Settore Scientifico-disciplinare: Biologia Molecolare (Bio/11)
Role of Unconjugated Bilirubin
in the Endothelial Dysfunction
Graciela Luján Mazzone
Coordinatore del Collegio Docenti:
Prof. Giannino Del Sal
Università degli Studi di Trieste
Prof. Claudio Tiribelli
Università degli Studi di Trieste
Dott. Igino Rigato
Università degli Studi di Trieste
Prof. Claudio Tiribelli
Università degli Studi di Trieste
Dr. Igino Rigato
Università degli Studi di Trieste
External Supervisor:
Dr. Helena Schteingart
Centro de Investigaciones Endocrinológicas - CONICET - Argentina
Thesis Committee:
Prof. Francesco Tedesco
Università degli Studi di Trieste
Prof. Stefano Gustincich
Scuola Internazionale Superiore di Studi Avanzati di Trieste
Prof. Massimo Levrero
Università degli Studi di Roma - La Sapienza
Prof. Franco Vittur
Università degli Studi di Trieste
Prof. Silvia Giordano
Università degli Studi di Torino
Dr. Claudio Brocolini
Università degli Studi di Udine
Prof. Giannino Del Sal
Università degli Studi di Trieste
Prof. Renato Gennaro
Università degli Studi di Trieste
This study was supported by a fellowship from the Italian Ministry of Foreign
Affairs (MAE) in Rome, Italy. In particular, I wish to thank Dr. Paola Ranocchia.
Bilirubin metabolism . . . . . . . . . . . . . . . . . . . . . . . .
Bilirubin and pathophysiology . . . . . . . . . . . . . . . . . . .
Vascular atherosclerosis . . . . . . . . . . . . . . . . . . . . . . .
Morphology of atherosclerotic lesions . . . . . . . . . . .
Atherogenesis - Response to the injury theory . . . . . . .
Endothelium . . . . . . . . . . . . . . . . . . . . . . . .
Pro-inflammatory cytokines . . . . . . . . . . . . . . . . . . . . .
Nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biosynthesis of nitric oxide . . . . . . . . . . . . . . . .
Endothelial Nitric Oxide Synthase (eNOS) . . . . . . . .
Inducible Nitric Oxide Synthase (iNOS) . . . . . . . . . .
Nitric oxide and pathophysiology . . . . . . . . . . . . .
Adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . .
The Selectins . . . . . . . . . . . . . . . . . . . . . . . .
Immunoglobulin superfamily adhesion molecules . . . . .
Signal transduction pathways . . . . . . . . . . . . . . . . . . . .
cAMP-response element(CRE)-binding protein (CREB) .
NF-κB . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aim of the Study
Materials and Methods
Endothelial Cells . . . . . . . . . . . . . . . . . . . . . . . . . .
Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
UCB solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . .
Cytokines treatment . . . . . . . . . . . . . . . . . . . .
Endothelial cell susceptibility . . . . . . . . . . . . . . . . . . . .
LDH release test . . . . . . . . . . . . . . . . . . . . . .
Mitochondrial toxicity by MTT test . . . . . . . . . . . .
Endothelial dysfunction analysis . . . . . . . . . . . . . . . . . .
Nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . .
Gene expression analysis . . . . . . . . . . . . . . . . . .
Western blot . . . . . . . . . . . . . . . . . . . . . . . .
Signal transduction pathways . . . . . . . . . . . . . . . . . . . .
cAMP-response element(CRE)-binding protein (CREB) .
Preparation of total nuclear extracts . . . . . . . . . . . .
Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of UCB on cell viability . . . . . . . . . . . . . . . . . .
UCB did not affect the LDH release induced by TNF-α
UCB reduced endothelial cell viability
. . . . . . . . . .
Nitric oxide analysis . . . . . . . . . . . . . . . . . . . . . . . .
Effect of UCB on NO levels in H5 V cells . . . . . . . . .
Effect of UCB on NOS mRNA expression . . . . . . . . .
NO levels in HUVEC cells . . . . . . . . . . . . . . . . .
UCB, the redox status and NO levels . . . . . . . . . . .
UCB reduced AM expression induced by TNF-α . . . . . . . . .
H5 V cells - mRNA relative expression . . . . . . . . . . .
HUVEC cells - mRNA relative expression . . . . . . . . .
AM protein expression . . . . . . . . . . . . . . . . . . . . . . .
UCB effects via NF-κB pathway . . . . . . . . . . . . . . . . . .
UCB and PDTC inhibit gene over-expression in an addictive pattern . . . . . . . . . . . . . . . . . . . . . . . . .
CREB phosphorylation is not influenced by UCB . . . . .
NF-κB nuclear translocation is inhibited by UCB . . . . .
Viability and UCB . . . . . . . . . . . . . . . . . . . . . . . . . 104
Nitric oxide and UCB . . . . . . . . . . . . . . . . . . . . . . . . 108
Adhesion molecules and UCB . . . . . . . . . . . . . . . . . . . 113
Signalling pathways and UCB . . . . . . . . . . . . . . . . . . . 114
List of Figures
Bilirubin metabolism . . . . . . . . . . . . . . . . . . . . . . . .
Hepatic heme metabolism of bilirubin . . . . . . . . . . . . . . .
Stages of atherosclerotic lesions . . . . . . . . . . . . . . . . . .
Leucocytes attachment, rolling and migration through endothelial
cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stepwise NO synthesis by NOS . . . . . . . . . . . . . . . . . .
Schematic Presentation of Nitric Oxide isoforms structure . . . .
Steps in the inflammatory process . . . . . . . . . . . . . . . . .
Structure of the Selectin domains . . . . . . . . . . . . . . . . . .
Structure of ICAM-1 domains . . . . . . . . . . . . . . . . . . .
1.10 Structure of VCAM-1 different isoforms domanins . . . . . . . .
1.11 Activation of the cAMP-CREB signalling pathway . . . . . . . .
1.12 Schematic Presentation of NF-κB and IκB Structure . . . . . . . .
Morphology of H5 V cells in vitro . . . . . . . . . . . . . . . . . .
Morphology of HUVEC cells in vitro . . . . . . . . . . . . . . .
Relationship of Bf-UCB with different albumin preparations . . .
Chemistry of the Griess Reagent . . . . . . . . . . . . . . . . . .
Effect of UCB on cell viability - MTT assay . . . . . . . . . . . .
Effect of different doses UCB on NO production . . . . . . . . .
TNF-α induces iNOS gene expression in H5 V cells . . . . . . . .
Effect of UBC on TNF-α-induced iNOS gene - H5 V cells . . . . .
NAC reverted TNF-α effects iNOS gene . . . . . . . . . . . . . .
Effect of NAC on NO production
. . . . . . . . . . . . . . . . .
UBC and NAC reverted TNF-α induction of iNOS gene . . . . . .
TNF-α induces AM gene expression in H5 V cells . . . . . . . . .
Effect of UBC on TNF-α-induced E-selectin gene - H5 V cells . .
4.10 Effect of UBC on TNF-α-induced Vcam-1 gene - H5 V cells . . .
4.11 Effect of UBC on TNF-α-induced Icam-1 gene - H5 V cells . . . .
4.12 Effect of UBC on TNF-α-induced E-selectin gene - HUVEC cells
4.13 Effect of UBC on TNF-α-induced VCAM-1 gene - HUVEC cells
4.14 Effect of UBC on TNF-α-induced ICAM-1 gene - HUVEC cells .
4.15 TNF-α induces E-selectin protein expression in H5 V cells . . . .
4.16 TNF-α induces Vcam-1 protein expression in H5 V cells . . . . . .
4.17 TNF-α induces Icam-1 protein expression in H5 V cells . . . . . .
4.18 Effect of UBC on TNF-α-induced AM protein expression - H5 V
cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.19 PDTC inhibits AM and iNOS mRNA over-expression by TNF-α .
4.20 UCB and PDTC inhibit additively the gene over-expression . . . .
4.21 Induction of CREB phosphorylation by TNF-α in H5 V cells . . .
4.22 UBC does not affect CREB phosphorylation in H5 V cells . . . . . 100
4.23 UCB inhibits TNF-α-induced nuclear translocation of NF-κB . . . 101
List of Tables
NF-κB inhibitors that demonstrate anti inflammatory activity in
experimental models . . . . . . . . . . . . . . . . . . . . . . . .
H5 V - Primer sequence designed for the mRNA quantification . .
HUVEC - Primer sequence designed for the mRNA quantification
Primary antibodies tested . . . . . . . . . . . . . . . . . . . . . .
Effect of UCB on cell viability - LDH release . . . . . . . . . . .
Effect of different doses of TNF-α on NO production . . . . . . .
Time dependent effect of TNF-α on NO production . . . . . . . .
Threshold cycle values of eNOS in HUVEC cells . . . . . . . . .
Threshold cycle values of genes studied in H5 V control cells . . .
Atherosclerosis, a progressive cardiovascular disease, is characterized by the accumulation of cholesterol in macrophage deposits (foam cells) and the formation
of atherosclerotic plaques in the walls large- and medium- sized arteries. The
earliest events in the development of atherosclerosis involve progressive modifications in the endothelial micro-environment. This endothelial dysfunction is
a complex of multi-step mechanisms, for which reduced NO levels have been
reported as a marker, is characterized by increasing expression of adhesion molecules (AMs), which mediate the diapedesis (migration) of inflammatory and immunocompetent cells through the endothelial layer into the arterial wall.
NO is synthesized intracellularly by nitric oxide enzymes (eNOS and iNOS)
and is regulated by a variety of stimuli. NO acts as an autocrine or paracrine
hormone, as well as intracellular messenger, with a critical role in vascular endothelial growth factor-induced angiogenesis and vascular hyper-permeability in
The over-expression of AMs is orchestrated by pro-inflammatory cytokines,
particularly TNF-α. The two major subsets of AMs participating in these processes are the selectins, in particular E-selectin, and the immunoglobulin gene
superfamily, in particular vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1).
Transcriptional regulation of these inflammatory genes requires the participation of several proteins, inducible activators, as: NF-κB and (CRE)-binding
protein (CREB). The most abundant form of NF-κB is an heterodimer of p50 and
p65; which is sequestered in the cytoplasm in an inactive form through interaction with the IκB inhibitor proteins. Signals that induce NF-κB release dimers
to enter to the nucleus and induce gene expression. Pyrridoline dithiocarbamate
(PDTC) a metal-chelating compound inhibits NF-κB by blocking ubiquitine ligase activity towards phosphorylated IκB, in turn downregulating the expression
of E-selectin, VCAM-1 and ICAM-1. CREB is a widely expressed DNA-binding
protein, downstream target of cAMP, activated by phosphorylation on serine 133.
A regulatory site, on the gene promoters of both E-selectin and VCAM-1, binds
both NF-κB and CREB transcription factors.
Unconjugated bilirubin (UCB), long considered to be simply a waste end product of heme metabolism and a marker for hepatobiliary disorders, is now thought
to function as an endogenous tissue protector by attenuating free radical-mediated
damage to both lipids and proteins. There is increasing epidemiological evidence
supporting an inverse association between cardiovascular disease and plasma levels of bilirubin. Recent studies indicated that bilirubin may be protective in the
development of atherosclerotic diseases by inhibiting the proliferation of vascular
smooth muscle cells by mechanisms yet to be established. It has been proposed
that UCB can interfere with the atherosclerotic disease development by inhibiting
the trans-endothelial vascular cell adhesion molecule (VCAM-1)-dependent migration of monocytes into the intima. The aim of this study is to investigate the
effect of the UCB in the endothelial dysfunction. Specifically UCB effects on NO
production, AMs expression and the regulatory transcription factors involve in the
inflammatory response.
Variable doses of free bilirubin (Bf) (the active form of UCB in plasma), simulating upper normal (15 nM) and modestly elevated levels (30 nM) for plasma,
were evaluated in two models of endothelial cells. A) H5 V, murine microvascular
endothelial cell line, and B) HUVEC (Human Umbilical Vein Endothelial Cells),
isolated from the vein of human umbilical cord. TNF-α (20 ng/mL) was added in
order to reproduce, in vitro, the endothelial dysfunction and describe UCB contribution on its effects.
UCB alone reduced the viability in H5 V cells by MTT assay in a dose dependent manner after 24 hours while no effect was observed in the LDH released.
In the first set of experiments NO production in H5 V cells was evaluated, a
time-depended increase on NO basal and a dose-dependent decrease on NO concentration after TNF-α (20 ng/mL) were observed. NO reduction related TNF-α
was seen at all times studied. The effect of UCB was studied in co-treatments
with TNF-α for 24 and 48 hours. UCB (Bf 15 and 30 nM) significantly reversed
the reduction of nitrite content induced by TNF-α at 48 hours.
The gene expression analysis was performed by Real Time PCR technology
with specific primers for eNOS, iNOS, E-selectin, VCAM-1 and ICAM-1. In H5 V
cells, TNF-α increased the expression of all the genes studied (except eNOS) at
2, 6 and 24 hours. The co-treatment with UCB , at a Bf that did not themselves
affect the expression of the three adhesion molecules, blunts the over-expression
of E-selectin, Vcam-1 and iNOS induced by a pro-inflammatory cytokine such as
TNF-α. The inhibitory effect of UCB was usually modest (20-30%) and detected
at 2 and/or 6 hours, but had disappeared 24 hours. Furthermore, a synergistic effect between TNF-α and UCB was seen on the expression of iNOS at 24 hours,
indicating a biphasic regulation. Moreover, no effects were seen on eNOS. Similar results were observed in the regulation of the gene expression of the AMs and
viability in HUVEC cells, indicating the lack of species specific effect. However,
no effect of TNF-α or UCB was seen in the expression of iNOS, eNOS or NO
Western blot analysis in H5 V cells confirmed that TNF-α induced the expression of E-selectin, VCAM-1 and ICAM-1 in a time-dependent manner. This effect
was blunted after 24 hours by the presence of UCB (Bf 15 and 30 nM).
The contribution of NF-κB pathway in UCB effects was investigated by addition of a specific inhibitor, PDTC. The co-treatment with PDTC and UCB for
2 hours produced an additive reduction of TNF-α effect on E-selectin, VCAM-1,
and iNOS in H5 V cells. In addition, UCB prevented the nuclear translocation of
NF-κB induced by TNF-α. Failure of UCB to alter TNF-α-induced phosphorylation of CREB (at Ser 133) suggested that the CREB pathway was not involved in
the UCB inhibition.
The results obtained in the present study shows that unconjugated bilirubin,
even at upper-normal physiological (15 nM) and mildly elevated (30 nM) Bf can
modulate gene expression and endothelial cell function. Furthermore, UCB may
regulate NO levels by a bi-phasic regulation of iNOS, and in addition influences
the expression of the endothelial adhesion molecules. In summary, these data indicates that bilirubin limits the over-expression of the adhesion molecules and regulates the NO metabolism in the pro-inflammatory state induced by the cytokine
TNF-α. Even though UCB alone does not alter these markers. UCB effects are
mediated in part by a modulation of the NF-κB transcription factor. These results
support the concept that modestly elevated concentrations of bilirubin may help
prevent atherosclerotic disease as suggested by epidemiological studies.
List of Publications relevant to the Thesis
• Multidrug resistance associated protein 1 protects against
bilirubin-induced cytotoxicity. Calligaris S, Cekic D, Roca-Burgos L,
Gerin F, Mazzone G, Ostrow JD, Tiribelli C.FEBS Lett. 2006 Feb 20;
580(5): 1355-9. Epub 2006 Jan 26.
• Unconjugated bilirubin prevents the TNF-α related induction of three
endothelial adhesion molecules via the NF-κB pathway. Mazzone G,
Rigato I, Ostrow DJ, Bortoluzzi A and Tiribelli C. Submitted.
List of other Publications
• FSH and bFGF stimulate the production of glutathione in cultured rat
Sertoli cells. Gualtieri AF, Mazzone GL, Rey RA, Schteingart HF.Int J
Androl. 2007 Nov 26; [Epub ahead of print.]
List of abbreviations
Adhesion Molecules
Human Vascular Cell Adhesion Molecule-1
Mouse Vascular Cell Adhesion Molecule-1
Human Intercellular Adhesion Molecule-1
Mouse Intercellular Adhesion Molecule-1
Human or Mouse Endothelial Selectin
Tumor Necrosis Factor alpha
TNF-α Receptor I
TNF-α Receptor II
Unconjugated Bilirubin
Free Unbound Plasma Bilirubin
Heme Oxygenase-1
Heme Oxygenase-2
Heme Oxygenase-3
Diphosphoglucuronate Glucoronosyltransferase
UDP-glucuronic Acid
Biliverdin Reductase
Canalicular Multispecific Anion Transporter
Adenosine Triphosphate
Nitric Oxide
Nitric Oxide Synthase
Endothelial Nitric Oxide Synthase
Inducible Nitric Oxide Synthase
cAMP-response Element(CRE)-binding Protein
Nuclear Factor κB
Signal Transducers and Activators of Transcription
cAMP-responsive Element(CREB)-binding Protein
Inhibitory Protein of NF-κB
Activating Transcription Factor (ATF)-binding Element
Activator Protein-1
CCAAT/enhancer Binding Protein
Mitogen-activated Protein Kinase
Globin Transcription Factor
Nuclear Factor I/C (CCAAT-binding transcription factor)
IFN-gamma-activating sites
Interferon-stimulated Response Element
Interferon Regulatory Factor Binding site
Pyrrolidine Dithiocarbamate
Reactive Oxygen Species
Extracellular Matrix
Human Umbilical Vein Endothelial Cells
H5 V
Murine Microvascular Endothelial Cells
Lactate Dehydrogenase
Chapter 1
Bilirubin metabolism
Bilirubin is produced as the end product of the degradation of hemoglobin from
senescent or hemolyzed red blood cells. The breakdown of hemoglobin, other
hemoproteins, and free heme generates 250 to 400 mg of bilirubin daily in humans (London et al., 1950). A number of studies indicate that hemoglobin is the
principal source of bile pigment in mammals, accounting normally for approximately 80% of daily bilirubin production (London et al., 1950; Ostrow et al.,
The heme degradation is enzymatic, mediated by the microsomal enzyme
heme oxygenase 1 (HO-1), which directs stereospecific cleavage of the heme
ring. This reaction requires a reducing agent, such as nicotinamide-adenine dinucleotide phosphate (NADPH) and three molecules of oxygen, and results in the
formation of the linear tetrapyrrole, biliverdin, carbon monoxide, and iron (Tenhunen et al., 1968). Three isoforms of HO have been described: two constitutively
expressed isoforms, HO-2 and HO-3 (Rublevskaya & Maines, 1994; McCoubrey
et al., 1997), and a inducible isoform, HO-1 (Elbirt & Bonkovsky, 1999). HO-1,
the rate-limiting enzyme in the catabolism of the heme, is ubiquitous and is transcriptionally inducible by a variety of agents, such as heme, oxidants, hypoxia,
endotoxin, and cytokines (Maines, 1997; Ishizawa et al., 1983). Following its
synthesis by HO1 or HO2, biliverdin is converted to bilirubin by the phosphoprotein biliverdin reductase (BVR), in the presence of NADPH (Figure 1.1).
