche Clinique en Athérothrombose,
Université Paris 7, Denis Diderot, Hôpital Bichat, Assistance Publique-Hôpitaux
de Paris, 9 Département d’Epidémiologie, Biostatistique et Recherche Clinique,
Hôpital Bichat, Paris, France, 10 Department of Internal Medicine, Aging and
Clinical Nephrology, University of Bologna, Bologna, Italy
Modest elevation of C-Reactive Protein in the high-sensitivity range (hsCRP)
identifies individuals with low-grade systemic inflammation at greater risk of
cardiovascular disease (CVD) events than those with similar conventional
risk factor profiles but lower hsCRP. The European Study on Cardiovascular
Risk Prevention and Management in Daily Practice (EURIKA, NCT00882336)
investigated the prevalence of elevated hsCRP levels in relation to CVD risk
factor profile.
This cross-sectional study conducted in 12 European countries recruited
patients free from clinical CVD, aged 50 years with 1 CVD risk factor. hsCRP
was measured by imunoturbidimetry (Roche P-Modular). 10 year cardiovascular
risk was assessed by Systematic COronary Risk Evaluation (SCORE) and
Framingham Risk Score (FRS) models.
7553 patients (63y, 48% male) were evaluated. 3608 (48%) were at high risk
by SCORE (5%) and 1737 (24%) by FRS (20%). 2717 (36%) had elevated
hsCRP levels (3 mg/L cutoff), including 1379 (35%) of those with SCORE < 5%
and 1948 (36%) with FRS < 20%. Of the 3463 patients without diabetes and
not receiving statins, 1568/3463 (45%) were at high risk by SCORE and
707/3297 (21%) by FRS. Elevated hsCRP was observed in 1352 (39%) of
these individuals, including 723 (38%) with SCORE < 5% and 1001 (39%) with
FRS < 20%). 3845 (51%) patients had hsCRP 2 mg/L, including approximately
half of each above subgroup.
Clinically relevant inflammation is common in European primary prevention
patients, identifying a population likely to be at higher CVD risk than expected.
This includes over a third of the sub-population at low-intermediate conventional
CVD risk who might otherwise not be considered for more intensive preventive
strategies.
103
484 DEVELOPMENT OF A NOVEL HOMOGENEOUS ASSAY FOR
REMNANT LIKE PARTICLE CHOLESTEROL
Y. Ito, Y. Hirao. Research and Development Department, Denka Seiken Co.,
Ltd., Niigata, Japan
Background and Aim: Remnant like particle (RLP), one of the lipoprotein
subclasses, has known as an atherogenic lipoprotein. Many epidemiological
and pathological studies have suggested RLP-cholesterol (C) level has close
relation to CHD and Type III hyperlipidemia. It is also known as a marker for
postprandial hyperlipidemia. We report the development of a fully automated
homogeneous assay for RLP-C quantification which does not require any offline sample pretreatment.
Methods: We screened enzymes and surfactants for our homogeneous
assay for RLP-C using CM-VLDL, LDL and HDL fractions isolated by
ultracentrifugation, and RLP fraction separated by anti-apoA and apoB affinity
gel.
Results: We found that a cholesterol esterase with no subunits of less than
40kDa (H Mol CHE) reacts lipoproteins except for RLP, whereas an enzyme
with a subunit of less than 40kDa (L Mol CHE) reacts also with RLP. We
then employed the H Mol CHE for the 1st step reaction to dissociate non-RLP
lipoproteins and degrade non-RLP-cholesterol to water and oxygen under the
presence of cholesterol oxidase and catalase. For the 2nd step, we applied the
L Mol CHE to release cholesterol from RLP, and then measured the released
RLP-C in the standard cholesterol oxidase and peroxidase system. Our new
homogeneous method exhibited a good correlation with a RLP-C method using
anti-apoA and apoB affinity gel (r > 0.9).
Conclusion: Our new homogeneous method gives a RLP-C result in 10 min
in a fully automated manner and thus allows the analysis of large number of
samples in routine laboratories.
485 RELATION OF CENTRAL AND BRACHIAL BLOOD PRESSURE TO
LEFT VENTRICULAR HYPERTROPHY. THE CZECH POST-MONICA
STUDY
P. Wohlfahrt1 , D. Wichterle2,3 , J. Seidlerová4 , J. Filipovský4 , J. Bruthans1 ,
V. Adámková1 , R. Cı́fková1 . 1 Department of Preventive Cardiology,
2
Department of Cardiology, IKEM, 3 Department of Cardiology and Angiology,
First Faculty of Medicine, Charles University, 4 2nd Department of Internal
Medicine, Charles University, Centre for Hypertension, Prague, Czech
Republic
Objective: Central blood pressure was shown to be a better predictor of target
organ damage and cardiovascular events than brachial blood pressure. Whether
central blood pressure is a better predictor of left ventricular hypertrophy (LVH)
determined by electrocardiography is not known.
Methods: Radial applanation tonometry and ECG were performed in 728
subjects from the Czech post-MONICA study (randomly selected 1% population
sample). LVH was determined using Sokolow-Lyon index and Cornell product;
central pressure was derived from radial pulse.
Results: Of 700 subjects included in the analysis 17 (9.4%) below 45 years
and 52 (10%) over 45 years had LVH. In multiple linear regression analysis
Sokolow-Lyon index in younger individuals was only associated with male sex
(b = 0.47, p < 0.001) and low BMI (b = −0.15, p < 0.05), while no association
with central or brachial blood pressure was found. In older individuals, LVH was
associated with higher central and brachial blood pressure. In separate binary
logistic regression analyses, the odds ratios for central systolic (1.08, 95% CI
1.06–1.11) and pulse pressure (1.09, 95% CI 1.06–1.12) were higher than for
brachial systolic (1.03, 95% CI 1.01–1.05) and pulse pressure (1.03, 95% CI
1.01–1.05) in LVH prediction.
Conclusion: Noninvasively determined central pressure in subjects over 45
years is more strongly related to ECG LVH than brachial pressure. This further
supports closer association of central pressure with target organ damage.
Voltage criteria of LVH are not independently associated with central or brachial
blood pressure in younger individuals.