Figure 1.1: Bilirubin metabolism. Bilirubin derives from heme metabolism by heme oxygenase and biliverdin reductase.
Bilirubin is tightly, but reversibly, bound to plasma albumin. Albumin binding
keeps bilirubin in solution and transports the pigment to different organs and to
the liver in particular. Albumin binding protects against toxic effects of bilirubin.
A small unbound fraction of bilirubin is thought to be responsible for its biological effects (Weisiger et al., 2001; Ahlfors, 2001; Wennberg et al., 1979).
At the sinusoidal surface of the hepatocyte, bilirubin dissociates from albumin
and is uptaken by hepatocyte, through mechanism not fully elucidated (Zucker
1.2 Bilirubin and pathophysiology
et al., 1999; Cui et al., 2001). Within the hepatocyte, bilirubin binds to a group
of cytosolic proteins, mainly to glutathione-S-transferases (GSTs) (Zucker et al.,
1995). GST binding inhibits the efflux of bilirubin from the cell (Figure 1.2).
A specific form of uridine diphosphoglucuronate glucoronosyltransferase (UGT,
also termed UGT1A1), located in the reticulum, catalyzes the transfer of the glucuronic acid moiety from UDP-glucuronic acid (UDPGA) to bilirubin. In these
step bilirubin diglucuronide and monoglucuronide is formed (Bosma et al., 1994;
Hauser et al., 1984). This conversion is critical for efficient excretion of bilirubin to the bile canaliculus. Conjugated bilirubin is excreted against concentration
gradient by a canalicular multispecific anion transporter (cMOAT) also known
as multidrug resistance-related protein (MRP2) (Kamisako et al., 1999). The
energy for the transport is provided by the hydrolysis of an adenosine triphosphate (ATP). Any abnormality causing slowing or blockage of this rather complicated metabolic pathway will lead to disturbances of bilirubin metabolism (Ostrow et al., 2003a).
Bilirubin and pathophysiology
Bilirubin has been implicated in the development of different diseases (Greenberg, 2002). Is well known that it is responsible for the yellow skin coloration in
physiological jaundice of the newborn (Gourley, 1997). In this case, jaundice is
the result of a combination of factors, such as the increased bilirubin production
and immaturity of the bilirubin disposal mechanism of the liver. In the great majority of the cases, neonatal hyperbilirubinemia is innocuous. But severe neonatal
hyperbilirubinaemia, in few cases, may cause kernicterus and ultimately death.
Kernicterus is a devastating, chronic, disabling neurological disorder (Shapiro,
2003). Furthermore, unconjugated hyperbilirubinemia is observed also as a consequence of mutations on UGT1A1 gene, leading the description of Crigle-Najjar
and Gilbert syndromes and in the hemolytic disorders (Bosma, 2003; Ostrow &
Tiribelli, 2001a).
Particularly, the kernicterus is an example that suggests the cytotoxic effect
Figure 1.2: Summary of hepatic metabolism of bilirubin. Bilirubin is strongly bound to albumin in the circulation. At the sinusoidal surface of the hepatocyte, this complex dissociates,
and the bilirubin enters hepatocytes. Within the hepatocyte, bilirubin binds to glutathione-Stransferases (GSTs). UGT1A1 (or UGT) catalyzes the transfer of the glucuronic acid forming
the diglucuronide and monoglucuronide forms. Canalicular excretion of bilirubin is mediated by
multidrug resistance related protein (MRP2).
1.2 Bilirubin and pathophysiology
of bilirubin. Several studies have shown such toxicity (Hansen, 2001; Ostrow &
Tiribelli, 2001b), which typically occurs at micromolar concentrations of bilirubin. In vitro studies demonstrated that bilirubin causes death of cultured neurons
(Rodrigues et al., 2002a) and cerebral microvascular endothelial cells (Akin et al.,
2002) by apoptotic pathways. N-methyl-D-aspartate receptor antagonists can protect cultured neurons from bilirubin toxicity (Grojean et al., 2000), suggests the
implications of this class of glutamate receptors in pathogenesis of bilirubin damage.
However, accumulating evidence points to a protective role of bilirubin at
physiologic levels (Dor et al., 1999; Tomaro & Batlle, 2002). Stocker et al.
(Stocker et al., 1987; Stocker & Peterhans, 1989) showed that bilirubin is an antioxidant that can scavenge peroxyl radicals. Furthermore, bilirubin has been reported to protect against a variety of pathological processes, including complementmediated anaphylaxis (Nakagami et al., 1993), myocardial ischemia (Clark et al.,
2000), pulmonary fibrosis (Wang et al., 2002), and cyclosporine nephrotoxicity
(Polte et al., 2002).
This raises a seeming paradox: how can such low concentrations of UCB
protect against the much higher levels of reactive oxygen species? (Ostrow &
Tiribelli, 2003). One possible explanation involves the mechanism of redox cycling. Biliverdin is reduced to bilirubin through the action of BVR. Bilirubin then
interacts with reactive oxygen species (ROS), which neutralize their toxicity and
oxidize bilirubin, thereby regenerating biliverdin. As this cycle is repeated, the
antioxidant effect of bilirubin is multiplied. In this manner, low concentrations
of bilirubin can be recycled to neutralize large amounts of ROS (Baranano et al.,
There is increasing epidemiological evidence supporting an inverse association between cardiovascular disease and plasma bilirubin levels (Rigato et al.,
2005). In a study involving 4156 individuals aged 5-30 years from a biracial
(black-white) community, bilirubin levels showed significant differences related
to races and sex. In males of both races the bilirubin level was higher than their
counterparts, except in the pre-adolescents age group of 5-10 years. However,
males in general have higher risk for cardiovascular diseases than females. This
apparent paradox may be due to the multifactorial nature of cardiovascular disease
(Madhavan et al., 1997).
Similarly, a cross-sectional prevalence study conducted on men aged 21 to 61
years found that the decrease in total serum bilirubin was associated with more
severe cardiovascular events (Schwertner et al., 1994).
Interestingly, the prospective Farmingham offspring study found that a higher
total serum bilirubin level was associated with a lower risk for myocardial infraction, coronary artery disease and any adverse cardiovascular events, particulary
in men (Djouss et al., 2001). It is not clear whether higher serum bilirubin concentrations in physiological ranges work in favor of the cardiovascular system in
younger persons with no cardiovascular risk factors (Breimer et al., 1994). Furthermore, another study demonstrated that patients with Gilbert syndrome (who
have mildly elevated bilirubin levels) have less ischemic heart disease than the
general population (Vitek et al., 2002).
On the contrary, in another study conducted on 7735 men, ages 40-59, in England, Wales and Scotland, it was demonstrated that low bilirubin concentration
is also strongly associated with several cardiovascular risk factors. Men with increased concentrations of serum bilirubin and liver enzymes appeared to be at
much increased risk of ischemic heart disease. Interestingly, after adjustment
for lifestyle factors, biological factors, and preexisting disease, the relationship
remained U-shaped. This analysis demonstrate that both low and high concentrations of serum bilirubin are associated with an increased risk of ischemic heart
disease (Breimer et al., 1995). However, Hopkins et al. (Hopkins et al., 1996)
demonstrated that significant lower levels of bilirubin were found in patients with
coronary artery disease.
The relevance of serum bilirubin as a risk factor inversely related to the coronary artery disease was suggested also in other studies (Gullu et al., 2005). How6
1.3 Vascular atherosclerosis
ever bilirubin correlation as a useful marker compared with others (such as apolipoprotein B) seems to be controversial (Levinson, 1997).
More recent studies indicated that bilirubin may be protective in the development of atherosclerotic diseases by inhibiting the proliferation of vascular smooth
muscle cells by mechanisms not well established yet (Stocker & Keaney, 2004).
Collectively, these data indicate that the relationship between UCB and coronary
heart disease is still to be elucidated.
Vascular atherosclerosis
Atherosclerosis is the major source of morbidity and mortality in the developed
world (Murray & Lopez, 1997). The magnitude of this problem is profound, as
atherosclerosis claims more lives than all types of cancer combined and the economic costs are considerable. Although currently a problem of the developed
world, the World Health Organization predicts that global economic prosperity
could lead to an epidemic of atherosclerosis as developing countries acquire Western habits.
The vascular atherosclerosis, a progressive cardiovascular disease, is characterized by the accumulation of cholesterol in macrophage deposits (foam cell) in
large- and medium- sized arteries and the formation of the so called atherosclerotic plaques in the arteries walls (Stocker & Keaney, 2004). Several clinical
studies provide important evidence between traditional risk factors (dyslipidemia,
hypertension, diabetes, obesity, among others) associated with atheromatous disease (Stocker & Keaney, 2004; Libby et al., 2002).
Morphology of atherosclerotic lesions
The arterial wall normally consists of three well-defined concentric layers that
surround the arterial lumen, each of which has a distinctive composition of cells
and extracellular matrix. The layer immediately adjacent to the lumen is called the
intima, the middle layer is known as the media, and the outermost layer comprises
the arterial adventitia. These three layers are demarcated by concentric layers of
elastin, known as the internal elastic lamina that separates the intima from the media, and the external elastic lamina that separates the media from the adventitia.
A single contiguous layer of endothelial cells lines the luminal surface of arteries.
These cells sit on a basement membrane of extracellular matrix and proteoglycans
that is bordered by the internal elastic lamina. Although smooth muscle cells are
occasionally found in the intima, endothelial cells are the principal cellular component of this anatomic layer and form a physical and functional barrier between
flowing blood and the stroma of the arterial wall.
Atherosclerosis manifests itself histological as arterial lesions known as plaques
that have been extensively characterized into six major types of lesions that reflect
the early, developing, and mature stages of the disease (Stary et al., 1995; Stary
et al., 1994). The lesions stages were described as:
• Type I lesions, represent the very initial changes and are recognized as
an increase in the number of intimal macrophages and the appearance of
macrophages filled with lipid droplets (foam cells);
• Type II lesions, include the fatty streak lesion, the first grossly visible lesion,
and are characterized by layers of macrophage foam cells and lipid droplets
within intimal smooth muscle cells and minimal coarse-grained particles
and heterogeneous droplets of extracellular lipid;
• Type III (intermediate) lesions, are the morphological and chemical bridge
between type II and advanced lesions. Type III lesions appear in some adaptive intimal thickening (progression-prone locations) in young adults and
are characterized by pools of extracellular lipid in addition to all the components of type II lesions;
• Type IV lesions, are defined by a relatively thin tissue separation of the lipid
core from the arterial lumen. Type V lesions exhibit fibrous thickening of
1.3 Vascular atherosclerosis
Figure 1.3: Stages of atherosclerotic lesion. Varying stages of atherosclerotic lesion as outline
by Stary et al. From (Stocker & Keaney, 2004)
this structure, also known as the lesion cap. These type IV and V lesions
can be found initially in areas of the coronary arteries, abdominal aorta, and
some aspects of the carotid arteries in the third to fourth decade of life;
• Mature type VI lesions exhibit architecture that is more complicated and
characterized by calcified fibrous areas with visible ulceration. These types
of lesions are often associated with symptoms or arterial embolization.
In summary, the cohort studies by Stary show that progression beyond the fatty
streak stage is associated with a sequence of changes starting with the appearance
of extracellular lipid which begins to form a core to a lesion that is becoming more
elevated (Figure 1.3). Smooth muscle cells migrate into and proliferate within the
plaque, forming a layer over the luminal side of the lipid core. More and more
collagen is produced and plaque size increases. The process culminates in what is
known as a raised fibrolipid or advanced plaque. In the aorta such plaques may be
a centimeter or more in length.
Atherogenesis - Response to the injury theory
Over the past 150 years, numerous efforts have been made to explain the complex events associated with the development of atherosclerosis. In this endeavor,
different hypothesis have emerged that emphasize different concepts as the necessary and sufficient events to support the development of the atherosclerosis lesions
(Stocker & Keaney, 2004).
These paradigms have been devoted to understand the molecular mechanisms
of atherosclerosis and the factors that predispose individuals to clinical events.
Ross et al. (Ross, 1999) proposed “the response to the injury” theory of atherosclerosis. In this hypothesis, the initial step in the atherogenesis is the endothelial
denudation leading to a number of compensatory responses that alter the normal
vascular homeostatic properties (Ross, 1999).
The earliest event that occurs in the development of atherosclerosis is characterized by a progressive modification in the physiological microenvironment
identified as endothelial dysfunction (Endemann & Schiffrin, 2004).
The endothelial dysfunction is a complex multi-step mechanisms that has been
implicated in the pathophysiology of different forms of cardiovascular disease
(Gimbrone et al., 1995). The endothelial injury or dysfunction is characterized by
enhanced endothelial permeability and low-density lipoprotein (LDL) retention
in the subendothelial space (Schwenke & Carew, 1989; Schwenke & Zilversmit,
1989). This event is followed by leukocyte adhesion and transmigration across
the endothelium (Petri & Bixel, 2006)(Figure 1.4).
1.3 Vascular atherosclerosis
In intermediate stages, atherosclerosis is characterized by foam cell formation
(macrophages filled with lipid droplets) and an inflammatory response including
T-cell activation, the adherence and aggregation of platelets and further entry of
leukocytes into the arterial wall along with migration of smooth muscle cells into
the intima (Bobryshev, 2006; Zernecke & Weber, 2005; Raines & Ross, 1993).
The oxidized lipids deposition leads to a cell proliferation within the arterial wall
that gradually impinges on the vessel lumen and impedes blood flow (Libby &
Aikawa, 2001).
Continued inflammation allows for cellular necrosis, with a concomitant release of cytokines, growth factors that set the stage for autocatalytic expansion of
the lesion (Raines & Ross, 1995). Finally, advanced atherosclerosis is characterized by continued macrophage accumulation, fibrous cap formation, and necrosis in the core of the lesion (Davies & Woolf, 1993; Bobryshev, 2006; Libby
& Aikawa, 2001). This process may be quite insidious lasting for decades until
an atherosclerotic lesion (plaque) becomes disrupted and deep arterial wall components are exposed to flowing blood, leading to thrombosis and compromised
oxygen supply to target organs such as the heart and brain (Libby, 2002; Davies
& Woolf, 1993).
Because of the silent and slow progression of atherosclerosis in humans, many
of the current concepts on the cellular and molecular mechanisms involved in the
formation and alteration of advanced lesions of atherosclerosis have come from
animal models.
The endothelium is strategically located between the wall of blood vessels and
the blood stream. Endothelial cells regulate a wide array of processes including
thrombosis, vascular tone, and leukocyte trafficking (Cook-Mills & Deem, 2005;
Zeiher, 1996). It senses mechanical stimuli such as pressure and shear stress, and
Figure 1.4: Attachment, rolling and migration of leucocytes through the arterial endothelial monolayer into the intima. In the human arterial intima, the majority of monocytes differentiate into macrophages but some differentiate into dendritic cells (A). The majority of macrophages
transform into foam cells (B), while others do not accumulate lipids in their cytoplasm (C). Nonfoam macrophages frequently contact other immuno-inflammatory cells (C). In (C), a non-foam
macrophage is marked by a black star, while white stars indicate lymphocytes. Transmission electron microscopy. Scale bars: 4 mm (B, C). From (Bobryshev, 2006)
1.3 Vascular atherosclerosis
hormonal stimuli, such as vasoactive substances (Galley & Webster, 2004; Cines
et al., 1998).
One of the most important vasodilating substances released by the endothelium is nitric oxide (NO)(Mann et al., 2003), which inhibits cell growth and
inflammation and has anti-aggregant effects on platelets (Bruch-Gerharz et al.,
1998). Reduced NO levels have often been reported in presence of impaired endothelial function (Endemann & Schiffrin, 2004). Different mechanisms may be
involved in the onset of this dysfunction:
1. reduced expression and/or functionality of nitric oxide synthase (Cai & Harrison, 2000);
2. shortage of co-substrates (Mann et al., 2003);
3. NO consumption by increased ROS production (Cai & Harrison, 2000; Fischer et al., 2003).
NO plays a pivotal role in anti-atherogenesis; in addition to being a vasodilator, it inhibits platelet adherence and aggregation, smooth muscle cells proliferation, and endothelial cell leukocyte interaction, all of which are key events in
atherogenesis (Cooke et al., 1992; Davignon & Ganz, 2004). Pathophysiological
states associated with a decrease in NO bio-availability and endothelial adhesion
molecules for monocytes are up-regulated (Caterina et al., 1995). This could enhance local inflammation of the vessel wall, which may play a critical role in
plaque rupture (van der Wal et al., 1994).
The endothelial dysfunction is an early event, characterize by markers of inflammation and endothelial activation. These markers become useful, by providing additional information about a patient’s risk of developing cardiovascular disease, as well as providing new targets for treatment (Endemann & Schiffrin, 2004;
Tardif et al., 2006). Moreover, the endothelial dysfunction may also precede the
development of other diseases not strictly associated cardiovascular disease (Engler et al., 2003; Raitakari et al., 2004; Virdis et al., 2001).