486 APOLIPOPROTEIN M (APOM) − A NOVEL BIOMARKER FOR ACUTE
KIDNEY INJURY
E.M. Madsen Svarrer1 , H.Ø. Andersen2 , M. Helvind2 , M.C.J. Slagman3 ,
G. Navis3 , R.P.F. Dullaart4 , B. Dahlbäck5 , L.B. Nielsen6,7 . 1 Department of
Clinical Biochemistry, 2 Department of Cardiothoracic Surgery, Rigshospitalet,
University of Copenhagen, Copenhagen, Denmark, 3 Department of
Nephrology, 4 Department of Endocrinology, University of Groningen
and University Medical Center Groningen, Groningen, The Netherlands,
5
Lund University, Department of Laboratory Medicine, Division of Clinical
Biochemistry, University Hospital, Malmö, Sweden, 6 Department of Clinical
Biochemistry, Rigshospitalet, 7 Department of Biomedical Sciences, University
of Copenhagen, Copenhagen, Denmark
Acute kidney injury (AKI) affects ~20% of hospitalized patients. The diagnosis
is currently made 24−72 hours after the kidney insult at a pathophysiological
point of no return where treatment is mainly supportive. We have examined
104
urinary excretion of apoM as an early biomarker of AKI which could facilitate
early intervention.
Liver-derived plasma apoM is bound to HDL preventing filtration into the
urine. The proximal tubules in the kidney also produce apoM, which normally
is secreted into the pre-urine and reabsorbed from the tubule lumen via
the megalin receptor. Hence, apoM is not detectable in urine from healthy
subjects.
We have collected urine every two hrs within 0−12 hrs post-operatively from
children undergoing cardiac surgery. Of 31 children examined, 14 developed
post-operative AKI (i.e. >50% increase of plasma creatinine). They all displayed
a transient increase of apoM in the urine from non-detectable to a peak
value of >6.5 nmol/L 0−4 hours postoperatively. Using the 2-hr values the area
under the receiver-operating characteristics curve for diagnosis of AKI was
0.89. The sensitivity and specificity were 0.80 and 0.94, respectively using an
apoM/creatinine cut-off value of 1.9 nmol/mmol.
In 10 adult patients with glomerular kidney disease and proteinuria only the two
with most severe proteinuria (>1.2 g/day) had detectable and very low levels of
apoM in the urine.
In conclusion, urinary apoM may be a novel biomarker for specific and early
detection of AKI in children undergoing cardiac surgery.
487 TARGETED PHOSPHOLIPID METABOLITE PROFILING IN THE
CLINICAL LABORATORY USING MALDI-QIT-TOF-MS/MS
G. Stübiger1 , E. Aldover-Macasaet1 , W. Bicker2 , K. Pock3 , V. Bochkov1 ,
K. Widhalm1 , O. Belgacem4 . 1 Medical University Vienna, 2 FTCForensic-Toxicological Laboratory Ltd., 3 Octapharma Pharmazeutika,
ProduktionsgesmbH, Vienna, Austria, 4 Shimadzu, MALDI Technologies Group,
Manchester, UK
Background and Aims: Phospholipids (PLs) and their metabolites (e.g. OxPCs
and LPCs) are increasingly recognized as key molecules with diagnostic
value in a number of pathologies including acute inflammation, CVD and
atherosclerosis. The aim of our investigation was to evaluate the potential of
a MALDI-MS/MS-based platform for screening of PL biomarkers in the clinical
laboratory.
Methods: Plasma from carefully phenotyped children and adolescents
(age 13±5 years, n = 20) with familial hyperlipidemias (e.g. FH and FCH)
representing high-CVD risk groups was evaluated. Lipids were isolated using
solvent extraction, purified by chromatography (SPE and HPLC) and subjected
to MALDI-QIT-TOF-MS/MS analysis. This approach allows direct targeted
monitoring of individual lipid molecules based on diagnostic ion monitoring from
MS/MS spectra. Lipid data were statistically evaluated and compared with the
laboratory parameters.
Results: PL-profiles were characterized by elevated PC/LPC and SM/PC
ratios in FH vs. FCH patients. Positive correlations between VLDL-C vs. LPC
(r = 0.669) and LDL-C vs. SM (r = 0.946) were observed. OxPC levels were in
the range of 2−20 mmol/L positively correlating with IMT (r = 0.952) and HDL-C
(r = 0.889) but negatively with LDL-C (r = −0.708). The preliminary results show
the potential of OxPLs as markers for early vascular disarrangements in patients
with familial hyperlipidemias.
Conclusions: MALDI-QIT-TOF-MS/MS serves as promising tool for targeted
screening of lipid biomarkers related to CVD and atherosclerosis. The technique
takes advantage of robustness, sensitivity, and accuracy for the quantitative
monitoring of PLs from clinical samples. Our study identified lipid parameters
of specific value to be validated in a broader clinical setting in the future.
488 EFFICACY BIOMARKER FOR PCSK9 INHIBITORS
R. Laaksonen1 , K. Koistinen1 , R. Huuhilo1 , K. Tarasov1 , R. Hurme1 ,
K. Ekroos1 , A. Prat2 , N. Seidah2 , M. Jänis1 . 1 Zora Biosciences Oy, Espoo,
Finland, 2 Clinical Research Institute of Montreal, Montreal, QC, Canada
Background and Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9)
is a potential therapeutic target for treatment of hyperlipidemia as inhibition of
its function increases hepatic uptake of LDL and thus reduces plasma LDLcholesterol levels. We aimed to define a an efficacy biomarker for PCSK9
inhibitors that would help in drug compound evaluations.
Methods: We analyzed the plasma and liver lipidomic profiles of wild-type
and (PCSK9)-deficient mice. Samples were extracted for lipids using roboticassisted method and analyzed on a hybrid triple quadrupole/linear ion trap
mass spectrometer (QTRAP 5500) equipped with a robotic nanoflow ion
source (NanoMate HD) and on a hybrid triple quadrupole/linear ion trap
mass spectrometer (4000 QTRAP) equipped with an ultra high pressure liquid
chromatography (UHPLC) system.
Results: PCSK9-inhibition induced significant changes in plasma and liver
expression of molecular lipid species. Ceramide and cerebroside lipid species
were particularly sensitive to PSCK9 inhibition. Western diet reduced the effect
of PCSK9 inhibition and induced hepatosteatosis in the homozygous knock-out
mice.
Conclusions: Ceramides and cerebrosides may serve as sensitive efficacy
biomarkers for PCSK9 inhibitors. Plasma lipidomic profiles will also be useful
in detecting off-target effects of novel lipid lowering compounds.
489 ENDOTHELIAL PROGENITOR CELL MOBILIZATION IS ENHANCED
IN HIGH PREFORMANCE ATHLETES
M.C. Izar, C.R. Bittencourt, C.N. Franca, H.R. Fonseca, H.T. Bianco,
V.L. Schwerz, F.A. Fonseca. Medicine, Federal University of São Paulo, São
Paulo, Brazil
Physical exercise is recommended to prevent cardiovascular disease. Exercise
stimulates the recruitment of endothelial-progenitor cells (EPC) from bone
marrow, improving endothelium turnover. However, studies evaluating high
performance runners (HPR) are less reported. The aim of this study was
to quantify EPC and endothelium-derived microparticles (EMP) in HPR and
controls.
Methods: HPR (n = 16), defined on basis of the time to perform a 10-km race
were compared with sedentary controls (n = 40), age and gender matched. The
quantification of three subpopulations of EPC was made using CD34, KDR and
CD133. EMP countings were assessed by CD51 by uL of platelet-poor plasma
using flow-citometry. Flow-mediated dilation (FMD%) and the intimal medial
thickness of the carotid artery (c-IMT) were also evaluated.
Results: Mean time to perform a 10-km race was 31 min and 40 sec for
men and 37 min and 37 sec for women in the HPR group, and they run an
average of 132 km/week. HPA presented higher HDL-C (p < 0.0001), lower body
mass index (p < 0.0001), LDL-C (p = 0.0001), triglycerides, apoB (p < 0.0001)
and C-reactive protein (p = 0.025). We observed a trend to higher EMP in
HPA (p = 0.088), and the percentage (SD) of EPC in HPA was higher than in
controls: CD34+/KDR+ [0.42 (0.34) vs 0.08 (0.03), p = 0.05], CD34+/CD133+
[0.07 (0.02) vs 0.01 (0.01), p = 0.002] e KDR+/CD133+ [0.29 (0.06) vs 0.01
(0.01), p < 0.0001]. FMD (SD) was higher in HPA group [30 (12)% vs 16 (10)%,
p < 0.0001], without differences in c-IMT.
Conclusions: High-performance exercise is associated with increased EPC
mobilization and vascular health.
490 CIRCULATING MICRORNAS: POTENTIAL BIOMARKERS IN
ARTHEROSCLEROSIS
D.S. Karolina, A. Armugam, K. Jeyaseelan. Biochemistry, Yong Loo Lin
School of Medicine, National University Health System, National University of
Singapore, Singapore, Singapore
Introduction: Artherosclerosis, the hardening and narrowing of the arteries, is
the usual cause of vascular diseases including stroke which ranks second in
causes of deaths. Diabetes, high cholesterol, high blood pressure and metabolic
syndrome are known major risk factors for atherosclerosis. MicroRNAs are short
noncoding RNAs that regulate >30% of protein-coding genes. The emerging
field of miRNAs has revealed its potential roles in the diagnosis, prognosis and
therapy of artherosclerosis.
Aim: In this study, we seek to understand the miRNA-mediated molecular
events in the development of artherosclerosis.
Method: Two hundred subjects were screened for this study and those with risk
factors of artherosclerosis were identified based on the National Cholesterol
Education Program criteria. All subjects were males with a mean age of
46.2±5.7 years. A separate pool of stroke patients (stroke considered as
secondary to artherosclerosis) was also recruited in the study. Total RNA was
extracted from these samples and screened on miRCURY LNA™ microarray
chip for miRNA profiling.
Result: Significant differences were observed between the miRNA profiles of
healthy individuals and subjects with diseases. In comparing the miRNA profiles
of those individuals with risk factors of artherosclerosis and the stroke patients,
we identified a handful of miRNAs involved in endothelial integrity, inflammation
and lipoprotein metabolism.
Conclusion: Our study has shown that miRNA profiling could prove as
a useful tool in understanding the molecular events and pathogenesis of
artherosclerosis, with a potential to serve as early biomarkers as well as
therapeutic targets for vascular diseases.
491 FACIAL WRINKLES, GREY HAIR AND BALDNESS PREDICT RISK OF
ISCHEMIC HEART DISEASE AND ISCHEMIC CEREBROVASCULAR
DISEASE INDEPENDENT OF CHRONOLOGICAL AGE
M. Christoffersen1 , R. Frikke-Schmidt1 , P. Schnohr2 , G.B. Jensen2,3 ,
B.G. Nordestgaard2,4 , A. Tybjærg-Hansen1,2 . 1 Dept. Clinical Biochemistry,
Rigshospitalet, Copenhagen University Hospital, 2 The Copenhagen City
Heart Study, Bispebjerg Hospital, Copenhagen University Hospital, 3 Dept.
Cardiology, Hvidovre Hospital, Copenhagen University Hospital, 4 Dept. Clinical
Biochemistry, Herlev Hospital, Copenhagen University Hospital, Copenhagen,
Denmark
Introduction: Cardiovascular disease is the most common cause of death,
and the primary risk factor for cardiovascular disease is age. Facial wrinkles,
greying of hair and baldness are common signs of aging, which might act as
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indicators of the biological age of an individual, and thus may be associated
with increased risk of cardiovascular disease.
Objective: We examined the hypothesis that presence of common aging signs
are associated with increased risk of ischemic heart disease (IHD), myocardial
infarction (MI), ischemic cerebrovascular disease (ICVD) and ischemic stroke
(IS) in the general population.
Methods: Facial wrinkles, greying of hair, and baldness were registered at
baseline in 10,928 individuals from the Danish general population. Of these,
3,360 developed IHD, 1,700 developed MI, 1,549 developed ICVD and 1,347
developed IS during up to 34 years of follow-up.
Results: Presence of common aging signs predicted hazard ratios of 1.21
(1.02–1.42) for IHD, 1.54 (1.22–1.94) for MI, 1.56 (1.23–2.00) for ICVD and
1.57 (1.20–2.04) for IS for individuals with 4 versus 0 common aging signs after
adjustment for chronological age and sex.