Clearly, understanding the mechanisms and mediators of endothelial dysregulation and inflammation may yield new targets to predict, prevent, and treat cardiovascular disease. Markers of endothelial dysfunction include soluble forms
of adhesion molecules, which can be assessed in plasma (Szmitko et al., 2003b).
However, several other markers such us oxidized low-density lipoprotein receptor1 (LOX-1), CD40 ligand, asymmetric dimethylarginine (ADMA), to name a few,
have been proposed (Castelli et al., 1986; Szmitko et al., 2003a).
In summary, the endothelium is a crucial vascular structure not only because
it serves as a barrier between flowing blood and vascular wall but also because
it produces mediators regulating vascular growth, platelet function, and coagulation. Thus, the endothelium is not only target but also mediator of inflammatory
Pro-inflammatory cytokines
Cytokines and growth factors constitute a potent set of multi-functional peptide
signalling molecules capable of regulating several cellular functions, including
chemotaxis or directed migration, proliferation, accumulation of lipid, and synthesis of matrix components all of which take place during atherogenesis.
Cytokine is the term that has been used to describe the family of peptides that
regulate immune function and the term growth factor has most often been applied
to stimulators and inhibitors of cell proliferation. These growth regulatory molecules are multi-functional and a single peptide can promote cellular changes at
several different levels (Nathan & Sporn, 1991).
In vivo, growth factors, cytokines and their specific cell-surface receptors are
expressed at low or undetectable levels, may be sequestered in inactive forms and
may be regulated differentially after activation. By binding to specific cell-surface
receptors on responsive cells, cytokines and growth factors may induce signals
that evoke a large number of biological responses (Nathan & Sporn, 1991).
1.4 Pro-inflammatory cytokines
Cytokines appear to orchestrate the chronic development of atherosclerosis by
mediating infiltration and accumulation of immunocompetent cells, or enhancing
foam cell formation and thrombogenicity of the lipid core. Several studies have
examined the expression of the various cytokines and growth factors that may be
involved in the cellular changes that accompany developing lesions. Increased
concentrations of IL-1, TNF-α, IFN-γ, and platelet-derived growth factor (PDGF)
have been observed in the lesions of atherosclerosis (Raines & Ross, 1993).
Pro-inflammatory cytokines, as TNF-α, increase leukocyte adhesion to culture
endothelium via the expression of adhesion molecules (AM) and the release of
chemokines, facilitating the attraction and diapedesis of immunocompetent cells
in vitro (Young et al., 2002).
In 1975, Lloyd et al. could demonstrated unambiguously that treatment of
mice or rabbit with bacille Calmette-Gurin (BCG) for 10-14 days, followed by
injection of lipopolysaccharide (LPS), led to the released into the circulation of
a protein, which they called Tumor Necrosis Factor or TNF-α (Carswell et al.,
On the basis of amino acid sequences data derived from purified human or rabbit TNF-α, different groups cloned the human TNF-α (hTNF) cDNA gene (Shirai
et al., 1985; Pennica et al., 1984). Subsequently, the cDNA from pig, cow, rabbit,
cat, rat and mouse have been reported (McGraw et al., 1990; Drews et al., 1990).
Both human and the murine cDNA could be expressed at very high efficiency in
Escherichia coli and became available for physico-chemical, biological, biochemical and preclinical research, as well as for clinical application.
The human genomic TNF-α gene is located on the short chromosome 6 and
the gene is interrupted by three introns. The TNF-α cDNA gene codes for a mature polypeptide of 157 amino acids in human and 156 amino acids in mouse
(Fiers, 1991). Interestingly, the polypeptide is preceded by a 76 amino acid long
pre-sequence that is much longer than a classical signal sequence. Furthermore, is
strongly conserved between different mammalian species as the mature sequence,
which suggest a specific and essential function. The native structure of TNF-α is a
trimmer with a total molecular mass of 52 kDa (Van et al., 1991). Unlike human,
mouse TNF-α (mTNF) is a glycoprotein.
TNF-α was originally thought to be produced exclusively by macrophages.
However, by immunohistochemistry and in situ hybridization was also detected in
mesenchymal, endothelial and smooth muscle cells of the atherosclerotic human
arteries (Barath et al., 1990).
Receptors for TNF-α are expressed in the majority of mouse and human cell
lines. The number of receptors vary from about 200 up to 10000, and the binding
constant is around 2X10−10 M. Although the presence of the TNF-α receptor is a
prerequisite for the biological effect, there is not correlation between the number
of receptors and the magnitude of the response, or even the direction of response.
Two distinct TNF-α receptor subtypes (type I and type II) have been identified.
The first TNF-α receptor has a molecular weight of about 55 kDa, and can be referred to as TNF-R55 or TNF-RI. The second receptor has a molecular weight of
about 75 kDa, and can be referred to as TNF-R75 or TNF-RII. Although both receptors bind TNF-α, different cellular responses can be activated (Fiers, 1991).
TNF-α has numerous biological functions, including hemorrhagic necrosis of
transplanted tumors, cytotoxicity, and an important role in endotoxic shock and
in inflammatory, immunoregulatory, proliferative, and antiviral responses (Clermont et al., 2003; Fiers, 1991).
Nitric oxide
The Nitric Oxide (NO), a diatomic radical, was originally recognized in connection with contraction and relaxation of blood vessels. In the meantime, it has
become clear that NO is an universal messenger substance that takes part in diverse forms of intercellular and intracellular communication. For example, NO is
formed with the help of specific enzymes systems activated by extracellular and
1.5 Nitric oxide
Figure 1.5: Stepwise NO synthesis by NOS. The two reactions of NO synthesis as catalyzed
by NOS. The NADPH and oxygen requirements of each reaction are shown.
intracellular signals (Lowenstein & Snyder, 1992). Indeed, NO is synthesized
intracellularly and reaches its effector molecules, which may be localized in the
same cell or in neighboring cells, by diffusion. Finally, NO is notable among
signals for its rapid diffusion, ability to permeate cell membranes, and intrinsic
instability, properties that eliminate the need for extracellular NO receptors or
targeted NO degradation (Nathan, 2003). Thus, NO has the character of an autocrine or paracrine hormone, as well as intracellular messenger (Bruch-Gerharz
et al., 1998). One way or another, practically every cell in mammals is subject to
regulation by NO.
Biosynthesis of nitric oxide
The NO is generated by the oxidation of L-arginine to citrulline exclusively by
the enzyme Nitric Oxide Synthase (NOS). NO is synthesized from L-arginine
through a five-electron oxidation step via the formation of the intermediate NG hydroxy-L-arginine (Hibbs et al., 1988; Palmer et al., 1988). Others substrates
for NOS-mediated NO production are the molecular oxygen and NADPH (Leone
et al., 1991)(Figure 1.5).
The NOS are enzymes of complex composition that are active as dimers but
can also exist as inactive monomers. Furthermore, three isoforms of NOS have
been identified (Moncada et al., 1991; Forstermann et al., 1998; Stuehr & Griffith,
1992). Two isoforms are constitutively expressed and are activated by Ca+2 intracellular levels (Nathan, 1992).
Among them, nNOS (NOS1) was found to have a widespread distribution in
specific neurons of the central and peripheral nervous system (Bredt et al., 1991;
Vincent & Kimura, 1992; Vincent & Hope, 1992). However, nNOS expression
is not confined to neuronal cells (Forstermann et al., 1998). eNOS (NOS3) was
first identified in endothelial cells (Frstermann et al., 1991), but the expression
has also been demonstrated in several nonendothelial cell types, including neurons and other rat brain regions (Abe et al., 1997; Dinerman et al., 1994), cardiac
myocyte (Balligand et al., 1995), blood platelets (Sase & Michel, 1995), hepatocyte (Zimmermann et al., 1996), smooth muscle cells (Teng et al., 1998) and
others (Forstermann et al., 1998). The third isoform is a calcium independent,
iNOS (NOS2) and is not constitutive expressed (Nathan, 1992) but is induced
in macropahges, as well as in others cells such as melanocytes (Fecker et al.,
2002), cardiac myocytes (Kacimi et al., 1997), hepatocytes (Moreau, 2002), rabbit
corneal epithelial, stroma and endothelial cells (O’Brien et al., 2001) in response
to cytokines and bacterial endotoxin .
NOS isoforms are highly homologous in their primary structure (Figure 1.6).
They differ in size (130 to 160 kDa), amino acid sequence (50 to 60% identity between any two isoforms) (Lamas et al., 1992), tissue distribution, transcriptional
regulation, and activation by intracellular calcium. Moreover, they share an overall three-component construction (Crane et al., 1997; Stuehr et al., 2001; Moncada
& Higgs, 1993):
• an NH2 -terminal catalytic oxygenase domain (residues 1 to 498 for iNOS)
that binds heme (iron protoporphyrin IX), BH4 , and the substrate L-Arginine;
• a COOH-terminal reductase domain (residues 531 to 1144 for iNOS) that
binds FMN, FAD, and NADPH;
1.5 Nitric oxide
Figure 1.6: Schematic Presentation of Nitric Oxide isoforms structure. eNOS and iNOS diffentes domains and homology regions. For eNOS, regions involved in acylation, binding of substrates and cofactors are indicated as well as the oxygenase and reductase domain. Arg, arginine;
BH4 , tetrahydrobiopterin; CaM, calmodulin; FMN, flavin mononucleotide; FAD, flavin adenine
• an intervening calmodulin-binding region (residues 499 to 530 for iNOS)
that regulates electronic communication between oxygenase and reductase
Although NO synthesis reaction by NOS is well understood, some aspects
have been question and still been controversial (Alderton et al., 2001). It is
well accepted that the biosynthesis of NO requires a number of essential cofactors such as tetrahydrobiopterin (BH4 ), flavin adenine dinucleotide (FAD), and
flavin mononucleotide (FMN). BH4 seems to be important in maintaining NOS
in its active dimeric form (Griffith & Stuehr, 1995). Furthermore, NOS contains
binding sites for heme and calmodulin, both being essential for the enzyme activity. Indeed, functionally and structurally NOS enzymes catalase, as expected,
a multi-electron transfer in order to generate NO. The FAD and FMN in the reductase domain accept electrons from NADPH and pass them on to the haem
domain. The essential role of the flavin cofactors is to allow a two-electron donor
(NADPH) to donate electrons to a one-electron acceptor (haem), by forming a
stable semiquinone radical intermediate (NG -hydroxy-L-arginine). These electron flow may result in the formation of the enzyme products citrulline and NO
(Abu-Soud & Stuehr, 1993).
Intriguingly the pathway of electron flow appears to cross over between different subunits of the dimer, i.e. the flavin domain of one polypeptide chain donates
its electron to the haem domain of the other. The physiological reason for this is
unclear, but it is clearly a major reason why the NOS monomer is inactive.
Endothelial Nitric Oxide Synthase (eNOS)
Regulation of eNOS expression
The eNOS promoter has been cloned from human (Marsden et al., 1993), bovine
(Venema et al., 1994), murine (Gnanapandithen et al., 1996), porcine (Zhang et al.,
1997) endothelial cells, and there is a high degree of homology in the promoter
sequence among the different species (Venema et al., 1994). This high sequence
homology suggests significant evolutionary conservation of transcriptional regulation. Like many so-called constitutively expressed proteins, the eNOS promoter
lacks a typical TATA box (Forstermann et al., 1998). In addition, eNOS promoter
possesses multiple potential cis-regulatory DNA sequences, including a CCAT
box, Sp1 sites, GATA motifs, CACCC boxes, AP-1 and AP-2 sites, a p53 binding region, NF-1 elements, NF-κB site, acute phase reactant regulatory elements,
sterol regulatory elements, and shear stress response elements (Marsden et al.,
1993; Cieslik et al., 1998; Karantzoulis-Fegaras et al., 1999; Laumonnier et al.,
2000; Tang et al., 1995; Grumbach et al., 2005). Deletion experiments revealed
that some binding sites are essential for eNOS promoter activity, in particular Sp1
and GATA (German et al., 2000).
Given the list of transcription factors that bind to the eNOS promoter it is
1.5 Nitric oxide
hardly surprising that eNOS mRNA levels in cultured and native endothelial cells
can be modulated by numerous stimuli (Searles, 2006). Although the term inducible has been restricted to iNOS (NOS 2), eNOS is also regulated by a variety of stimuli (Bruch-Gerharz et al., 1998; Govers & Rabelink, 2001). For instance, TNF-α is known to lower eNOS expression by decreasing the half-life of
its mRNA (Lai et al., 2003). In addition, TNF-α induced destabilization of eNOS
message has been observed by others. Yoshizumi et al. (Yoshizumi et al., 1993)
demonstrated that there was a dramatic decrease in steady-state levels of eNOS
mRNA and protein in human umbilical vein endothelial cells (HUVECs) treated
with the cytokine TNF-α. This finding was consistent with earlier work showing
impaired endothelium dependent vasorelaxation in isolated arteries treated with
TNF-α (Aoki et al., 1989). In nuclear run-on analysis of cells treated with TNF-α,
there was no difference in the rate of eNOS transcription compared with untreated
cells . However, TNF-α treatment resulted in a reduction of eNOS mRNA halflife from 48 h at baseline to 3 h (Yoshizumi et al., 1993).
Co-translational modification and post-translational regulation of eNOS
It was also demonstrated that there is a marked discrepancy among the amounts
of eNOS mRNA, protein expression and activity, strongly suggesting a regulatory
mechanisms at post-transcriptional (Yoshizumi et al., 1993; Forstermann et al.,
1998) and post-translational level (Govers & Rabelink, 2001).
In contrast to the other NOS isoforms, eNOS contains a myristoyl group that is
covalently attached to the glycine residue at its NH2 terminus. The turnover of the
myristoyl group is as slow as that of eNOS itself, demonstrating the irreversibility of myristoylation (Liu et al., 1995). Myristoylation renders eNOS membrane
bound, whereas iNOS and nNOS are predominantly, if not exclusively, cytoplasmic. Indeed, the myristoyl moiety is and absolute requirement for the membrane
localization and activity of eNOS (Sakoda et al., 1995).
The monomers that compose the active eNOS dimer are also palmitoylated
(Sessa et al., 1995). This post-translational modification does not modify eNOS
activity, is reversible, requires myristoylation and stabilizes the association with
intracellular membranes. The membrane association is required for the phosphorylation and activation of eNOS (Patterson, 2002). Functional eNOS can be detected in at least three membrane compartments: the Golgi apparatus (O’Brien
et al., 1995; Sessa et al., 1995), the plasma membrane (Hecker et al., 1994) and
the plasmalemmal caveolae, a specialized plasma membrane domain principally
composed by caveolins proteins (Feron et al., 1996; Garca-Cardea et al., 1996;
Liu et al., 1996; Govers et al., 2002).
The co-localization of the signal transduction molecules and proteins that
comprise the eNOS signaling complex within the different membrane compartments facilitates enzyme activation, NO production, and the activation of downstream effector pathways (Govers & Rabelink, 2001).
Another determinant of eNOS expression is NO itself. NO has been shown
to be involved in a negative-feedback regulatory mechanism and decreases eNOS
expression via a cGMP-mediated process (Vaziri & Wang, 1999). In agreement,
the decrease in glomerular filtration rate after administration of LPS could be attributable to inhibition of eNOS function, most likely by NO auto-inhibition via
activation of iNOS (Schwartz et al., 1997).
Inducible Nitric Oxide Synthase (iNOS)
In contrast to eNOS and nNOS, iNOS, once expressed, is present in much greater
amounts and is continuously active due to the tight binding of calmodulin even at
basal levels of cytosolic Ca2+ . These properties result in the production of much
greater amounts of NO by iNOS, typically within the micromolar range, as compared with eNOS and nNOS (Cho et al., 1992; Stuehr et al., 2001).
Thus, the relatively large amounts of NO and its reaction products produced
by iNOS are capable of killing bacteria, viruses, and other infectious organisms
1.5 Nitric oxide
and are also capable of causing tissue damage (Hobbs et al., 1999). Indeed, NO is
extremely reactive and short-lived. A variety of reactive products of NO formed
in tissues, including peroxynitrite, NOX , and N2 O3 , are likely the molecules responsible for tissue damage (Bruch-Gerharz et al., 1998; Stuehr et al., 2001).
Regulation of iNOS
The iNOS gene is quiescent in most tissues until it is transcriptionally activated
by diverse stimuli to produce large amounts of NO (Kone & Baylis, 1997). Accordingly, both positive and negative modulators have evolved to control tightly
iNOS expression and to prevent untoward effects of excessive NO production.
The 5’ flanking region of iNOS gene was cloned and sequenced for mouse
(Xie & Nathan, 1993; Lowenstein et al., 1993), rat (Zhang et al., 1998) and human (Nunokawa et al., 1994). The large size of these region suggested a complex
regulation of induction. iNOS transcription is regulated in a complex manner by
several constitutive and inducible transcription factors, including CREB (Eberhardt et al., 1998), C/EBPbeta (Eberhardt et al., 1998), NF-κB (Beck & Sterzel,
1996; Neufeld & Liu, 2003) and many others cytokines responsive elements such
as: AP-1, γ-IRE, NF-IL6, GAS, IRF-E, ISRE, TNF-RE, and X box (Chu et al.,
1998). Indeed, the promoter region of human iNOS contains multiple binding
sites for NF-κB (Taylor et al., 1998; Xie & Nathan, 1994). Various stimuli in a
wide variety of cells induce iNOS (Taylor & Geller, 2000; Rao, 2000; Frstermann
& Kleinert, 1995). It was demonstrated that iNOS can be regulated by stimuli
including cytokines, e.g., IFN-γ, IL-1β, and TNF-α that, have been shown to activate NF-κB (Chu et al., 1998; Neufeld & Liu, 2003). In contrast, transforming
growth factor-β (Pfeilschifter & Vosbeck, 1991), interleukin (IL)-13 (Saura et al.,
1996), and STAT3 (Yu et al., 2002b) suppress iNOS transcription.