Conclusions: Common aging signs predict risk of IHD, MI, ICVD and IS in the
general population independent of chronological age. In societies where other
cardiovascular risk factors cannot be readily measured, presence of common
aging signs may therefore be useful in identifying individuals with increased risk
of cardiovascular disease.
492 FABP4 PREDICTS ATHEROGENIC DYSLIPIDEMIA DEVELOPMENT.
THE PREDIMED STUDY
A. Cabré1 , N. Babio2 , I. Lazaro1 , M. Bulló2 , A. Garcia-Arellano3 , L. Masana1 ,
J. Salas-Salvadó2 . 1 Unitat de Medicina Vascular i Metabolisme, Unitat de
Recerca de Lı́pids i Arteriosclerosi, Hospital Universitari Sant Joan, IISPV,
Universitat Rovira i Virgili, CIBERDEM, 2 Human Nutrition Unit, IISPV, Hospital
Universitari de Sant Joan, Universitat Rovira i Virgili, CIBER Fisiopatologia
de la Obesidad y Nutrición (CIBERObn), Instituto de Salud Carlos III, Reus,
3
Department of Preventive Medicine and Public Health, University of Navarra,
Pamplona, Spain
Aims: Atherogenic dyslipidemia (AD), characterized by high plasma triglycerides and low HDL particles, is considered one of the main effectors of vascular
damage associated with obesity, metabolic syndrome (MS) and type 2 diabetes
(T2D). The adipose Fatty Acid Binding Protein (FABP4) plasma concentrations
have been linked to metabolic alterations that are associated with adiposity,
such as MS, T2D and AD. The aim of this work was to prospectively study the
predictive value of baseline FABP4 plasma concentrations on the development
of AD.
Methods and Results: A nested study within the PREDIMED trial, a multicenter
dietary interventional project. Prospectively, we have analyzed the baseline
plasma FABP4 levels and the incidence of AD over a six-year follow-up
period (median 4 [IQR, 3−5 years]). We included 578 volunteers who visited
their general practitioners because of their cardiovascular risk factors. During
follow-up, 103 individuals developed AD. Baseline plasma FABP4 levels were
associated with new onset AD over the follow-up period. An increase of 1 unit
in baseline plasma FABP4 concentrations was associated with a 3% higher risk
of developing AD (OR 1.03 [95% IC, 1.01–1.05], P= 0.008). This increased risk
was observed in women but not in men. Among women, those in the highest
tertile of FABP4 had a 92% increased relative risk of developing AD compared
to the lowest tertile (HR 1.92 [95% CI, 1.03–3.58], P for trend = 0.039).
Conclusion: Elevated plasma FABP4 concentrations should be considered as
a marker of metabolic derangement, which may predict the development of AD
in women.
493 THE APPLICATION OF RESEQUENCING MICROARRAY IN THE
SCREENING OF FAMILIAL HYPERCHOLESTEROLEMIA
M.-J. Charng1 , K.-R. Chiou2 . 1 Medicine, Taipei Veterans General Hospital/
National Yang-Ming University, Taipei, 2 Medicine, Kaohsiung Veterans General
Hospital, Kaohsiung, Taiwan R.O.C.
Background: Familial hypercholesterolemia (FH) is a heterogeneous autosomal dominant disease with a prevalence of 1 in 500. To date, over 1200 unique
pathogenic mutations have been identified in at least 3 genes. The large allelic
and genetic heterogeneity of FH requires high-throughput, rapid, and affordable
mutation detection technology to efficiently integrate molecular screening into
clinical practice. We developed an array-based resequencing assay to facilitate
genetic testing in FH patients.
Methods and Results: We designed a custom DNA resequencing array
to detect mutations on all 3 FH-causing genes − LDL receptor (LDLR),
apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 gene
(PCSK9) − and 290 known insertion/deletion mutations on LDLR. We verified
FH array performance by analyzing 35 previously sequenced subjects (21 with
point mutations, 2 insertions, 7 deletions, and 5 healthy controls) and blindly
screening 125 FH patients. The average microarray call rate was 98.45% and
the agreement between microarray and capillary sequencing was 99.99%. The
FH array detected mutations by using automated software analysis, followed
by manual review in 28 of the 30 subjects (pickup rate, 93.3%). In the blinded
study, the FH array detected at least 1 mutation in 77.5% of patients clinically
diagnosed with definite FH according to Simon Broome FH criteria and in 52.9%
with probable FH diagnosis.
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Conclusions: The high-throughput FH resequencing array detects LDLR,
APOB, and PCSK9 with high efficiency and accuracy and identifies diseasecausing mutations cost-effectively. Thus, it facilitates large-scale screening of
the heterogeneous FH populations.
494 ASSOCIATION OF ANTIBODY AGAINST HEAT SHOCK PROTEIN-27
AND PRO-OXIDANT-ANTIOXIDANT BALANCE WITH THE SEVERITY
OF CORONARY ARTERY DISEASE AND METABOLIC SYNDROME
M. Ghayour-Mobarhan1 , H. Pourghadamyari2 , M. Moohebati2 ,
S.M.R. Parizadeh2 , S. Tavallaie2 , A. Sahebkar2 , A. Rahsepar2 , R. Paydar2 ,
G. Ferns3 . 1 Biochemistry and Nutrition Research Center & Cardiovascular
Research Center, Faculty of Medicine, Mashhad University of Medical
Sciences, 2 Mashhad University of Medical Sciences, Mashhad, Iran, 3 Institute
for Science and Technology in Medicine, University of Keele, Guy Hilton
Research Centre, Thornburrow Drive, Stoke on Trent, Staffordshire, UK
Objectives: Antibody titers to several heat shock proteins (anti-Hsps) and
oxidative stress have been reported to be associated with the severity and
progression of cardiovascular disease. However, there are little data regarding
anti-Hsp27 titers and pro-oxidant antioxidant balance (PAB) in patients with
coronary artery disease (CAD) and metabolic syndrome.
Methods: A total of 400 patients with suspected CAD were recruited. Based on
the results of coronary angiography, these patients were classified into CAD+
(n = 300) and CAD− (n = 100) groups defined as patients with 50% and <50%
stenosis of any major coronary artery, respectively. Eighty three healthy subjects
were also recruited as the control group.
Results: CAD+ patients had significantly higher anti-Hsp27 titers and PAB
compared to both CAD- and control groups. Anti-Hsp27 titers and PAB were
also higher in the CAD− group compared to the control group. With regard to the
number of affected vessels in the CAD+ group, patients with 3 vessel disease
(3VD) had higher anti-Hsp27 titers compared to both 2VD and 1VD subgroups,
PAB did not differ significantly among patients with different vascular disease
(SVD, 2VD and 3VD).
Furthermore, the CAD+ patients were divided into metabolic syndrome and nonmetabolic syndrome patients. Metabolic syndrome patients had significantly
higher anti-HSP27 titer than non metabolic syndrome patients (P = 0.021).
However, PAB did not differ significantly between them (P = 0.85).
Conclusions: Anti-Hsp27 titers may be associated with the presence and
severity of CAD and metabolic syndrome. However, PAB may be associated
with the present of CAD, but not for the severity of CAD.
495 ARTIFICIAL NEURAL NETWORK SYSTEM PREDICTS 6-YEAR
INCIDENCE OF METABOLIC SYNDROME USING SERUM MARKERS
FOR ATHEROSCLEROSIS
H. Hirose1,2 , T. Takayama2 , S. Hozawa2 , I. Saito1,2 . 1 Health Center, Keio
University, 2 Internal Medicine, Keio University School of Medicine, Tokyo,
Japan
Aims: Metabolic syndrome (MetS) is known as an important risk factor for
atherosclerosis and cardiovascular diseases. Although insulin resistance and
serum adiponectin level are closely related to the occurrence of MetS, it is
difficult to predict the onset of MetS in clinical practice. The aims of this study
were to predict the 6-year incidence of MetS using an artificial neural network
(ANN) system based serum markers for atherosclerosis, including the insulin
resistance index calculated by homeostasis model assessment (HOMA-IR),
HDL-cholesterol and adiponectin levels.
Methods: Subjects were recruited among the participants of annual health
check-ups in 2000 and 2006. A total of 410 Japanese male teachers and
workers at our Institute, aged 30 to 59 years at baseline, were participated
in this retrospective cohort study. Clinical parameters were randomly divided
into a training data set and a validation data set, and the ANN system and
multiple logistic regression (MLR) analysis were used to predict the individual
incidences.
Results: The sensitivity of the prediction was 0.27 for the MLR models and
0.93 for the ANN, while the specificity was 0.95 for the MLR and 0.91 for
the ANN. Sensitivity analysis of the ANN identified the BMI, age, diastolic
blood pressure, HDL-cholesterol, LDL-cholesterol, and HOMA-IR as important
predictive factors, suggesting that these factors are non-linearly related to the
outcome.
Conclusion: We successfully predicted the 6-year incidence of MetS using an
ANN system based on clinical data, including HOMA-IR, serum HDL-cholesterol
and adiponectin levels, in Japanese male subjects.
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496 MOLECULAR INSIGHTS INTO CARDIOMYOCYTE APOPTOSIS:
TREATMENT WITH PIOGLITAZONE IN LOW-DOSE
STREPTOZOTOCIN AND HIGH FAT DIET FED OBESITY IN MURINE
MODEL
U. Bhandari1 , V. Kumar1 , C.D. Tripathi2 , G. Khanna3 , C. Hemantkumar1 ,
K.K. Pillai1 . 1 Pharmacology, Hamdard University, 2 Pharmacology, 3 Pathology,
Vardhman Mahavir Medical College, Safdarjung Hospital, New Delhi, India
Cardiomyocyte apoptosis is one of the pathologic phenomenon associated with
obesity and related conditions including insulin resistance & hyperlipidemia.
Combination of low dose of streptozotocin (STZ) & high fat diet is an effective
model for screening of antiobesity activity in diabetic rats. Male Wistar rats (150–
200 g, body wt) were injected with a low-dose streptozotocin (STZ, 45 mg/kg,
i.v., single dose) and orally fed with a high fat diet (HFD, 20 gm/d/rat) for
a period of 28 days. Pioglitazone (20 mg/kg/p.o.) treatment along with HFD
for a period of 21 days (from 8th day to 28th day) in diabetic rats resulted
in decrease in cardiomyocyte apoptosis as revealed by decrease in cardiac
caspase-3 & LDH levels & DNA fragmentation (decrease in DNA laddering was
observed by Agarose gel electrophoresis) & increase in Na+ K+ ATPase levels.
Further, cardiac sections (stained with haematoxylin/eosin) of obese diabetic
rats showed dense focal fatty infiltration in the myocardial cells whereas normal
architecture with regular morphology & well preserved cytoplasm was observed
with pioglitazone treatment. In addition, treatment with pioglitazone significantly
(p < 0.01) reduced the heart rate, mean arterial blood pressure, BMI and levels
of serum leptin, insulin, total cholesterol & triglycerides, apolipoprotein B and
glucose; atherogenic index and increased the levels of serum HDL-C and
cardiac antioxidant enzymes as compared to the obese diabetic control rats.
The present study suggests for the first time that the pioglitazone possesses
antiobesity & cardiac antiapoptotic potential against the HFD-induced obesity &
hyperlipidemia in diabetic rats.
497 DEVELOPMENT OF A NOVEL HOMOGENEOUS ASSAY FOR SMALL
HDL
M. Higuchi1 , T. Hirano2 , Y. Ito1 . 1 Research and Development Department,
Denka Seiken Co., Ltd., Niigata, 2 Department of Diabates, Metabolism and
Endocrinoligy, Showa University School of Medicine, Tokyo, Japan
Background and Aim: Some studies have shown that different HDL
subclasses play different roles in progression and regression of arteriosclerosis.
The aim of this study was to develop a fully automated assay for quantification of
small HDL cholesterol with easier techniques than currently available methods.
Materials: TG rich lipoprotein (TRL), and LDL and 26 HDL subfractions were
isolated by ultracentrifugation. Each fraction was used for screening of enzymes
and surfactants to specifically measure large HDL.
Results: We found that sphingomyelinase reacts with lipoproteins except HDL,
and phospholipase C and D preferentially react with large HDL lipoproteins than
small HDL. By applying such enzymes, we constructed an automated assay for
small HDL consisting of the following reaction steps: Sphingomyelinase and
Phospholipase in the 1st reagent react with non-small HDL lipoproteins to
degrade the cholesterol from such non-small HDL to water and oxygen under
the presence of cholesterol oxidase, cholesterol esterase and catalase. 2nd
reagent reacts with remaining small HDL to measure the small HDL cholesterol
in the standard cholesterol oxidase and peroxidase system with surfactant. The
reactivity with fraction No. 7 (large HDL) and No. 20 (small HDL) were 17.3%
and 86.1%, respectively, proving that the assay specifically measures small
HDL. The reactivity against the other lipoproteins such as TRL and LDL were
negligible.