Epigenetic controls on iNOS transcription are also operative, and it was shown
that hyperacetylation (Yu et al., 2002b) and DNA methylation (Yu et al., 2002a)
limit iNOS activation. Although much is known about the cis and trans regula23
tory factors controlling activation of iNOS transcription by cytokines and bacterial
LPS, relatively little is known about how iNOS transcription might be constrained
and how local changes in chromatin structure might participate in this process.
On the other hand, iNOS is also regulated post-transcriptionally. Different and
multiple levels may affected iNOS activity (Nathan & Xie, 1994). Among them:
• mRNA and protein stability (Vodovotz et al., 1993);
• binding of calmoduling (Cho et al., 1992);
• activity of kinase and phosphatase regulating the protein phosphorylation
(Dawson et al., 1993; Michel et al., 1993);
• availability of subtracts and cofactors (Vodovotz et al., 1994; Albina et al.,
1988; Gross & Levi, 1992);
• NO itself (Assreuy et al., 1993; Griscavage et al., 1993);
• subcellular localization (Vodovotz et al., 1993).
Complex regulation of iNOS at multiple levels may reflect the dual role of
iNOS in host defense and autotoxicity (Bogdan, 2001a).
Nitric oxide and pathophysiology
Excessive NO production has been associated to several pathologies. The concentration of NO produced within a cell has also significant implications for the
ultimate signals produced. Under certain pathological conditions such as inflammation, up-regulation of inducible NOS affords production of NO at low micromolar concentrations (Xie & Nathan, 1994). In this concentration range, NO
competes effectively with the enzyme superoxide dismutase (SOD) for O2– , facilitating formation of ONOO− and other reactive nitrogen species (RNS) (Koppenol
et al., 1992). This increase in tissue RNS, a condition termed “nitrosative stress”
(Hausladen et al., 1996), leads to the modification of cellular targets such as thiols, proteins, and lipids, many of which have implications for cellular signaling
1.5 Nitric oxide
(Davis et al., 2001).
Excessive NO production was linked to pathologies including: immune-type
diabetes, inflammatory bowel disease, rheumatoid arthritis, carcinogenesis, septic
shock, multiple sclerosis, transplant rejection and stroke. On the other hand, several pathologies were linked to insufficient NO production, including: hypertension, impotence, arteriosclerosis and susceptibility to infection (Bogdan, 2001a;
Bruch-Gerharz et al., 1998; Nathan, 1992).
In the immune system, the use of NO donors and NOS inhibitors and the analysis of NOS knock out mice have provided evidence that NO governs a broad
spectrum of processes (Bogdan, 2001a). These include the differentiation, proliferation and apoptosis of immune cells, the expression adhesion molecules, the
production of cytokines and other soluble mediators, and the synthesis and deposition of extracellular matrix components (Marshall et al., 2000; Bogdan, 2001b;
Pfeilschifter et al., 2001). Many molecular targets for NO have been identified
whose contribution to a specific phenotype.
High-input NO release may strongly affect endothelial cell functions. Increased NO production likely plays an important role in different steps of angiogenesis, modulating migration, proliferation, and endothelial cell organization
into a network structure (Papapetropoulos et al., 1997; Shizukuda et al., 1999;
Fukumura & Jain, 1998).
NO is a principal factor involved in the anti-atherosclerotic properties of the
endothelium (Endemann & Schiffrin, 2004). It has been documented that NO
plays a critical role in vascular endothelial growth factor-induced angiogenesis
(Hood et al., 1998), vascular hyper-permeability mediated by eNOS and iNOS expression in vitro (Papapetropoulos et al., 1997) and down-regulation of cytokineinduced endothelial cell adhesion molecule expression (Jiang et al., 2005). In
agreement with these findings, inhibition of the NO-producing enzyme eNOS
caused accelerated atherosclerosis in experimental models (Davignon & Ganz,
NO interferes in vitro with key events in the development of atherosclerosis,
such as monocyte and leukocyte adhesion to the endothelium (Landmesser et al.,
2006). Also, high concentrations of NO have been implicated in the modulation
of leukocyte recruitment by the regulation of adhesion molecule expression on endothelial cells (Zadeh et al., 2000) and in the microbicidal activity of endothelial
cells (Jiang et al., 2005; Bogdan, 2001a). NO also decreases endothelial permeability and reduces vessel tone, thus decreasing flux of lipoproteins into the vessel
wall (Rubbo et al., 2002). Finally, NO has been shown to inhibit vascular smooth
muscle cell proliferation, migration (Endres & Laufs, 1998) and platelet aggregation (Radomski et al., 1987). It has been proposed that eNOS has a dual role
in the pathogenesis of atherosclerosis: under normal conditions, it generates low
concentrations of NO and probably peroxynitrite (Koppenol et al., 1992), which
favor an anti-atherosclerotic environment (Endemann & Schiffrin, 2004; Wever
et al., 1998). However, during hyperlipidemia and atherosclerosis, it may contribute to the formation of oxidative stress by a reduction in BH4 -dependent NO
formation and unopposed superoxide formation by the enzyme (Schillinger et al.,
2002). Particularly, in the setting of local induction, iNOS could favor the development of local toxic concentrations of peroxynitrite in atherosclerotic plaques
(Lee et al., 2004). This concept further emphasizes the role of redox state as a
determinant of vascular integrity in atherosclerosis (Stocker & Keaney, 2004; Endemann & Schiffrin, 2004; Davignon & Ganz, 2004).
In summary, NO production plays a role in the physiology or pathophysiology
of almost every organ system. Thus, it should come as little additional surprise
to learn that the production of NO is regulated by means as diverse and complex
as NOS functions. The diversity of ways in which NO production can be timed,
confined, augmented, or suppressed, combined with the wide spectrum of NO’s
molecular targets, helps explain how one molecule can serve many functions.
1.6 Adhesion molecules
Adhesion molecules
Most eukaryotic cells have the ability to recognise and react functionally to extracellular matrices. This is true not only for actively migrating cells that use
adhesive contact for traction and guidance, but also for stationary cells that require a platform for support and orientation.
Cells in vivo must form contacts with their neighbours or with the extracellular matrix (ECM) in order to form tissues or organs. The macromolecular components of ECM, which are secreted by resident cells, include proteglycans, glycoproteins and collagens that are secreted and assembled locally into an organised
network to which cells adhere (Hay, 1981). Other members of the ECM, including adhesive molecules such as laminin, vitronectin and fibronectin, facilitate the
adherence of cells to their substratum (Hay, 1981; Humphries, 1990).
ECM not only fills intercellular spaces, shaping and strengthening many tissues. The ECM offers structural support for cells, and can also act as a physical
barrier or selective filter to soluble molecules. On the other hand, ECM can influences cellular functions such as state of differentiation and proliferation (Wylie
et al., 1979; Adams & Watt, 1993; Springer, 1990). ECM components regulate
differentiation and development by mechanisms involved intracellular events that
may transduce signals between ECM receptors and the nucleus (Adams & Watt,
1993; Aplin et al., 2002).
Cell adhesion receptors identified to date mediate both homophilic adhesion,
which involves binding of an adhesion molecule on one cell to the same adhesion molecule on a second cell and heterophilic adhesion, in which an adhesion
molecule on one cell type binds to a different type of cell adhesion molecule on a
second cell. The T-cell interaction with antigen-presenting target cells in the immune system is the best known example of heterophilic adhesion (Springer, 1990).
Diversity in the composition of ECM in different tissues and at different stages of
development arises not only through expression of different matrix molecules, but
also from the existence of multiple forms of individual molecules.
Many different molecules have been identified by using specific monoclonal
antibodies and the subsequent identification of genes responsible for encoding
these molecules has shown that they are structurally different from each other
(Wylie et al., 1979).
The cell adhesion molecules can be divided into 4 major families: A) the
cadherin superfamily (Takeichi, 1988), B) the selectins (Bevilacqua & Nelson,
1993), C) the immunoglobulin superfamily (Hogg et al., 1991) and D) the integrins (Hynes, 1987) (Figure 1.7). The interactions of the adhesion molecules
with the ECM has a homeostatic function in promoting tissue regeneration during
wound healing (Eliceiri, 2001), while aberrant adhesion contributes to the etiology and pathogenesis of a number of major human diseases including asthma,
allergy, cardiovascular disease and cancer (Kelly et al., 2007; Blankenberg et al.,
2003; Juntavee et al., 2005).
Adhesion molecules mediate many other different functions, acting as receptors for growth factors and mediating cell-cell adhesion rather than cell extracellular matrix interactions (Lster & Horstkorte, 2000). Adhesion molecules are
important on early phase of atherosclerosis involving the recruitment of inflammatory cells from the circulation and their transendothelial migration (Kelly et al.,
2007; Jang et al., 1994; Petri & Bixel, 2006).
The two major subsets of adhesion molecules participating in the inflammatory disease are: the selectins, in particular E and P selectins and the immunoglobulin gene superfamily, in particular vascular cell adhesion molecule 1 (VCAM-1)
and intercellular adhesion molecule 1 (ICAM-1) (Blankenberg et al., 2003; Jang
et al., 1994). Selectins, belong to a family of Ca+2 dependent carbohydrate binding
proteins, mediate the earlier adhesion of leukocytes to the endothelium during the
rolling step of leukocyte extravasations in inflammation. VCAM-1 is a glycoprotein expressed on the surface of activated endothelium and on a variety of cell
types. ICAM-1 is a counter receptor for the leukocyte β2 integrin, LFA-1. ICAM1 is expressed on leukocytes, fibroblast, epithelial cells and endothelial cells. The
1.6 Adhesion molecules
Figure 1.7: Steps in the inflammatory process. The five distinct steps leading to leukocyte
accumulation and tissue damage during inflammatory processes. Selectins, VCAM and ICAM
interaction with leukocyte integrins. From (Jackson, 2002).
expression of this adhesion molecules is also regulated by several cytokines, such
as IL-1β, IL-4, TNF-α and IFN-γ (Dustin et al., 1986; Shimizu et al., 1992a).
The Selectins
The selectins, a family of Ca+2 dependent carbohydrate binding proteins, mediate
the initial attachment of leukocytes to the endothelium on the blood vessel wall
during the rolling step of leukocyte extravasation in inflammation (Abbassi et al.,
1993; Petri & Bixel, 2006).
Selectins recognise fucosylated carbohydrate ligands, especially structures containing Sialyl-LewisX (sLeX ) and Sialyl-Lewisa (sLea ), which are heavily ex29
pressed on neutrophils and monocytes and also found on natural killer cells. These
selectin/carbohydrate interactions permit leukocytes to roll along the vascular endothelium in the direction of blood flow as a prelude to integrin-mediated adhesion
(Munro et al., 1992).
All selectins have a unique and characteristic extracellular region composed of
an amino-terminal calcium dependent lectin-like binding domain which is formed
by a 120-amino acid. This region determines the ability of each selectin to bind
to specific carbohydrate ligands (Drickamer, 1988). This domain is followed by a
sequence of 35-40 amino acids similar to a repeat structure, which was first found
in epidermal growth factor (EGF). The lectin and EGF-like domains are shown
to have 60% to 70% homology at the nucleotide and protein level. There is also
a region composed by two to nine short consensus repeat sequences (SCR), similar to those found in complement regulatory proteins. The size variation of the
selectins is due to the different numbers of SCR domains, each ∼60 amino acids
long. This is followed by a single transmembrane region and a short cytoplasmic
tail (Vestweber & Blanks, 1999) (Figure 1.8).
The selectins family consists of three closely related cell-surface molecules:
L-selectin (MEL-14, LAM-1, CD62L), E-selectin (ELAM-1, CD62E), and Pselectin (PADGEM, GMP-140, CD62P). P-, L-, E-selectin are most closely related in amino acid sequence within lectin and EGF like domains. Moreover,
these domains mediate specific interactions with similar, if not identical, carbohydrate determinants displayed on diverse ligands (Tedder et al., 1995).
All selectins participate in different, though overlapping, ways to the early
steps of leukocyte recruitment at the endothelial surface under shear forces: leukocyte rolling and tethering. By interactions with their ligands, selectins create weak
bonds between activated endothelial cells (E- and P-selectin) and leukocytes (Lselectin). P-selectin/PSGL-1 binding triggers leukocyte activation, integrin mobilization and induces inflammation and thrombosis (Blankenberg et al., 2003;
Vestweber & Blanks, 1999).
1.6 Adhesion molecules
Figure 1.8: Structural organization of selectins. Selectins are composed of an amino-terminal
lectin domain, a single epidermal growth factor (EGF)-type repeat and various numbers of consensus repeats or so called complement binding domains, which share sequence homology with
a domain structure often found in proteins with complement binding activity. Proteins have a
single transmembrane region and a short cytoplasmic tail. E-selectins have different numbers of
complement binding domains in different species.
Unlike E- and P-selectins, L-selectin is found only on leukocytes and is expressed continuously throughout myeloid differentiation and on early erythroid
progenitor cells but not on mature erythrocytes (Tedder et al., 1995). L-selectin
was originally reported to mediate lymphocyte binding to high endothelial venules
of peripheral lymph nodes during lymphocyte homing. Subsequently, it was
shown to be expressed on most of other peripheral blood leukocytes and is thought
to be involved in regulating leukocyte traffic in the systemic microcirculation
(Warnock et al., 1998).
P-selectin is another type of selectin adhesion protein that was initially found
in platelets and also is constitutively expressed in endothelial cells (Johnston et al.,
1989). In both cell types, P-selectin is synthesised and stored in cytoplasmic
granules. In platelets P-selectin is contained in the α-granules (Wagner, 2005),
whereas in endothelial cells it is found in Wiebel-Palade bodies (McEver et al.,
1989). P-selectin is mobilized rapidly to the external plasma membrane of endothelial cells and platelets in response to activation with cytokines such as thrombin (Tedder et al., 1995). Expression of P-selectin on the cell surface generally
is short-lived. This supports the idea that P-selectin mediates early leukocyteendothelial interactions and also mediates the binding of activated B-cells and a
subset of T-cells, to stimulated endothelium in vitro (Wagner, 2005). Since Pselectin an E-selectin can bind to the tetrasaccharide sLeX and both mediate the
binding of PMNs and monocytes, the function of the endothelial selectins appears
to be redundant (Larsen et al., 1992). The rapid transport of P-selectin to the cell
surface (McEver et al., 1989) and the more slowly acting up regulation by de novo
synthesis of E-selectin (Bevilacqua et al., 1987) had served as an explanation for
this redundancy, arguing for similar functions of both selectins at different time
points. At the same time, the parallel expression of both selectins after induction
with TNF-α might argue for redundancy. However, indirect evidence has emerged
recently, suggesting that the physiological ligands for both endothelial selectins on
the same leukocytes might be different (Hahne et al., 1993; Larsen et al., 1992).
E (endothelial)-selectin is specific from endothelial cells. This adhesion molecule is almost absent from non activated endothelial cells and become induced
1.6 Adhesion molecules
upon the exposure of the endothelium to various pro-inflammatory stimuli. Eselectin synthesis is increased rapidly after cell stimulation by cytokines such as
TNF-α or IL-1β (Invernici et al., 2005) and lipopolisaccharide (LPS). Induction
occurred on the transcriptional level, and within 34 h after stimulation, maximal
levels of E-selectin protein are expressed at the cell surface. Basal levels are
reached again after 16-24 h, in contrast to other cytokine-inducible adhesion molecules such as ICAM-1 and VCAM-1. A similar mechanism and similar kinetics
of the regulation of mouse E-selectin were found on mouse endothelioma cells
(Hahne et al., 1993).
The 5’-flanking regions of human E-selectin were cloned and sequenced, and
the regulatory elements of the gene were studied intensively. Four regulatory elements were found in the human E-selectin promoter, three of them are NF-κB
binding sites and one is an activating transcription factor (ATF)-binding element
(Kaszubska et al., 1993). NF-κB elements are not sufficient, but necessary, for the
cytokine-stimulated induction of E-selectin transcription (Whelan et al., 1991).
Furthermore, proteosome inhibitors block the degradation of IκB, consequently
block NF-κB activation and inhibit transcriptional activation of E-selectin (Read
et al., 1997). In addition to the NF-κB elements, the ATF element is involved
in cytokine-stimulated expression of E-selectin as well. These two pathways are
rapidly activated and converge on the E-selectin promoter to result in full cytokine
responsiveness of this gene (Read et al., 1997).
Immunoglobulin superfamily adhesion molecules
The immunoglobulin superfamily is the most abundant family of cell surface adhesion molecules, accounting for 50% of leukocyte surface glycoprotein. The
structure of this family is characterized by repeated domains, similar to those
found in immunoglobulins. These 70-100 aminoacid domains are composed of
two β sheets and give rise to immunoglobulin folds that participate to adhesion
sites (Blankenberg et al., 2003; Petri & Bixel, 2006).
Alternative splicing is frequent in the genes of this family and allows the
production of multiple isoforms. By mutation and deletion analysis these immunoglobulin domains have been shown to mediate many different functions, including acting as receptors for growth factors and mediating cell-cell adhesion
rather than cell- extracellular matrix interactions (Holness & Simmons, 1994;
Shimizu et al., 1992b). Though not all immunoglobulin-superfamily adhesion
molecules mediate cell-cell interactions. Many glycoprotein which belong to
this family do function as adhesion receptors, including: intercellular adhesion
molecule-1 (ICAM-1; CD54), intercellular adhesion molecule-2 (ICAM-2), vascular cell adhesion molecule-1 (VCAM-1; CD106), platelet-endothelial cell adhesion molecule-1 (PECAM-1; CD31) and the mucosal addressin cell adhesion
molecule-1 (MAdCAM-1). ICAM-1, ICAM-2 and VCAM-1 are involved in the
adhesion of T-cells to endothelial cells by serving as surface ligands for the integrins LFA-1 (leukocyte-function antigen-1), αLβ2 and α4 β1 (Shimizu et al.,
Intercellular Adhesion Molecule-1 (ICAM-1)
The adhesion molecules ICAM-1 (CD54) and ICAM-2 (CD102) are counterreceptors for the leukocyte β2 integrin, LFA-1 (CD11α/CD18) (Diamond et al.,
1991; van de Stolpe & van der Saag, 1996). ICAM-1 molecule mediate adhesion of leukocytes to activated endothelium by establishing strong bonds with
integrins and inducing firm arrest of inflammatory cells at the vascular surface,
and participate to leukocyte extravasation (Petri & Bixel, 2006; Katagiri et al.,
1996). Linkage with the cytoskeleton, ICAM-1 may localize within regions of the
endothelial cell membrane in order to facilitate leukocyte adherence and transmigration (van der Wal et al., 1994). Conversely, blocking ICAM-1 function with
antibodies prevents leukocytes to firmly adhere to the endothelium, resulting in a
significant reduction in leukocyte trans-endothelial migration in various animals’
models. In line with these experimental findings, ICAM-1 knock-out mice show
an impaired inflammatory response exemplified by reduce tissue infiltration of
neutrophils (Sligh et al., 1993).