Conclusion: Our new assay enables the measurement of small HDL in a fully
automated manner and can be useful for research on the clinical significance
of HDL subfractions.
498 ROLE OF MITOCHONDRIAL DNA DAMAGE IN ATHEROSCLEROSIS
J. Mercer, L. Hurst, M. Bennett. Medicine, University of Cambridge,
Cambridge, UK
Introduction: Atherosclerotic plaques are associated with nuclear and
mitochondrial DNA damage which lead to cell cycle arrest, senescence
and apoptosis; all combine to produce plaque destabilisation and increased
vulnerability leading to clinical events. The common mitochondrial deletion
(D4977bp) has been documented as a sign of plaque instability in murine
models of atherosclerosis and metabolic syndrome.
Aim and Objective: Our study compared the relative abundance of nuclear
and mtDNA damage between normal and diseased human atherosclerosis
samples that may represent loss of function, loss of viability and increased
plaque vulnerability.
Methods: Using quantitative qPCR, normal human arterial and atherosclerotic
diseased tissue were used to calculate the abundance of D4977bp.
Immunocytochemical staining for DNA repair foci and the COMET assay were
used to estimate DNA repair activity and capacity in VSMC lines of both young
and old individuals to examine the presence and extent of DNA damage.
Results: Data from qPCR revealed a greater abundance of D4977bp present
in the plaque-derived DNA (p= < 0.04). Moreover, sequencing data uncovered a
novel 5031bp deletion variant. DNA damage assays revealed VSMCs from aged
individuals possessed greater levels (p= < 0.0001) of basal damage compared
to young isolates, indicating temporal accumulation of mis-repaired DNA.
Conclusion: This novel variant may be a specific biomarker in atherogenesis.
The higher levels of nuclear DNA damage witnessed in older cell isolates
confirms compromised repair capacity, presumably through higher levels of
oxidative stress leading to senescence. Future work will focus on mitochondria
respiratory complex activity, ROS&ATP production correlated to mtDNA damage
phenotype.
499 CIRCULATING CD4+CD25+FOXP3+ REGULATORY T CELLS DO
NOT PREDICT RISK FOR ACUTE CARDIOVASCULAR EVENTS
M. Wigren1 , H. Björkbacka1 , B. Hedblad1 , G.N. Fredrikson1,2 , J. Nilsson1 .
1
Clinical Sciences Malmö, Lund University, Skane University Hospital, 2 Faculty
of Health and Society, Malmö University, Malmö, Sweden
Regulatory T cells (Tregs) have been shown to protect against atherosclerosis
in experimental models but their association with cardiovascular disease in
man remains to be elucidated. To determine if the level of Tregs in the
circulation reflects the severity of atherosclerosis and predict the development
of acute cardiovascular events in man. The study included 350 subjects
with incident acute cardiovascular events (coronary heart disease or stroke)
and 350 matched controls recruited from the Malmö Diet and Cancer study.
Mononuclear leukocytes stored at −140ºC at the baseline investigation in
1991−94 were thawed and Tregs analyzed by flow cytometry. Release of
IL-10 from activated leukocytes was measured by Mesoscale. There was no
difference in Tregs (defined as CD4+CD25+FoxP3+ or CD4+CD25+ cells) or
IL-10 release between cases and controls. There was also no association
between Tregs and severity of atherosclerosis as assessed by the carotid
intima-media thickness at baseline. Our observations show that circulating
Tregs, defined as CD4+CD25+FoxP3+ or CD4+CD25+ cells, do not reflect
the severity of atherosclerosis and do not predict acute cardiovascular events.
However, it should be emphasized that these findings do not exclude a role for
Tregs in human atherosclerosis.
500 INFLAMMATION-SENSITIVE PROTEINS AND RISK OF ATRIAL
FIBRILLATION: A POPULATION-BASED COHORT STUDY
S. Adamsson Eryd1 , J.G. Smith2 , O. Melander1 , G. Engström1 , B. Hedblad1 .
1
Department of Clinical Sciences, Lund University, Malmö, 2 Department of
Cardiology, Lund University, Lund, Sweden
Background and Aims: Low-grade inflammation has repeatedly has been
associated with cardiovascular diseases but the relationship with incidence of
atrial fibrillation (AF) remains unclear. We explored the association between
elevated plasma levels of inflammation-sensitive proteins (ISPs) and incidence
of AF in a population-based cohort.
Methods: Plasma levels of five ISPs (fibrinogen, haptoglobin, ceruloplasmin,
a1 -antitrypsin and orosomucoid) and two complement factors (C3 and C4) were
measured in 6031 men (mean age 46.7 years) without history of myocardial
infarction, heart failure, stroke or cancer. Incidence of hospitalizations due to
AF during a mean follow-up of 25 years was studied both in relation to individual
inflammatory proteins and the number of elevated ISPs.
Results: During follow-up, 667 patients were hospitalized with a diagnosis of
AF. After adjustment for potential confounding factors, the hazard ratios for AF
were 1.00 (reference), 1.08 (95% CI: 0.88–1.31), 1.07 (CI: 0.84–1.36), 1.40 (CI:
1.12–1.74), respectively, in men with none, one, two and three or more ISPs in
the 4th quartile (p for trend = 0.007). Ceruloplasmin was the only individual ISP
significantly associated with incidence of AF after adjustment for confounding
factors (HR 1.17 per standard deviation, 95% CI: 1.08–1.26).
Conclusion: A score of five ISPs was associated with long-term incidence
of hospitalizations due to AF in middle-aged men. Of the individual ISPs, a
significant association was observed for ceruloplasmin, a protein previously
associated with copper metabolism and oxidative stress.
501 DECREASED PLASMA LEVELS OF SRAGE ARE ASSOCIATED
TO CORONARY FIBRO-LIPIDIC WALL VOLUME IN PATIENTS WITH
PROVEN OR SUSPECTED CAD
G. Basta1 , S. Del Turco1 , T. Navarra1 , A. Mazzarisi1 , F. Cocci2 , M. Coceani2 ,
M. Bianchi2 , M. Schlueter2 , P. Marraccini1 . 1 Consiglio Nazionale delle
Ricerche, Institute of Clinical Physiology, 2 Fondazione G. Monasterio
CNR-Regione Toscana, Pisa, Italy
Objective: Low levels of soluble receptor for advanced glycation end-products
(sRAGE) were reported to be associated with coronary artery disease (CAD).
This study explored the relationship between circulating levels of sRAGE with
the characteristics of coronary plaques using 64-slice computed tomography
angiography (CTA).
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Methods and Results: The study population consisted of 127 consecutive
patients with proven or suspected CAD, but without acute coronary syndrome,
who underwent 64-slice CTA. CTA was used to assess
1. coronary calcification;
2. stenosis severity;
3. non-calcified plaque volume;
4. plaque composition.
Sixty-eighth patients (age 59.9±12.5 years; 50% men) exhibited non-calcified
plaques while 59 patients (age 65.8±10.4 years; 80% men) had calcified
plaques and prevalence of stenotic plaques.
Specification of plaque morphology revealed a significant inverse association
between sRAGE levels and percent fibro-lipidic wall volume and a direct
association between sRAGE and percent luminal volume in the patients
with non-calcified plaques (r = −0.254, p = 0.0444 and r = 0.296, p = 0.014
respectively) but not in the patients with calcified plaques (r = −0.064, p = 0.637
and r = −0.101, p = 0.450 respectively).
In a fully adjusted multivariate model sRAGE levels remained predictive of
percent fibro-lipidic wall volume (b = −0.292, p = 0.0271).
Conclusions and Significance: Decreased sRAGE levels were significantly
associated with fibro-lipidic wall burden identified by CTA. Low levels of sRAGE
can help identify those patients with subclinical atherosclerosis who are at risk
for near-term atherothrombotic events.
502 ASSOCIATION OF BLOOD ACTIVE MATRIX
METALLOPROTEINASE-3 WITH CAROTID PLAQUE SCORE IN
MALE FROM A COMMUNITY POPULATION IN TAIWAN
L.-M. Lien1 , Y.-C. Hsieh2 , C.-H. Bai2 , W.-H. Chen1 , H.-C. Chiu1 , F.-I. Hsieh2 ,
K.-G. Shyu3 , H.-Y. Chiou2 . 1 Department of Neurology, Shin Kong Wu Ho-Su
Memorial Hospital, 2 School of Public Health, Taipei Medical University,
3
Department of Cardiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei,
Taiwan R.O.C.
Objective: Blood levels of active matrix metalloproteinases-3 (MMP-3) are
reported to be associated with the extent of carotid atherosclerosis based on
carotid plaque score (PS) in a community population recently. However, the
blood level of total MMP-3 is usually lower in female as compared with male. In
this study, we assessed blood levels of active MMP-3 in relation to PS in male
and female.
Methods: In 433 subjects from a community primary stroke prevention program,
blood levels of active MMP-3 and total MMP-3 were determined using enzymelinked immunosorbent assays and carotid plaque score (PS) by high-resolution
B-mode ultrasonography.
Results: The blood levels of active MMP-3 (8.4±4.1ng/ml vs. 4.0±2.4ng/ml,
p < 0.0001) and total MMP-3 (13.1±5.6ng/ml vs. 6.6±3.3ng/ml, p < 0.0001) are
both higher in male than in female, respectively. Study subjects were separated
into 3 groups based on PS: group 1 (PS = 0), group 2 (PS = 1−2) and group 3
(PS 3). Blood levels of active MMP-3, but not total MMP-3, bear a highly
significant relationship with PS in male, but not in female, (active MMP-3 level
in group 1: 7.5±3.2ng/ml, group 2: 8.4±3.9ng/ml, and group 3: 10.7±5.5ng/ml;
p for trend = 0.0004). A stepwise logistic regression analysis after adjustment of
potential covariates including age, hypertension and cigarette smoking revealed
blood levels of active MMP-3 are correlated with PS (OR, 1.5; 95% CI, 1.1 to
2.0; p = 0.006) in male.
Conclusion: Blood levels of active MMP-3 are associated with the extent of
carotid atherosclerosis in this community population in male but not in female
in Taiwan.
503 ASSOCIATION OF COPY NUMBER VARIATIONS AND SINGLE
NUCLEOTIDE POLYMORPHISMS IN METALLOTHIONEIN GENES
WITH PATHOGENESIS OF DIABETES AND CORONARY ARTERY
DISEASE
R. Kozarova1 , A. Postadzhiyan2 , B. Finkov2 , M. Apostolova1 . 1 Institute of
Molecular Biology, Bulgarian Academy of Sciences, 2 Clinic of Cardiology, St.
Anna Hospital, Sofia, Bulgaria
Introduction: Copy number variations (CNVs) and single nucleotide
polymorphisms (SNPs) are widely distributed in the human genome. SNPs have
allele frequency of 1%, while CNVs involve DNA fragment which is >1 kb.
Methods: QPCR was used to detect the MT1X gene variations. SNPs
+1245A/G in MT1A and −209A/G in MT2A gene were analyzed. Molecular
analyses of CNVs and SNPs were done in 142 patients with coronary artery
disease (CAD), 101 diabetic patients without clinical evidence for cardiovascular
disease and 117 aged and sex matched controls.
Results: 334 different CNVs were detected using 105K array CGH (Agilent
Technologies). QPCR analyses for MT1X gene copy number variations
confirmed the data from CGH array and showed significant association with
developing of CAD (c2 = 21.036; OR = 4.787; 95% CI = 2.382–9.620; p < 0.001)
and diabetes (c2 = 21.632; OR = 5.448; 95% CI = 2.565–11.570; p < 0.001).
The polymorphism +1245A/G MT1A was significant associated with CAD
(c2 = 9.492; p = 0.002). A binary logistic regression showed that +1245 AA
107
genotype (comparing to AG + GG-genotypes) was independent predictor only
for CAD after adjustment for the conventional risk factors (adjusted OR = 6.712,
95% CI = 1.818–24.775; p = 0.004). The polymorphism −209 A/G MT2A showed
significant association of −209 AG+GG genotypes compared to −209 AA
genotypes (c2 = 3.825; OR = 4.130; 95% CI = 0.904–18.862; p = 0.050) only in
patients with diabetes.