1.6 Adhesion molecules
Figure 1.9: Structure of ICAM-1 domains. Intercellular adhesion molecule-1 (ICAM-1) has
five immunoglobulin like domains followed by a transmembrane region and a short cytoplasmic
ICAM molecules are able to bind more than one ligand by using different
binding domains. The dimerisation or formation of larger protein multimers is
commonly observed for such molecules and may increase binding affinities with
ligands. Amino acid substitutions in the extracellular domains have indicated that
the primary binding site for LFA-1 is located in the NH2 -terminal first domain of
ICAM-1 (Stanley & Hogg, 1998; Staunton et al., 1990). A second ligand-binding
site for another β2 integrin on leukocytes (CD11b/CD18, Mac-1) is localized to
the third immunoglobulin-like domain. However, it is clear that the NH2 -terminal
two domains in both cases do not contribute equally to the binding site (Figure
ICAM-2 has only two extracellular immunoglobulin-like domains and the
binding site for Mac-1 is localized to the third immunoglobulin-like domain of
ICAM-1. The second domain has a less critical role (Holness & Simmons, 1994),
it appears that ICAM-2 contribution as an endothelial ligand for this leukocyte
integrin is rather limited (Staunton et al., 1989; Casasnovas et al., 1999).
ICAM-1 is expressed on leukocytes, fibroblasts, epithelial cells and endothelial cells (Dustin et al., 1986). ICAM-2 also has a similar tissue distribution
(Blankenberg et al., 2003). ICAM-1 displays molecular weight heterogeneity in
different cell types with a mature form of of 97 kDa on fibroblasts, 114 kDa on
the myelomonocyte cell line U937, and 90 kDa on the B lymphoblastoid cell JY.
ICAM-1 biosynthesis involves a 73 kDa intracellular precursor which is converted
to the mature form in 20 to 30 min. The maturation, in the Golgi complex, is followed by transport to the cell surface within a few minutes (Dustin et al., 1986).
In vitro, ICAM-1 expression can be up regulated in responses to proinflammatory cytokines such as IFN-γ, TNF-α and IL-1β (Invernici et al., 2005; Sawa
et al., 2007). The induction is dependent on protein and mRNA synthesis and is
reversible. The up-regulation of ICAM- 1 by IL-1β involved a rapid mRNA and
protein synthesis-dependent, which is apparent within 1 hr (Dustin et al., 1986).
On the contrary, ICAM-2 apparently is expressed constitutively and is not regulated by cytokines (van Buul et al., 2007).
It is therefore interesting the characterization to the genomic structure of the
5’-flanking region for the human ICAM-1 gene. It was identified to be a functional potent promoter region. Structural analysis revealed that contained potential
interferon responsive elements, glucocorticoid receptor-binding sites, an NF-κB
consensus element, and AP1 and AP2 sites, regions which may be involved in the
regulation of this gene expression (Voraberger et al., 1991; Muller et al., 1995; Degitz et al., 1991). The exact biologic roles played by these potential elements, as
well as other regions involved in the constitutive and tissue-specific regulation of
ICAM-1 gene expression, are currently under investigation (Degitz et al., 1991).
Vascular Cell Adhesion Molecule-1 (VCAM-1)
Another member of the immunoglobulin gene superfamily, VCAM-1, is a 90–110
kDa glycoprotein which supported the adhesion of mononuclear leukocytes (Petri
1.6 Adhesion molecules
& Bixel, 2006) and certain tumor cells (Osborn et al., 1989; Rice & Bevilacqua,
1989; Rice et al., 1990). In vitro studies demonstrated that VCAM-1 is expressed
on the surface of isolated human fetal endothelial cells deriving from different organs such as: brain, heart, lung, liver and kidney. In this way, it was supported the
notion that VCAM-1 expression can be up-regulated by interferon-γ (IFN-β) and
several cytokines, such as IL-4, IL-1β and TNF-α (Li et al., 1993; Invernici et al.,
2005; Sawa et al., 2007).
VCAM-1 interacts with the leukocyte integrin α4β1 on many different cells
including eosinophils, monocytes and with α4β7 on activated peripheral T-cells.
Thus α4β1/VCAM-1 interactions, like LFA-1/ICAM-1 interactions, may regulate
the movement of lymphocytes out of blood vessels to cross the endothelium in the
inflammatory sites (Petri & Bixel, 2006). Furthermore, α4β1/VCAM-1 interaction has been shown to be crucial for the binding of hematopoietic precursor cells
to a bone marrow derived adherent cell population (Ryan et al., 1991).
Osborn et al. (Osborn et al., 1989) demonstrated on HUVEC cells treated
with IL-1, that VCAM-1 contains six immunoglobulin domains . On the other
hand, Cybusky et al. (Cybulsky et al., 1991), reported that VCAM-1 contained
an additional 276 base-pair domain, located between domains 3 and 4. Together,
these data indicate that the two forms of mRNA arise by alternative splicing, although the seven-domain form appeared predominant. On the surface of HUVEC
cells only a 110 kDa polypeptide was detectable by immunoprecipitation. This is
consistent with the seven-immunoglobulin like domain form of VCAM-1 (Cybulsky et al., 1991). Alternative splicing of the VCAM-1 gene, in cytokine activated
endothelium, may generate functionally distinct cell-surface adhesion molecules
(Figure 1.10). In this way, it was demonstrated by functional analysis that the
major form of VCAM-1 has seven extracellular immunoglobulin like domains
(VCAM-7D). Moreover, the three NH2 -terminal domains (domains 1-3) are similar in amino acid sequence to domains 4-6. However, on the minor form of
VCAM-1 (VCAM-6D), the domain 4 is deleted by an alternative splicing (Osborn et al., 1992).
Figure 1.10: Structure of VCAM-1 different isoforms domains. Vascular adhesion molecule1 (VCAM-1) has either six or seven immunoglobulin domains followed by a transmembrane region and a short cytoplasmic tail.
It was determined that either the first of the homologous fourth domain of
VCAM-1 are required for VLA-4-dependent adhesion (Jackson, 2002). These
binding sites can function in the absence of the other. When all are present, cell
binding activity is increased relative to monovalent forms of the molecule. Thus,
VCAM-1 exemplifies evolution of a functionally bivalent cell-cell adhesion molecule by intergenic duplication (Osborn et al., 1992).
The characterization to the genomic structure of the 5’-flanking sequences
of the human VCAM-1 promoter was also performed. It was identified a functional potent promoter cis-acting sequences that direct the cytokine-induced transcription. Within the cytokine-responsive region of the core promoter were functional NF-κB and GATA elements. Upstream of the core promoter, the VCAM-1
5’flanking sequence contained a negative regulatory activity. NF-κB mediate in
this way activation of VCAM-1 gene expression (Neish et al., 1992).
Cell adhesion and adhesion molecules have been shown to contribute to the
pathogenesis of a large number of common human disorders and tumor cell metastasis in cancer. Several studies have demonstrated that cell adhesion molecules are
1.7 Signal transduction pathways
involved in signal transduction pathways (Adams & Watt, 1993). These molecules
transmit signals from the extracellular matrix on the cell interior (outside-in) and
from the inside of the cell to the outside of the cell (inside-out) similar to those
transduced by growth factors, hormones and cytokines. These results might be
extremely significant in metastatic spread and the treatment of a large number of
human disorders.
Signal transduction pathways
It has become clear over the last few years that, in addition to enabling leukocytes
to adhere to endothelium, adhesion molecules are also involved in intracellular
signal transduction. Leukocyte responses to integrin engagement have been extensively studied, while responses of endothelial cells have received much less attention. Nevertheless, leukocyte adhesion is known to be associated with alterations
in the functional state of endothelium, affecting surface protein expression, secretory function, permeability to macromolecules, and leukocyte transmigration.
These responses are associated with intracellular signals, including cytoskeletal
modification, protein phosphorylation, and calcium influx.
Transcriptional regulation, a critical basal mechanism in fundamental biologic
processes, requires the participation of several classes of proteins: those that binds
specific DNA sequences, those associate with transcriptional regulators through
protein-protein interactions (coactivators or corepressors) and those that perform
an architectural function. Collectively, these proteins interact with the components of the basal transcription apparatus to affect gene transcription. In addition,
it has been widely shown that most cytokines action involves the activation of
transcription factors (e.g. NF-κB, AP-1) and protein kinases (e.g. PKA and PKC)
that in turn, regulate the expression of many target genes, indispensable to the
maintenance of the inflammatory state and are involved in the pathophysiology of
inflammatory diseases (Kleinert et al., 1998; Hanada & Yoshimura, 2002).
cAMP-response element(CRE)-binding protein (CREB)
Another important transcription factor is the cAMP-response element (CRE) binding protein (CREB). CREB is a 43 kDa nuclear transcription factor member of a
family of cAMP-responsive activators. In mammalian systems this family includes also the activating transcription factor 1 (ATF1) and the cAMP response
element modulator (CREM)(Mayr & Montminy, 2001).
As indicates by its name, CREB is activated by phosphorylation in response
to, among other signals, cAMP. The accumulation of cAMP in response to activation of guanine-nucleotide-binding (G)-protein-coupled receptors induces most
cellular responses through the cAMP-dependent protein kinase (PKA).
The primary structure of the CREB family reveals a centrally located 60 amino
acid kinase inducible domain (KID). This domain contains the a PKA phosphorylation site (RRPSY) as well as several potential phosphorylation sites for casein
kinase I and II (Brindle et al., 1993; de Groot et al., 1993). There is also a basic
region of leucine zipper (bZIP) dimerization domain located at the carboxy terminally site in all members of the family.
Phosphorylation of the serine residue at 133 (Ser 133), promotes recruitment
of the transcriptional co-activator CBP and its paralogue p300 (Kwok et al., 1994;
Arias et al., 1994). It was demonstrated that Ser 133 phosphorylation is response
to cAMP stimulation is sufficient to induce target gene expression through promoters containing only CRE site. At the same time additional promoter bound
factors seems to be required for gene activation by CREB in response to mitogen
and stress signals. This cooperative interaction permits the efficient recruitment
of CBP (Mayr et al., 2001).
At the basal state, PKA resides in the cytoplasm as an inactive heterotetramer
of paired regulatory (R) and catalytic (C) subunits. Induction of cAMP liberates
the C subunits, which passively diffuse into the nucleus and induce cellular gene
expression by phosphorylating CREB at serine residue 133 (Figure 1.11).
1.7 Signal transduction pathways
The mechanism by which the cAMP-signaling pathway can achieves specificity include:
• compartmentalization of PKA via binding to scaffolding proteins;
• regulated expression of the distinct regulatory and catalytic subunit according cell and tissue;
• differential combinations of the regulatory and catalytic subunit isoforms.
It has been reported that specific localization and association of PKA type I is
activated on the cytoplasm and by a downstream pathway activated CREB phosphorylation that induced gene transcription (Constantinescu et al., 2002).
Furthermore, it has been demonstrated this transcription factor is necessary
for the activation and induction of several targets genes. Among them, VCAM-1
(Ono et al., 2006), E-selectin were reported to be regulated by this transcription
factor on endothelial cells (Gerritsen et al., 1997).
Recent results in HUVEC cells demonstrated that after TNF-α stimulation the
E-selectin gene activation is dependent of CBP and the closely related factor p300
that can interact with p65. The induction is dependent of chromatin remodeling by
selective histone modification involving hyper-acetylation, phosphorylation and
methylation (Edelstein et al., 2005).
NF-κB is an important transcription factor that plays a central and evolutionary
conserved role in many cellular responses to environmental changes. Several
pro-inflammatory genes involved in controlling, for example, cell adhesion, immune stimulation, apoptosis, chemoattraction, differentiation, extracellular matrix
degradation, redox metabolism, and production of mediators have been shown to
Figure 1.11: Activation of the cAMP-CREB signalling pathway. Induction of adenylyl cyclase (AC) by ligand (L)-bound receptor (R) proceeds through activation of the heterotrimeric G
protein (G). Increases in the levels of cellular cAMP promote dissociation of the protein kinase A
(PKA) heterotetramer, which consists of paired regulatory (R) and catalytic (C) subunits. Liberated C subunits migrate into the nuclear compartment by passive diffusion and phosphorylate the
cyclic AMP response element (CRE)-binding protein (CREB) at a single phospho-acceptor site,
Ser133. The phosphorylation promotes transcription by recruitment of the co-activator CREBbinding protein (CBP). CBP mediates transcriptional activation through its association with RNA
polymerase II (Pol II) complexes and through intrinsic histone acetyltransferase activity. Target
gene activation is terminated by the serine/threonine phosphatase PP-1-mediated dephosphorylation of CREB. From (Mayr et al., 2001)
1.7 Signal transduction pathways
be regulated by NF-κB (Kempe et al., 2005).
NF-κB exists in the cytoplasm of the majority of cell types as homo (Fujita
et al., 1992) or heterodimers (Inoue et al., 1991; Urban et al., 1991) of a family
of structurally related proteins. Five proteins belonging to the NF-κB family have
been identified in mammalian cells: p65 (RelA), c-Rel, RelB, p50/p105 (NF-κB1)
and p52/p100 (NF-κB2). The first three are produced as transcriptionally active
proteins; the latter are synthesized as longer precursor molecules of 105 and 100
kDa respectively, which are further process to the smaller, transcriptional active
forms by processed that are not fully understood (Ghosh et al., 1998).
Each member of this family contains a conserved N-terminal region called
the Rel-homology domain (RHD) and the nuclear localization signal (NLS). The
RHD domain is responsible for DNA binding (Schreck et al., 1990), dimerization
and association with the inhibitory proteins (Verma et al., 1995)(Figure 1.12).
NF-κB dimers are sequestered in the cytosol of unstimulated cells via noncovalent interactions with a class of inhibitory proteins called IκB. These proteins
also comprise a structurally and functionally related family of molecules (Verma
et al., 1995).
Seven IκB molecules have been identified: IκBα, IκBβ, IκBγ, IκBε, Bcl-3,
p100 and p105 (Baeuerle, 1998b; Link et al., 1992). All known IκB proteins contain multiple copies of a 30–33 amino acid sequence called ankyrin repeats; and
the specific interaction between the ankyrin repeats and the RHD is the defining
feature of the association between NF-κB and IκB. Through these associations,
IκB molecules mask the NLS of NF-κB. Thus, IκB degradation would simply
lead to unmasking of the NLS, allowing free NF-κB dimers enter the classical
nuclear import pathway (Verma et al., 1995).
The nuclear translocation of this protein complex may be due to cellular stimulation with inflammatory cytokines, phorbol esters, UV radiation. The phosphorilation, ubiquitination, and proteosomal degradation of IκB causes the nuclear
Figure 1.12: Schematic Presentation of NF-κB and IκB Structure. The numbers refer to the
ankyrin repeats. Right circles represent p50 and left circles p65 with their two Ig-like domains.
Dashed lines indicate sequences missing from the structures. RHD, rel homology domain; P,
phosphate groups on serines 32 and 36; C and N, C and N termini of the three proteins. Shown 1
to 6 IκBα domains. From (Baeuerle, 1998b)
1.7 Signal transduction pathways
translocation and is involved into the inflammatory response by induction of different genes (Ghosh & Karin, 2002).
TNF-α and IL-β act as a primary endogenous inducers of NF-κB. When cells
are exposed to those pro-inflammatory cytokines, a cascade of events leads to the
phosphorylation and subsequent degradation of IκB. As the result, NF-κB is liberated and can enter to the nucleus for gene expression activation. The stimulation
and activation of NF-κB do not require protein synthesis, there is a rapid and efficient induction of target genes (May & Ghosh, 1998).
Activation of NF-κB through IκB phosphorylation and degradation depends
on IκB kinases (IKKs) activity (May & Ghosh, 1998). The IKK complex is composed of three subunits, the catalytic subunits IKKα (IKK1) and IKKβ (IKK2)
and the regulatory subunit IKKγ (IKKAP or NEMO, NF-κB essential modulator),
and was originally identified as a high-molecular-weight kinase complex able to
phosphorylate serines 32 and 36 of IκBα (Verma et al., 1995).
There is evidence that IkBα, which is very rapidly resinthesized after degradation, can enter to the nucleus and remove IκBα from DNA. The discovery of
the leucin rich nuclear export sequences (NES) supported this idea (ArenzanaSeisdedos et al., 1997). The inactive complex is then transported back into the
cytoplasm or degraded in the nucleus thereby completing a cycle of activation and
inactivation of NF-κB. However, NES sequence was not found in IκBβ (Malek
et al., 2001), protein that was shown to be functional equivalent to IκBα (Cheng
et al., 1998). Futhermore, the mechanism responsible for the nuclear uptake remained controversial. Moreover, the biological significance of this process its yet
to be establish.
On the other hand, it was demonstrated a basal phosphorylation of IκBα in
un-stimulated cells. This basal phosphorylation occurs at the carboxy-terminal
casein kinase II sites. The presence of free IκBα in un-stimulated cells would
prevent rapid induction and reduce the sensitivity of the NF-κB system (Barroga
et al., 1995). It is important to note that IκBα in its un-stimulated state is con45
tinuously turning over (Rice & Ernst, 1993; Henkel et al., 1993; Miyamoto et al.,
1994). The half-life of IκBα is ∼2.5 hr in the 70Z/3 murine pre-B cell line, making it a very unstable protein (Miyamoto et al., 1994). Moreover, the half-life of
Rel/NF-κB is much longer in the same cells (Miyamoto et al., 1994). This is in
agreement with an earlier observation that NF-κB is regulated by a labile inhibitor
(Sen & Baltimore, 1986). Additionally, it explains why inhibition of protein synthesis results in NF-κB activation (Sen & Baltimore, 1986). If IκBα turning over
faster than NF-κB, the lack of IκBα synthesis will eventually lead to the presence
of free NF-κB.
Recently, it was shown that IκBα was able to regulate other pathways such
as p53, a tumor suppressor protein, by preventing the p53 nuclear translocation.
The C terminal of IκBα enhanced cell dead, which suggests that may be a proapoptotic protein. Interestingly, the relationship of NF-κB, p53 and IκBα and the
mechanism remains to be determined (Li et al., 2006).
An aspect of the NF-κB system that has not been extensively studied is the
kinetics of nuclear translocation of NF-κB proteins following activation. Although complete IκBα degradation and maximum DNA-binding activity appears
in <10 min following stimulation in some cells, the amount of NF-κB proteins
that translocate into the nucleus within the same period is <l0%-20% of total NFκB proteins (Miyamoto et al., 1994). A possible explanation for this effect is that
some NF-κB proteins may be associated with other IκB proteins, such as IκBβ,
Bcl-3, p105, and p100. Also, the nuclear translocation machinery may reach a
saturation point. Additionally, there may be others unexplored regulatory steps
important for the nuclear translocation.
Anti-inflammatory inhibition of NF-κB
Inhibitors of NF-κB activation are useful tools for elucidating molecular mechanism involved in gene expression. The regulatory role of NF-κB in inflammatory
pathways can be further characterized when several mechanistically distinct in46
1.7 Signal transduction pathways
hibitors are studied in the same model of gene expression.
Several steps of the NF-κB signal transduction pathway can be targeted by
various inhibitors:
• IKK activation;
• IκB phosphorylation and degradation;
• NF-κB nuclear translocation and transcriptional activity.
Several studies demonstrated that different molecules were able to inhibit NFκB pathway. This molecules were classified according the structure and activity include: aspirin, salicylates, nonsteroidal anti-inflammatory drugs, glucocorticoids, antioxidants, proteasome inhibitors, antisense oligodeoxynucleotides, natural compounds and cell penetrating peptides (Delhalle et al., 2004). All of them
were reported to function through distinct mechanisms in vivo and in vitro as are
summarized in Table 1.1.
The generation of reactive oxygen species (ROS) by phagocytic leukocytes
(neutrophils, monocytes, macrophages, and eosinophils) is one of the most important hallmarks of the inflammatory process. By oxidizing essential cellular
components of invading pathogens, reactive radicals and oxidants also represent
the first line of defense against microorganisms (Hensley et al., 2000). In addition, to promoting general cytotoxicity, ROS may also act to up-regulate proinflammatory gene expression by activating NF-κB, a process that is itself sensitive to the cellular redox state (Schoonbroodt & Piette, 2000). Diverse agents that
cause oxidative stress can activate NF-κB (Schreck et al., 1991; Zhang & Chen,
2004) and numerous stimuli that activate NF-κB, including cytokines, phorbol esters, LPS, and CD3 engagement, increase the levels of intracellular ROS (Bowie
& O’Neill, 2000a). Although evidence for the role of ROS in pro-inflammatory
NF-κB activation remains circumstantial, more convincing studies demonstrated
that a variety of antioxidant molecules, such as N-acetylcysteine (NAC), dithiocarbamates, vitamin E derivatives, and glutathione peroxidase, can inhibit NF-κB
Table 1.1: NF-κB inhibitors that demonstrate anti inflammatory activity in experimental
aspirin, sulfasalazine, triflusal
ibuprofen, sulindac, tepoxalin
dexamethasone, hydrocortisone
Anti-sense oligodeoxynucleotides
anti-p50, anti-p65
Transcription factor
Natural compounds
flavanoids, polyphenols, sesquiterpene lactones, curcumin, sesterterpene
PDTC, N-acetylcysteine, Vitamin
E, Vitamin C
Proteasome inhibitors
lactacystin, MG132, TLCK, TPCK,
PSI, PS-519, PS341
In vitro
In vivo
Synovial fibroblast, lung epithelial
cells, dendritic cells, monocytes,
macrophages, T-cells, endothelial
cells, vascular smooth muscle cells.
Contact hypersensitivity, zymosan-induced
paw inflammatio
Macrophages, endothelial cells, Tcells
Zymosan-induced inflammation in the paw
and spinal cord
Macrophages, endothelial cells,
pulmonary epithelial cells, T-cells
Carrageenin-induced air pouch, peritoneal
sepsis, myocardial contractile depression
Fibroblast, B-cells, T-cells
Graft rejection, septic shock
Endothelial cells, vascular smooth
muscle cells, macrophages
Rheumatoid arthritis, ischemia-reperfusion
injury, nephritis, carrageenin-induced paw
inflammation, Arthus reaction
T-cells, macrophages, fibrosarcoma
and epithelial cells
Macrophages, monocytes, T-cells
Septic shock, neutrophilic alveolitis, multiple organ injury, experimental allergic
Macrophages, monocytes, T-cells,
Asthma, septic shock, neutrophilic alveolitis, cerebral and myocardial ischemiareperfusion injury
Macrophages, T-cells, endothelial
cells, vascular smooth cells
Septic shock, zymosan-induced peritonitis, PMA-induced ear edema, carrageenininduced paw inflammation, Arthus reaction,
inflammatory bowel disease
1.7 Signal transduction pathways
activation (Szotowski et al., 2007; Bowie & O’Neill, 2000b).
Indeed, dithiocarbamates are widely used in basic and clinical resarch and
seams to be more potent and effective than others antioxidants as NAC and glutathione (GSH) (Zhu et al., 2002). In agree with different studies, the antioxidant
molecular mechanism of NAC and dithiocarbamates is different. In a study conducted in human pulmonary vascular endothelial cells that had been pre-incubated
with NAC and stimulated with TNF-α. NAC attenuated TNF-α induced activation of the mitogen-activated protein (MAP) kinase cascades and in these way
intracellular GSH levels were increased (Hashimoto et al., 2001). However NAC
effects as antioxidants on endothelial cells through NF-κB pathway seams to be
controversial an not fully understood (Schubert et al., 2002).
Pyrrolidine dithiocarbamate (PDTC) is a NF-κB inhibitor (Schreck et al., 1992).
It was demonstrated that PDTC treatment prevents IκBα degradation, thereby
blocking NF-κB activation (Tamada et al., 2006). Indeed, PDTC does not lead
to the appearance of a newly phosphorylated IκBα variant, suggesting that the
drug blocked phosphorylation. In addition to IκBα degradation, phosphorylation
is necessary for NF-κB activation but not for the direct release of IκBα. The
modification seems to dramatically enhance the rate of proteolytic breakdown by
proteosome (Traenckner et al., 1994). Additionally, PDTC can inhibits NF-κB
induction by lipopolisaccharide. In this way regulates the expression of endogenous tissue factor, a glycoprotein receptor for coagulation factors VII and VIIa on
HUVEC cells (Orthner et al., 1995). Moreover, PDTC is able to inhibit specifically the production of IL-6, IL-8 and granulocyte macrophage colony stimulating
facto in response to inflammatory mediators on HUVEC cells (Muoz et al., 1996).
As described previously, E-selectin, ICAM-1 and VCAM-1 expression are
under the control of NF-κB signalling (Hanada & Yoshimura, 2002; Lin et al.,
2007) . The combined treatment of cytokines such as TNF-α and IL-1β induced
the expression of these genes on HUVEC cells. The NF-κB role was confirm by
over-expression of dominant negative inhibitor IκB protein and also by combinatory treatment with several inhibitors (PDTC, dexametasone and others)(Kuldo
et al., 2005). However, NF-κB contribution for VCAM-1 and ICAM-1 expression
is still controversial (Zerfaoui et al., 2008). Others inflammatory markers such as
NO levels and the inducibel nitric oxide synthase are under the control of NF-κB
signalling (Beck & Sterzel, 1996).
Thus, different antioxidants inhibit NF-κB activation via multiple mechanisms,
which may depend on the properties of the antioxidant, its specific target in the
treated cell, or the origin of the treated cells. Development of novel NF-κB inhibitory drugs bares an important significance in the prevention and treatment of
cardiovascular diseases. Natural antioxidants originated have a huge advantage
over existing drugs being non-toxic and inexpensive. Their potency as NF-κB
inhibitors on endothelial cells, and the novel mechanism of activation, provide a
strong rational for further studies both in vitro and in vivo.
Chapter 2
Several clinical observations pointed out the critical role of bilirubin on the risk
of cardiovascular atherosclerotic disease. It remains unclear whether modestly
elevated or high normal levels of serum bilirubin (as in Gilbert’s syndrome) are
protective or harmful in non-hepatic diseases.
It has been proposed that the antioxidant properties of bilirubin against atheromatous disease might be exerted at multiple steps preventing: the peroxidation of
lipoproteins in the intima, the oxidation of membrane phospholipids in the endothelial cells and macrophages or even the activation of metalloproteinases in
the intima (Rigato et al., 2005).
Indeed, bilirubin might also act as a second messenger and not merely as a
pharmacological compound. It was shown that bilirubin might have a direct regulatory effect by binding the aryl hydrocarbon receptor (Seubert et al., 2002) or
indirectly by activation of constitutive androstane receptor (Huang et al., 2004).
Bilirubin, that so far was regarded as a waste product of heme metabolism,
must be consider as an active molecule with many unexplored functions and therapeutic potential.
The aim of this study is to investigate the effect of the unconjugated bilirubin
(UCB) in the endothelial dysfunction, as the earliest event in the development of
the atherosclerotic disease. Specifically, UCB effects on the nitric oxide metab51
Aim of the Study
olism, the vascular adhesion molecules expression and the main signaling pathways involved in the inflammatory response. The main goal of the present work
is to elucidate the bilirubin molecular mechanism involved in the atheromatous
diseases that correlates with the previous epidemiological evidence.
Chapter 3
Endothelial Cells
The vascular endothelium is a single layer of cells lining the inside face of all
blood vessels and constitute an important metabolic organ which is critically involved in the generation and the regulation of multiple physiological and pathological process such as inflammation, atherosclerosis and angiogenesis (Pratico,
Endothelial cells are dynamic and have both metabolic and synthetic functions. They exert significant autocrine, paracrine and endocrine actions and influence smooth muscle cells, platelets and peripheral leucocytes (Cines et al., 1998).
The endothelium is sensitive to growth factors and exchanges messengers with
blood and sub-endothelium, the extracellular matrix and the smooth muscle cell
of the media in large vessels (Nathan & Sporn, 1991).
Even cells from the same part of the vasculature can have varied responses.
It is also important to note that responses of cultured endothelial cells may not
reflect responses seen in the same cells in vivo, and the immortalized endothelial
cell lines used in many laboratory studies may, in particular, have altered expression patterns of key markers compared with cells studied in vivo.
In the present study a murine microvascular endothelial cells (H5 V) was used.
This cell line is a transformed endothelial cell line from heart mice with a retro53
Materials and Methods
Figure 3.1: Morphology of H5 V cells in vitro. H5 V 10X Normal Light
viral construct encoding polyoma middle-sized T antigen (Garlanda et al., 1994)
(Figure 3.1). To further confirm our data, a human umbilical vein endothelial cells
(HUVEC), isolated from the vein of the umbilical cord (Booyse et al., 1981; Jaffe
et al., 1973) (Figure 3.2) was also considered.
Unconjugated bilirubin (UCB)(Sigma Chemical Co, St. Louis MO), was purifeied as described by McDonagh and Assisi (McDonagh & Assisi, 1972). Dulbecco’s Phosphate saline, Dulbecco’s modified Eagles’s medium high glucose
(DMEM/High glucose), penicillin and streptomycin were purchased from Euroclone U.K. and Fetal calf serum was obtained from Invitrogen Carlsbad, California.
Chloroform (99%) was obtained from Carlo Erba Milan, Italy. Fatty acid free
bovine serum albumin (BSA), tetrazolium salt (MTT), DMSO, TNF-α, and all
other reagents and chemicals were purchased from Sigma-Aldrich Italy Milan,
3.3 UCB solutions
Figure 3.2: Morphology of HUVEC cells in vitro. HUVEC 40X Normal Light
UCB solutions
The free (unbound) plasma bilirubin concentration (Bf), a little fraction of total
bilirubin concentration, is the principal determinant of tissue uptake and toxicity.
However, methods to estimate the Bf from medium has rarely been performed
(Nelson et al., 1974; Jacobsen & Wennberg, 1974). Indeed, in most of the in vitro
studies of cellular toxicity the UCB levels were higher than those seen in physiological and pathophysiological conditions (Ostrow et al., 2003b).
Recently, in our group the Bf bilirubin levels in tissue culture media were evaluated by a standardization of peroxidase method (Roca et al., 2006). The methods
involves minimal dilution of the sample, minimizing the effect of dilution of the
albumin concentration on the binding affinity (Ahlfors, 1981). The effects of albumin concentration on bilirubin-albumin binding measured were evaluated by the
peroxidase method in order to reproduce different physiologic Bf levels. The molar ratio of UCB and bovine serum albumin (BSA 30 µM) was tested in DMEM
high glucose in order to obtain variable doses of Bf (Figure 3.3). Similar results
were obtained with M199 medium used in HUVEC culture.
Materials and Methods
Figure 3.3: Relationship of Bf to UCB with three different albumin preparations.(N) FCS
10%, (2) BSA 30 µM, () HSA 30 µM. Data represent the mean ±SD of three independent
experiments in triplicate. From (Calligaris et al., 2007)
3.4 Culture conditions
Purified UCB was dissolved in chloroform at a concentration of 0.85 mM
and aliquots were dried under nitrogen. Immediately before each incubation, an
aliquot was dissolved in DMSO (0.3 µL of DMSO per µg of UCB, and diluted
with serum free medium containing 30 µM bovine serum albumin (BSA).
Experiments were performed with two final UCB concentrations of 2.5 and
5 µM, yielding unbound UCB concentrations (Bf) calculated to be respectively
15 and 30 nM. In order to standardize DMSO-related effects, a further volume
of DMSO was added to the final solution to reach an equal total amount in all
treatment groups. To minimize photo-degradation, all the experiments with UCB
were performed under light protection (dim lighting and vials wrapped in tin foil).
Culture conditions
H5 V cells were grown up to ∼80% of confluence in Dulbecco’s Modified Eagle’s Medium High Glucose (DMEM) containing fetal calf serum (10% vol/vol),
penicillin (100 U/mL) and streptomycin (100 g/mL). After confluence cells were
washed three times with PBS and then incubate in six different combinations of
• Control group: serum free medium containing BSA (30 µM) and DMSO
(0.29% v/v).
• TNF alone group: add TNFα 20 ng/mL, serum free medium, BSA, DMSO.
• UCB 15 alone: add UCB at Bf 15 nM, serum free medium, BSA, DMSO.
• UCB 30 alone: add Bf 30 nM, serum free medium, BSA, DMSO.
• Co-treatment UCB 15-TNF: add Bf 15 nM, TNFα 20 ng/mL, serum free
medium, BSA and DMSO.
• Co-treatment UCB 30-TNF: add Bf 30 nM, TNFα 20 ng/mL, serum free
medium, BSA and DMSO.
Materials and Methods
Human Umbilical Vein Endothelial Cells (HUVEC) were kindly gifted by
Prof. F. Tedesco from Dept. of Physiology an Pathology University of Trieste.
Cells were cultured in medium M199 with Hanks’ salt and NaHCO3 (SIGMA
M7653) enriched with fetal calf serum (20%), bovine cerebral extraction (50
µg/mL, gifted by Prof. F. Tedesco) (Maciag et al., 1979), Na-heparin (50 µg/mL,
EPSOCLAR, Biologici Italia Laboratory Srl, Milan, Italy), penicillin (100 U/mL)
and streptomycin (100 µg/mL). Cells were grown up on 25 cm 2 plastic flasks covered with gelatine (1%, SIGMA G-9391) in sterile bidistilled water (v/v). Cells
were used for experiments between the 4th and 6th cell passage. Cells were treated
in the same conditions as described previously for H5 V cells.
Cytokines treatment
Cytokines represent a group of multi-functional substances that could be involved
in the initiation and amplification of the inflammatory process regulating the expression of many target genes. Human TNF-α, one of the pro-inflammatory cytokine, was added to the culture in order to describe UCB contribution on its
effects. TNF-α time and dose response were determined as indicated in Table 4.2
and Table 4.3.
Endothelial cell susceptibility
In this part of the study, different endothelial susceptibility to UCB and TNF-α in
the two cell lines (HUVEC and H5 V) was analyzed. The approaches used for this
point to test cytotoxicity were:
• assess of Lactate Dehydrogenase (LDH) release, to evaluate the presence
and degree of membrane damage;
• analysis of Mitochondrial Toxicity by MTT test (Liu et al., 1997).
LDH release test
Lactate dehydrogenase (LDH) is a stable cytoplasmic enzyme rapidly released
into the cell culture supernatant upon the damage of the plasma membrane (Hu &
3.5 Endothelial cell susceptibility
E., 1970).
The Cytotoxicity Detection Kit (LDH, ROCHE Applied Science, Penzberg,
Germany) was used to detect the cell damage. This kit allows to measure the LDH
activity by a colorimetric reaction. In the first step NAD+ is reduced to NADH/H+
by the LDH-catalyzed conversion of lactate to pyruvate. In the second step the
catalyst (diaphorase) transfers H/H+ from NADH/H+ to the tetrazolium salt INT
(2-[4-iodophenyl]-3- [4-nitrophenyl]-5-phenyltetrazolium chloride) which is reduced to formazan. The formazan formed during the reaction is proportional to
the number of lysed cells and shows a maximum absorption at about 500 nm light
The H5 V monolayers cells were cultured on 24-well plates and treated for 24
hours, as indicated in the Table legend 4.1, with different UCB concentrations
with or without TNF-α (20 ng/mL).