Conclusion: Changes in copy numbers of MT1X gene possibly contribute to
developing of CAD and Diabetes mellitus. SNP analyses showed that +1245
AA genotype is independently associated with an increased risk of CAD after
adjusting for classical risk factors where the −209 AG+GG MT2A genotype is
predominantly associated with diabetes.
504 SMALL ARTERIES DILATION AND ENDOTHELIAL MARKERS IN
CARDIOVASCULAR RISK PATIENTS
G. Aragonès, R. Ferré, J. Girona, N. Plana, J. Merino, M. Heras, L. Masana.
Vascular Medicine and Metabolism Unit, Research Unit on Lipids and
Atherosclerosis, Universitat Rovira i Virgili, Internal Medicine Department, Sant
Joan University Hospital, IISPV. CIBER of Diabetes and Associated Metabolic
Diseases (CIBERDEM), Reus, Spain
Background: The use of methods based on reactive hyperaemia of small
distal arteries to assess endothelial function (EF) is increasing at the clinical
level; however, the mechanisms regulating vascular function in large and
small arteries are different and still not completely understood. We studied
the correlations between hyperaemia reactivity of small peripheral arteries
determined by peripheral artery tonometry (PAT) and the levels of serum
biomarkers of EF, inflammation and oxidation in patients with cardiovascular
(CV) risk factors.
Methods: 407 patients without previous CV disease but at intermediate CV
risk were recruited into a cross-sectional study to examine whether soluble
endothelial, inflammatory and lipid oxidative biomarkers correlate with small
arteries reactive hyperaemia index (saRHI) values, which were measured by
PAT.
Results: A significant correlation was found between saRHI values and the
concentrations of soluble E-selectin (sE-selectin) (r = −0.184, P < 0.0001) and
soluble VCAM-1 (sVCAM-1) (r = −0.119, P = 0.018). The correlation remained
significant after adjustment for confounding variables. Patients with a lower
saRHI (saRHI < 1.70) had higher concentrations of sE-selectin, sVCAM-1
and oxLDL/LDL (P < 0.0001, P = 0.023 and P = 0.048, respectively). Using a
stepwise multivariable linear regression model, we found that sE-selectin was
the only biomarker that significantly correlated with saRHI values (P < 0.0001).
Conclusion: Elevated levels of sE-selectin, sVCAM-1 and oxLDL/LDL are
associated with lower postischemic reactivity in the small distal arteries, which
suggests that similar mechanisms are involved in the vascular function of large
and small arteries. Therefore, methods based on the saRHI could be useful
tools to assess EF at the clinical level.
505 THE RELATIONSHIP BETWEEN HDL-CHOLESTEROL AND HDL
FUNCTION
L. Wade1 , M. Widdowson2 , A. Mcgown2 , J. Gibney2 , F. Thies3 , A. Mcginty1 ,
I. Young1 , J. McEneny1 . 1 Centre for Public Health, Queen’s University Belfast,
Belfast, UK , 2 Endocrinology and Diabetes, Adelaide and Meath Hospital,
Dublin, Ireland, 3 School of Medicine and Dentistry, Aberdeen University,
Aberdeen, UK
Background: Epidemiological evidence has revealed that HDL protects against
cardiovascular disease (CVD). However, CVD can be present in individuals with
normal or even high HDL-cholesterol, suggesting that HDL-cholesterol levels
may not predict its functionality.
Objectives: To assess if HDL-cholesterol levels can predict the activity of HDL
associated enzymes, and therefore, predict HDL function. Furthermore, the role
of obesity and the adipocyte derived inflammatory molecule serum amyloid-A
(SAA) on HDL function were also assessed.
Design: Serum was collected from 118 overweight control subjects (48 males,
70 females, (mean±SEM) 50±0.07years: HDL-cholesterol, 1.54±0.00 mmol/l:
BMI, 27.4±0.04 Kg/m2 ). HDL was subfractionated into HDL2&3 by rapid
ultracentrifugation and the activity of paraoxonase-1 (PON-1; spectrophotometrically) and lecithin cholesteryl acyl transferase (LCAT) & cholesterol-ester
transfer protein (CETP; fluorometrically) were assessed within HDL2&3 . SAA
was measured within serum and HDL2&3 by an ELISA method.
Results: As serum HDL-cholesterol levels increased there was a concomitant
increase in PON-1 activity within HDL2&3 (HDL2 r = 0.290, p = 0.002; HDL3
r = 0.301, p = 0.001) and a decrease in CETP activity within HDL2 (r = −0.343,
p = 0.000). Additionally, as BMI increased, serum HDL-cholesterol (r = −0.362,
p = 0.000), HDL2 -protein (r = −0.199, p = 0.036) and HDL2&3 PON-1 activity
(r = −0.222, p = 0.019; r = −0.263, p = 0.005, respectively) decreased, while
HDL2 CETP activity increased (r = 0.215, p = 0.023). BMI positively correlated
with serum-SAA (r = 0.270, p = 0.005) and SAA-associated with HDL2&3 (HDL2
r = 0.334, p = 0.002; HDL3 r = 0.240, p = 0.012).
108
Discussion: These results demonstrate that HDL-cholesterol is weakly associated with HDL function, suggesting that complete compositional/functional
analyses may be required to fully establish HDL’s anti-atherogenic properties.
506 BIOMARKERS OF INFLAMMATION AND OXIDATIVE STRESS
INDEPENTLY PREDICT THE PROGRESSION OF SUBCLINICAL
ATHEROSCLEROSIS
D.J. Mulder1 , J.D. Lefrandt1 , E. Tremoli2,3 , D. Baldassarre2,3 , A.J. Smit1 , on
behalf of the IMPROVE Study Group. 1 Department of Internal Medicine,
University Medical Center Groningen, Groningen, The Netherlands,
2
Department of Pharmacological Sciences, University of Milan, 3 Monzino
Cardiology Center, IRCCS, Milan, Italy
Objectives: Prediction of cardiovascular events by inflammation and oxidative
stress biomarkers reflects their pivotal role in development of atherosclerosis.
However, it is unclear whether such biomarkers are associated with an
accelerated progressi

Ankle-brachial index as a marker of cognitive impairment and

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