The culture media was kept for this assay and cells were lysated with 1% Triton X-100. 50 µl of the supernatant and equal amount of the lysate cells were
incubated with the reaction mix for 20 minutes protected from light at room temperature. The absorbance of the samples at 490 nm was determined in a LD 400C
Luminescence Detector (Beckman Coulter S.p.A, Milan, Italy). Results were expressed as percentage of the maximum amount of releasable LDH, obtained by
lysing cells.
Mitochondrial toxicity by MTT test
One of the most frequently used methods for measuring cell proliferation and cytotoxicity is the reduction of 3(4,5-dimethyltiazolyl-2)-2,5 diphenyl tetrazolium
(MTT), a monotetrazohum salt (Mosmann, 1983).
H5 V and HUVEC cells were cultured on 24-well plates and treated for variable periods of time, as indicated in the Figure legend 4.1, with different UCB
concentrations with or without TNF-α (20 ng/mL).
Materials and Methods
A stock solution of MTT was dissolved in PBS pH 7.4 at 5 mg/mL. MTT solution was further diluted to 0.5 mg/mL in DMEM/High Glucose without phenol
red to avoid interference with the plate reading. Cells were incubated with DMEM
containing MTT for 2 hours at 37 ◦ C. At the end of incubation period, the medium
was replaced with the addition of 1 ml isopropanol/HCL 0.04 M, to dissolve MTT
formazan crystals. Samples were then gently shacked in an orbital shaker for 2
hours at 37 ◦ C. After centrifugation at 10,000 RPM for 3 min, absorbance values,
at a length light of 570 nm, were determined in a LD 400C Luminescence Detector (Beckman Coulter S.p.A, Milan, Italy). Results were expressed as percentage
of control cells, not exposed to UCB, which was considered as 100% viability.
Endothelial dysfunction analysis
The different markers for endothelial dysfunction were evaluated by:
• measurement of Nitric Oxide levels;
• gene expression analysis of the adhesion molecules and Nitric oxide Synthase enzymes;
• protein expression analysis of the adhesion molecules.
Nitric oxide
Nitric oxide (NO) is unstable in an aerobic environment. The most commonly
employed methods for analysis of NO in aqueous solutions are the colorimetric
assays by Griess reagent. Through the years, modifications to the original reaction
described by Griess in 1879 have been reported (Nims et al., 1996; Cook et al.,
This methodology is based on the fact that free NO, reacts with oxygen to
yield reactive nitrogen oxide intermediates that can subsequently oxidize or nitrosate various substrates. In aerobic aqueous solution several stable and nonvolatile breakdown products can be detected, among them nitrate NO3– and sub60
3.6 Endothelial dysfunction analysis
Figure 3.4: Chemistry of the Griess Reagent. Chemical reactions involved in the measurement
of NO2– using the Griess Reagent system
sequently nitrite NO2– (Green et al., 1982).
The colorimetric assay for evaluating NO concentration depends on the nitrosative properties of the NO intermediates NO2– . The nitrosation of sulfamide by
acidic nitrite solutions in the presence of naphthylethylenediamine dihydrochloride (NEDD) results in an azo dye with absorption maximum at 540 nm light
length (Figure 3.4).
H5 V and HUVEC cells were cultured on 6-well plates and treated for variable
periods of time, with different UCB concentrations with or without TNF-α (20
ng/mL) as indicate in Table 4.3 and Figure 4.2.
Culture media was kept for the assay, the absorbance was determined at 540
nm in a spectrophotometer Beckman DU 640 (Beckman Coulter S.p.A, Milan,
Italy). Values were compared against a standard curve with increasing concentrations of nitrite (1.56 to 100 µM). Cell lysates were stored for protein determination by Bicinchoninic Acid Protein Assay (BCA)(Smith et al., 1985) following
the procedure’s instructions (B-9643, SIGMA). Results were expressed as nmol
NO2– mg/mL protein.
Materials and Methods
Gene expression analysis
RNA extraction
H5 V and HUVEC cells were cultured on 6-well plates and treated for 2, 6 and
24 hours, with different UCB concentrations with or without TNF-α (20 ng/mL).
Total RNA was isolated by Tri Reagent solution according to the manufacture’s
suggestions (SIGMA, Missouri, USA. T9424). The total RNA concentration and
the purity were quantified by spectrophotometric analysis in a Beckman DU640.
For each sample the A260 /A280 ratio comprised between 1.8 and 2.0 was considered as good RNA quality criteria. The integrity was determined by agarose
gel electrophoresis and staining with ethidium bromide, indicating that the RNA
preparations were of high integrity. Isolated RNA was dissolved in RNAse free
water and store at −80 ◦ C until analysis.
mRNA Quantification by Real-Time RT-PCR
Expression analysis of target gene were performed by Real Time RT-PCR technology, using specific primers for detection of the following markers of endothelial
dysfunction: eNOS, iNOS, ICAM-1, VCAM-1, E-selectin.
Retrotranscription using 1µg of total RNA was performed with an iScriptT M
cDNA Synthesis Kit (BIO-RAD Laboratories, Hercules, CA, USA Catalog # 1708891) according to the manufacture’s suggestions. The reaction was run in a Thermal Cycler (Gene Amp PCR System 2400, Perkin -Elmer, Boston, MA, USA) at
25 ◦ C per 5 min, 42 ◦ C for 45 min, 85 ◦ C for 5 min. The final cDNA was conserved
at −20 ◦ C until used.
Real-time RT-PCR was performed according to the iQ SYBR Green Supermix
protocol (Bio-Rad Laboratories). The selected genes and their primer sequences
for mouse and human are reported in Table 3.1, and Table 3.2, respectively. The
primers were designed using Beacon Designer 4.02 software (PREMIER Biosoft
International, Palo Alto, CA, USA). All primer pairs were synthesized by Sigma
Genosys (Cambridgeshire, UK).
3.6 Endothelial dysfunction analysis
Table 3.1: H5 V - Primer sequence designed for the mRNA quantification
Mouse - Gene
Accession number
Primer Forward
Primer Reverse
NM 08713.2
NM 010927.1
NM 010493.2
NM 011693.2
NM 011345.1
NM 007393.0
PCR amplification was carried out in 25 µL reaction volume containing 25 ng
of cDNA, 1 x iQ SYBR Green Supermix (100 mM KCL; 40 mM Tris-HCl; pH:
8.4; 0.4 mM each dNTP; 50U/mL iTaq DNA polymerase; 6 mM MgCl2 ; SYBR
Green I; 20 mM fluorescein; and stabilizers)(BIO-RAD Laboratories) and 250 nM
gene specific sense and anti-sense primers and 100 nM primers for 18S. Reactions
were run and analyzed on a Bio-Rad iCycler iQ Real-Time PCR detection system
(iCycler IQ software, version 3.1; Bio-Rad).
Cycling parameters were determined, and resulting data were analyzed by using the comparative Ct method as means of relative quantification. The relative
quantification was made using the Plaffl modification of the ∆∆Ct equation (Pfaffl,
2001; Tichopad et al., 2004). The relative gene expression levels of each transcript
were determined by comparison with a standard curve. The genes were normalized by dividing the expression value of a housekeeping gene βactin for H5 V cells,
hypoxanthine guanine phosphoribosyltransferase (HPRT) and βactin for HUVEC
cells. Melting curve analysis and gel electrophoresis were performed to check
product specificity. Results reported as indicated in the Figure legends represent
the mean of 3 different experiments.
Western blot
H5 V cells were treated as previosly described for 24 hours with different UCB
concentrations with or without TNF-α (20 ng/mL). Cells were then washed once
with PBS at room temperature and dissolved in cell lysis buffer, PBS containing
Materials and Methods
Table 3.2: HUVEC - Primer sequence designed for the mRNA quantification
Human - Gene
Accession number
Primer Forward
Primer Reverse
NM 000603
NM 000625.3
NM 000201
NM 001078
NM 000450
NM 001101
NM 000194
1% v/v of a protease inhibitor cocktail from Sigma (P-8340) and 2 mM PMSF
(phenylmethylsulfonylfluoride). Cells were placed on ice and disrupted by ultrasonic sonication (Bandelin Sonoplus, HD2070, Berlin, Germany; 3 times at 5 s at
30% of power).
Protein concentration in the lysate was determined by the Bicinchoninic Acid
Protein Assay (BCA)(Smith et al., 1985) following the instructions reported by
the supplier (B-9643, SIGMA).
Equal amounts of protein (60 µg) were subjected to sodium dodecyl sulphatepolacrilamide gel electorphoresis (SDS-PAGE). Molecular weight standards (Precision Plus Protein dual color standards, Bio-Rad) were used as marker proteins.
Samples were immersed in a boiling water bath for 5 min and then immediately
settled on ice. Proteins were loaded on 10% polyacrylamide gel by electrophoresis in a Mini Protein III Cell (Bio-Rad, Hercules, CA, USA). After SDS-PAGE,
gels were electrotransferred with a semi-dry blotting system at 100 V for 120
min onto immune-blot PVDF membranes (Bio-Rad) using a Mini Trans-Blot Cell
Membranes were incubated overnight at 4 ◦ C with commercial antibodies (Table 3.3) that allow the specific recognition of Vcam-1 (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA), Icam-1 (Santa Cruz Biotechnology), E-selectin (Santa Cruz
3.6 Endothelial dysfunction analysis
Table 3.3: Primary antibodies tested
Primary Antibody
(Catalog #)
Antibodies were dissolved in a solution containing skim milk (5%) and TTBS buffer (Tris/HCL 20 mM, Tween 20, 0.2%, NaCl 500 nM, pH 7.5) at the
dilution reported in Table 3.3. After three washes with T-TBS, membranes were
incubated for 1 hour at room temperature with a secondary antibody (SigmaAldrich Italy Milan, Italy). The following antibodies were used: IgG-anti-goat
(dilution 1:4000) for Vcam-1 or Icam-1 and IgG-anti-rabbit (dilution 1:1000) for
E-selectin, all conjugated with peroxidase.
Proteins bands were detected by peroxidase reaction and visualized by exposure of membrane in the ECL-Plus Western Blot detection system solutions (ECL
Plus Western Blotting Detection Reagents, GE-Healthcare Bio-Sciences, Italy).
Membranes were incubated with a stripping buffer (0.5 mM Tris/HCl pH 6.8,
0.2 %v/v SDS, 0.68%v/v β-Mercaptoethanol) for 30 min at 55 ◦ C followed by
overnight incubation with a commercial antibody specific for Actin recognition
(Table 3.3, SIGMA) according to the procedure previously described. After repeated washes, the membranes were incubated with secondary antibody IgG-antirabbit peroxidase conjugated (dilution 1:5000), for 1 hour at room temperature.
The immunoreactivity was visualized as previously described, by ECL-Plus detection kit.
The intensities of the autoradiographic bands were estimated by densitometric
scanning using NIH Image software (Scion Corporation Frederick, MD, USA).
Materials and Methods
Signal transduction pathways
The transcription factors involved in the expression of markers for endothelial
dysfunction were evaluated by:
• use of Inflammatory inhibitors (PDTC - NAC);
• evaluation of of CREB phosphorylation;
• assessment of NF-κB (p65 subunit) nuclear translocation.
To study the role of UCB on NF-κB pathway, cells were treated with PDTC
(Pyrrolidine dithiocarbamate) a specific inhibitor NF-κB. H5 V cells were treated
for 2 hours with PDTC (10 µM) alone or with UCB, as described above, in the
presence or absence of TNF-α. Cells were pre-treated with PDTC 1 hour before
incubation with TNF-α. PDTC was dissolved in serum free medium on the day
of treatment. Cells were then collected and the mRNAs were extracted. The expression of AMs was evaluated by Real Time RT-PCR.
UCB effects on NO levels were also evaluated after 24 hours treatment with
NAC in the same culture conditions described previously. NAC solution was
freshly prepared on the day of treatment and adjusted to pH 7.4 by the addition of
8 M NaOH. NAC dose response were determined as indicate in Figure legends 4.5.
cAMP-response element(CRE)-binding protein (CREB)
The H5 V monolayers cells were cultured on 6-well plates and pre-treated for variable periods of time, as indicated in the Figure legends 4.21, with different UCB
concentrations with or without TNF-α (20 ng/mL). Proteins were collected and
a gel electrophoresis (SDS-PAGE) was performed as described in Western Blot
The phosphorylated CREB at Ser 133 was detected by the PhosphoPlus CREB
antibody Kit (Catalog # 9190, Cell Signaling). The kit allowed the specific recog66
3.7 Signal transduction pathways
nition of the phosphorylation status of CREB at serine 133. Phospho-CREB specific antibody was used (dilution 1:500). The membranes were reprobed with an
antibody against total CREB (recognized phosphorylated and non phosphorylated
form) (dilution of 1:500). Secondary antibody IgG-anti-rabbit (dilution 1:1000)
conjugated with peroxidase was used. All antibodies were analyzed by the same
procedure previously described.
Preparation of total nuclear extracts
The total cytoplasmic and nuclear extracts were obtained by using minor modification of the Dignam’s method (Dignam et al., 1983). H5 V cells were seeded at
a density of 5x107 on 75 − cm 2 flasks and were treated with different UCB concentrations with or without TNF-α (20 ng/ml) for 30 minutes.
After treatment, the cells were collected by centrifugation at 800Xg for 10
min. The cells were resuspended in 400 µL cells ice-cooled solution A (10 mM
Hepes, pH 7.9, 0.1 mM MgCl2 , 10 mM KCl, 0.1 mM EDTA, pH 8, 0.1 mM
dithiotreitol, 0.5 mM phenylmethylsulphonyl fluoride, 1 mM Na orthovanadate
and 1 mM Na Fluoride). After 10 min ice incubation the cells were centrifuged at
800Xg for 5 min at 4 ◦ C. The supernatant containing the cytoplasm was collected
and stored at −80 ◦ C. The pellet containing nuclei was resuspended with solution
A and was centrifugated at 800Xg for 5 min at 4 ◦ C. The nuclear fraction was
resuspended in ice-cooled solution B (20 mM Hepes, pH 7.9, 420 mM NaCl, 1.5
mM MgCl2 , 0.2 mM EDTA, 5% glycerol, 0.1 mM dithiotreitol, 0.5 mM phenylmethylsulphonyl fluoride, 1 mM Na orthovanadate and 1 mM Na Fluoride). After
30 min ice incubation with constant stirring, the suspension was vortexed for 10
s, then centrifuged at 15,000Xg for 20 min at 4 ◦ C. The supernatant containing
nuclear extract was recovered and stored at −80 ◦ C.
The protein content of the extracts was determined by BCA method as described before. The nuclear and cytoplasmic extract fractions were analyzed by
SDS-page Western Blot.
Materials and Methods
Commercial antibody that allow the specific recognition of NF-κB p65 subunit(Catalog # SC-109, dilution 1:1000, Santa Cruz Biotechnology, Santa Cruz,
CA, USA) was used. To test nuclear enrichments, the presence of a nuclear matrix
protein p84 (Catalog # ab487, dilution 1:1000, Abcam, Abcam Inc., Cambridge,
MA, USA) was evaluated as marker (Portal et al., 2006). Secondary antibodies
conjugated with peroxidase (both from Sigma-Aldrich) IgG-anti-rabbit (dilution
1:2000), for NF-κB, and IgG-anti-mouse (dilution 1:2000), for p84, were used.
Both antibodies were analyzed by the same procedure previously described on
Western blot section.
Statistical analysis
All experiments were run in triplicate and repeated three times. Results are expressed as mean±SD. Oneway ANOVA with Tukey-Kramer post test was performed using GraphPad InStat version 3.00 for Windows 95 (GraphPad Software,
San Diego, CA, USA). Probabilities, ≤ 0.05 were considered statistically significant.
Chapter 4
Effects of UCB on cell viability
Bilirubin has been found to be toxic to many cell examined in vitro, including fibroblasts (Nelson et al., 1974), hepatocytes, erytrocytes, leukcocytes, liver (Czernobilsky & Dubin, 1965), HeLa (Shimabuku & Nakamura, 1983) and platelets(Amit
et al., 1992).
The effect of UCB and TNF-α on endothelial cell viability was evaluated.
Two different methods to analyzes cell viability were used, Lactate Dehydrogenase (LDH) release and Mitochondrial Toxicity (MTT) assay.
UCB did not affect the LDH release induced by TNF-α
The plasma membrane integrity was unchanged in presence of different doses of
UCB (Bf, 15, 30 and 100 nM). As expected, the addition of TNF-α (20 ng/mL)
significantly increased the extracellular LDH activity. However, no further effects
were observed when co-treatment TNF-α plus UCB were performed. Table 4.1
summaries the results obtained in H5 V cells.
UCB reduced endothelial cell viability
Based on the negative results of LDH and since MTT assay was demonstrated
to be a method more suitable for studying bilirubin cytotoxicity in a human liver
LDH release (%)
UCB (Bf nM)
- TNF-α
+ TNF-α
Table 4.1: Effect of UCB on cell viability - LDH release. H5 V cells were incubated with different doses of UCB (Bf 15, 30 and 100 nM), with or without treatment with TNF-α. Control cells
(UCB, Bf 0 nM) were treated as described in Materials and Methods. Cells were collected after 24
h of treatment, LDH released (%) into the cell medium was calculated. Results are expressed as
mean percentage values (%) of three independent experiment performed in triplicate. *: p< 0.05
versus control.
cell line (Ngai et al., 1998), H5 V cells were exposed to different doses of UCB
(Bf 15, 30 and 100 nM) with or without TNF-α (20 ng/mL) for 24 hours. Control cells were treated as described in Materials and Methods. UCB significantly
decreased H5 V vitality in a dose-dependent manner (Figure 4.1, D). However,
the co-treatment with TNF-α(20 ng/ml) did not modify UCB effects. Same results were obtained when cells were exposed to UCB with or without TNF-α (20
ng/ml) for 48 hours (data not shown).
In order to verify UCB effects on endothelial cell viability, HUVEC cells were
treated with different doses of UCB (Bf 15, 30 and 100 nM) with or without TNFα (20 ng/mL) for 2, 6 and 24 hours. Similarly to H5 V cells, UCB was able to
reduce endothelial cell viability in a dose dependent manner at 2 and 6 hours (Figure 4.1, A and B). Treatment for 24 hours greatly decreased the cell viability even
in control cells (Figure 4.1, C). Indeed, HUVEC cells have an initial reduction on
cell viability, even at 2 and 6 hours, due to the absence of fetal calf serum and
bovine cerebral extraction, conditions of control group (UCB, Bf 0 nM). This initial reduction of cell viability was significantly increased by treatment with TNF-α
alone. However, once UCB was add to the cell medium culture, co-treatment with
TNF-α(20 ng/mL) did not cause further effects.
4.2 Nitric oxide analysis
(NO2– nmol/mg
Table 4.2: Effect of different doses of TNF-α on NO production. H5 V cells were incubated
with different doses of TNF-α (0, 2.5, 5, 10, 20, 40 ng/mL). Cells were collected after 24 h of
treatment. Results are expressed as NO2– nmol/mg protein and represent means±SD, n=3. *: p<
0.05 versus control group (TNF-α 0 ng/mL).
Based on these results, in the following experiments we decided to removed
the treatment with high doses of UCB (Bf 100 nM), in order to avoid excessive
loss of cell survival. To summarize, UCB reduced in a dose dependent manner
the cell viability in both cell lines. UCB toxicity was manifested by impaired
mitochondrial function (MTT activity). However, UCB did not cause change in
the cellular permeability or necrosis, base on LDH release assay.
Nitric oxide analysis
The effect of increasing concentrations of TNF-α on NO production in H5 V cells
was evaluated. While TNF-α up to a concentration of 10 ng/mL did not influence
the NO production at 24 hours, when the concentration was over 20 a significant
decreased on the NO concentration in the cell medium was observed. Table 4.2
summaries the results obtained in H5 V cells.
On the other hand, the secretion profile of NO to cell medium, in cells treated
with or without TNF-α (20 ng/mL) was also evaluated. A time depended NO
basal increased was reveled. The NO reduction with TNF-α was seen at all times
studied (12h 86%, 24h 76% and 48h 78%, p< 0.05). Table 4.3 summaries the
results obtained in H5 V cells.
Figure 4.1: Effect of UCB on cell viability - MTT assay. HUVEC (A, B and C) and H5 V cells
(D) were incubated with different doses of UCB (Bf 15, 30 and 100 nM), with or without TNF-α.
Control cells (UCB, Bf 0 nM) were treated as described in Materials and Methods. Results are
expressed as mean percentage values (%) of three independent experiment performed in triplicate.
*: p< 0.05 versus control. #: p< 0.05 versus complete medium.
4.2 Nitric oxide analysis
NO (NO2– nmol/mg protein)
- TNF-α
+ TNF-α
Table 4.3: Time dependent effect of TNF-α on NO production. H5 V cells were incubated
with TNF-α (20 ng/mL) for 12, 24 and 48 hours. Cell medium was collected after treatment. Results are expressed as NO2– nmol/mg protein and represent means±SD, n=3. *: p< 0.05 compared
to respective cells without TNF-α.
Effect of UCB on NO levels in H5 V cells
To study the putative role of UCB in NO concentration, nitrite (NO2– ) production
in culture supernatant was measured. Treatments with UCB at two different undersaturation concentrations of free bilirubin (Bf 15 and 30 nM) with or without
TNF-α (20 ng/mL), for 24 and 48 hours were performed. The time periods (24
and 48 hours) were considering eNOS half life, which is about 20 hours (Govers &
Rabelink, 2001). As demonstrated before, TNF-α at a concentration of 20 ng/mL,
significantly reduces nitrite levels. The significant reductions of nitrite content,
by treatments with TNF-α (20 ng/mL), was not reversed by the presence of any
doses of UCB after treatments for 24 hours (Figure 4.2, A). Interestingly, the
significant reductions of nitrite content in culture supernatant induced by TNF-α
was reversed after 48 hours by the presence of UCB, either a Bf of 15 or 30 nM
(Figure 4.2, B).
Effect of UCB on NOS mRNA expression
We investigated, by Real Time RT-PCR, whether or not UCB modifies the gene
expression of the two Nitric Oxide Synthases (NOS), constitutive (eNOS) and inducible form (iNOS) in endothelial cells.
The expression of eNOS was not influenced by the treatment with TNF-α (20
ng/mL) at 2, 6 and 24 hours (Figure 4.3, A). On the other hand, the addition of
TNF-α (20 ng/mL) increased the expression of iNOS at 2, 6 and 24 hours (Figure
Figure 4.2: Effect of different doses UCB on NO production. H5 V cells were incubated
with different doses of UCB (Bf 15 and 30 nM), with or without TNF-α. Control cells (UCB,
Bf 0 nM) were treated as described in Materials and Methods. Cell medium was collected after
24 h (A) or 48 h (B) of treatment for NO2– levels determination. Results are expressed as mean
percentage values (%)±SD of control cell group, from three independent experiment performed
in triplicate.*: p< 0.05 versus control. #: p< 0.05 versus TNF-α alone group (UCB, Bf 0 nM plus
4.2 Nitric oxide analysis
4.3, B).
Co-treatments with TNF-α and UCB (Bf 15 nM) determined a slightly but significant reduction of mRNA expression at 2 hours (74%, p< 0.05). However, no
effect was seen in treatments with UCB alone. Interestingly, at 24 hours, the cotreatment with both UCB (Bf 15 and 30 nM) and TNF-α was able to increase the
levels of iNOS expression (160% and 126%, respectively) if compared to TNF-α
alone (considered as 100%) treatments (Figure 4.4).
According to this data we can postulate that UCB is able to modulate the
TNF-α effect on the induction of iNOS expression while as well as TNF-α, UCB
has been shown not be involved in the regulation of eNOS. These results suggest
that iNOS expression is affected by UCB treatments in biphasic regulation, which
could modify NO concentration at 48 hours. Such mechanism would constitute
a self-regulating pathway by which NO production from this NOS could be finetuned (Schwartz et al., 1997; Bogdan, 2001a).
NO levels in HUVEC cells
The same experiments were conducted in HUVEC cells. The NO levels were
undetectable by Griess’ assay. This result was also confirmed by an ion chromatography with suppressed conductivity detection (data not shown). Indeed, no
effects of either TNF-α (Table 4.4) or UCB (data not shown) were seen on eNOS
expression. Moreover, the mRNA expression of iNOS evaluated by Real Time
RT-PCR was undetectable in all the experimental conditions described in Materials and Methods.
UCB, the redox status and NO levels
ROS levels can be considered as molecular second messengers that could activate
or inhibit cell functioning depending on the intensity and duration of the oxidative
stress produced in the cell. As described previously, NO levels result from an imbalance between the synthesis and consumption. The quenching of NO by ROS
lead to the formation of reactive nitrogen species (Endemann & Schiffrin, 2004;
Figure 4.3: TNF-α induces iNOS gene expression in H5 V cells. Effect of TNF-α (20 ng/mL)
on eNOS (A) and iNOS (B) mRNA gene expression on H5 V cells at 2, 6 and 24 hours. Bars represent fold of expression, obtained by real time RT-PCR, compared to control cells and normalized
to β-actin used as housekeeping gene. Results are representative of three independent experiments.
Values are mean ±SD. *: p< 0.05 versus control group.
4.2 Nitric oxide analysis
Figure 4.4: Effect of UBC on TNF-α-induced iNOS gene expression in H5 V cells. Effect of
different doses of UCB (15 and 30 nM) with or without TNF-α (20 ng/mL) on H5 V cells. Cells
were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by Real Time
RT-PCR. Results are expressed as mean percentage of folds of expression (%), related to TNF-α
(20 ng/mL) alone treatment, ±SD, n=3. *: p< 0.05 versus TNF-α alone treatment group.
eNOS expression
- TNF-α
+ TNF-α
Table 4.4: Threshold cycle values of eNOS in HUVEC cells. Effect of TNF-α (20 ng/mL) on
eNOS mRNA gene expression in HUVEC cells at 2, 6 and 24 hours. Values are a representative set
of mean±SD threshold cycle values (Ct) obtained by Real Time RT-PCR reaction from samples
in triplicate. Data represent control group cells at 2, 6 and 24 hours.
Madamanchi et al., 2005).
In order to verify if the UCB effects on NO production were influences by
ROS, a set of experiments using NAC, a well known antioxidant, were done as
described in Materials and Methods. Nitrite (NO2– ) production in culture supernatant was measured as described before. iNOS and eNOS expression, were evaluated by Real Time RT-PCR.
H5 V cells were incubated for 2 hours with TNF-α (20 ng/mL) with or without NAC (10 mM). As expected, the expression of eNOS was not influenced by
TNF-α or NAC treatment (Figure 4.5, A). However, TNF-α induction on iNOS
mRNA expression was reverted in a dose-dependent manner by NAC. Moreover,
treatment with NAC 5 mM completely abolished TNF-α effects (Figure 4.5, B).
As previously established, TNF-α (20 ng/mL), significantly reduces nitrite
levels. In addition, significant reductions of nitrite content, was also observed after treatment with NAC (10 mM) alone. Interestingly, an additive inhibitor effect
was observed by co-treatments with TNF-α and NAC (Figure 4.6, A). These results can partially be explained by the regulation of iNOS expression. Treatments
with TNF-α (20 ng/mL) induced iNOS mRNA, as demonstrated before. However
when NAC, alone or in co-treatment with TNF-α, was added to the cell medium
a significant reduction of the iNOS expression was observed (Figure 4.6, B).
4.2 Nitric oxide analysis
Figure 4.5: NAC reverted TNF-α effects on iNOS gene expression. H5 V cells were incubated
with different doses of NAC (50, 20, 10 and 5 mM) with or without TNF-α (20 ng/mL). eNOS (A)
and iNOS (B) mRNAs gene expression were evaluated after 2 hours of treatment. Bars represent
fold of expression, obtained by Real Time RT-PCR, compared to control cells and normalized to
β-actin used as housekeeping gene. Results are representative of two independent experiments.
Values are mean±SD.
Table 4.5: Threshold cycle values of genes studied in H5 V control cells. Values are a representative set of mean±SD threshold cycle values (Ct) obtained by Real Time RT-PCR reaction
from samples in triplicate. Data represent control group cells at 2, 6 and 24 hours.
Interestingly, the significant reduction of iNOS expression, induced by TNFα, was additively reversed by the presence of UCB (at Bf of 15 nM) and NAC,
after 2 hours (Figure 4.7).
UCB reduced AM expression induced by TNF-α
H5 V cells - mRNA relative expression
In order to characterize unconjugated bilirubin (UCB) effects on adhesion molecules (AM) gene expression, mRNAs levels were quantified by Real Time RTPCR. H5 V cells were incubated for 2, 6 and 24 hours with TNF-α (20 ng/mL), as
As expected, TNF-α alone engendered a significant increase of all three AM
genes at 2, 6 and 24 hours, with lower, but still elevated mRNA levels at 24 hours
(Figure 4.8). In addition, no changes were observed in control cells group at different time points. Table 4.5 summaries the threshold cycle (Ct) values of AM
genes studied in the control cell group.
Therefore, later experiments were designed to evaluate the direct role of physiological doses of UCB in co-treatment with TNF-α, as previously described.
Co-treatment with UCB (Bf 15 and 30 nM), significantly blunted the TNF-αinduced expression of E-selectin at 2h by 31% and 43% (respect to TNF-α alone
group, considered as 100%; p<0.05). However, no statistically significant differ80
4.3 UCB reduced AM expression induced by TNF-α
Figure 4.6: Effect of NAC on NO production. H5 V cells were incubated with NAC (10 mM),
with or without TNF-α. Control cells were treated as described in Materials and Methods. Cell
medium was collected after 24 h of treatment for NO2– levels determination. Results are expressed
as NO2– nmol/mg protein and represent means±SD, n=3. (A) *: p< 0.05 compared to respective
group. (B) *: p< 0.05 versus control group. #: p< 0.05 versus TNF-α alone treatment group. &:
p< 0.05 versus NAC alone treatment group.
Figure 4.7: TNF-α induction of iNOS gene expression was reverted by UBC and NAC.
Effect of different doses of UCB (Bf 15 and 30 nM) with NAC (10 mM) on H5 V cells. Cells were
incubated with TNF-α (20 ng/mL) and collected after 2 h of treatment. The mRNAs were analyzed
by Real Time RT-PCR. Results are expressed as mean percentage of folds of expression (%),
related to TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus TNF-α alone treatment
group. &: p< 0.05 versus NAC alone treatment group.
4.3 UCB reduced AM expression induced by TNF-α
Figure 4.8: TNF-α induces AM gene expression in H5 V cells. Effect of TNF-α (20 ng/mL) on
AM mRNAs gene expression in H5 V cells at 2, 6 and 24 hours. Bars represent fold of expression,
obtained by Real Time RT-PCR, compared to control cells and normalized to β-actin used as
housekeeping gene. Results are representative of three independent experiments. Values are mean
±SD. *: p< 0.05 versus control group.
ences were recorded at 6 and 24 hours (Figure 4.9).
TNF-α-induced Vcam-1 gene expression was significantly blunted by 28%,
by co-treatment with UCB 15 nM at 2 hours (p<0.05) and by 28% & 35% with
UCB 15 nM and 30 nM at 6 hours (p<0.05); no differences were observed at 24
hours (Figure 4.10).
Interestingly, the induction of Icam-1 gene expression by TNF-α was not affected by co-treatment with UCB over any time period (Figure 4.11). It should be
noted that the gene expressions of all genes studied were not modified when cells
were treated with UCB alone at both concentrations.
HUVEC cells - mRNA relative expression
Further experiments were done in order to assess whether UCB also regulated the
adhesion molecules mRNA expression on HUVEC cells.
Adhesion molecules mRNA levels gene expression were analyzed by Real
time RT-PCR. HUVEC cell were incubated for 2, 6 and 24 hours with different
doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL), as previously described.
The results were similar to those observed in H5 V cells. TNF-α alone determined a significant increase of E-selectin, VCAM-1 and ICAM-1 at all times
studied, respect to the control group (Figure 4.12, 4.13, 4.14). Moreover, mRNA
gene expression was unchanged in control cells at all time points (data not shown).
Indeed, no significant changes in AM gene expression were seen in UCB treatment alone (data not shown).
TNF-α induced over-expression (considered as the 100%) of E-selectin. This
was blunted 25–30% by co-treatment with either dose of UCB (Bf 15 and 30 nM)
at 2 and 6 h (p< 0.05) but the 20% reduction at 24 hours was not significant (Figure 4.12).
4.3 UCB reduced AM expression induced by TNF-α
Figure 4.9: Effect of UBC on TNF-α-induced E-selectin gene expression in H5 V cells. Effect
of different doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) on H5 V cells.
Cells were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by Real
Time RT-PCR. Results are expressed as mean percentage of folds of expression (%), related to
TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus control group. #: p< 0.05 versus
TNF-α alone treatment group.
Figure 4.10: Effect of UBC on TNF-α-induced Vcam-1 gene expression in H5 V cells. Effect
of different doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) on H5 V cells.
Cells were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by Real
Time RT-PCR. Results are expressed as mean percentage of folds of expression (%), related to
TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus control group. #: p< 0.05 versus
TNF-α alone treatment group.
4.3 UCB reduced AM expression induced by TNF-α
Figure 4.11: Effect of UBC on TNF-α-induced Icam-1 gene expression in H5 V cells. Effect
of different doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) on H5 V cells.
Cells were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by Real
Time RT-PCR. Results are expressed as mean percentage of folds of expression (%), related to
TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus control group.
By contrast, TNF-α induced over-expression of VCAM-1 was decreased 20–
25% by co-treatment with UCB (Bf 15 and 30 nM) only at 6 hours (p< 0.05,
Figure 4.13).
Interestingly, unlike H5 V cells, the ICAM-1 gene over expression caused by
TNF-α treatment (D) was blunted by UCB co-treatment (40% for Bf 15 nM and
48% for Bf 30 nM) at 6 h (p< 0.05), but not at 2 or 24 hours (Figure 4.14).
AM protein expression
H5 V cells were harvested after 24 hours treatment under the different conditions
as described in Materials and Methods. AM protein expression was determined
by SDS-PAGE Western Blot analysis.
TNF-α was able to induce protein expression of E-selectin (Figure 4.15),
Vcam-1 (Figure 4.16) and Icam-1(Figure 4.17), in a time-dependent manner, confirming data previously obtained from other endothelial cells (Cook-Mills & Deem,
When cells were treated with TNF-α and UCB for 24 hours, the protein expression was reduced if compared with TNF-α treatment alone (Figure 4.18).
Similar results were obtained at 6 hours of treatment, whereas not significant differences were observed (data not shown). Moreover, UCB alone, at Bf of 15 and
30 nM did not affect protein expression (data not shown).
Due to the low antibodies cross-reactivity observed, protein profiles in HUVEC
cells were unable to be obtained.
4.4 AM protein expression
Figure 4.12: Effect of UBC on TNF-α-induced E-selectin gene expression in HUVEC cells.
Effect of different doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) on HUVEC
cells. Cells were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by
real time RT-PCR. Results are expressed as mean percentage of folds of expression (%), related
to TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus control group. #: p< 0.05 versus
TNF-α alone treatment group.
Figure 4.13: Effect of UBC on TNF-α-induced VCAM-1 gene expression in HUVEC cells.
Effect of different doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) on HUVEC
cells. Cells were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by
Real Time RT-PCR. Results are expressed as mean percentage of folds of expression (%), related
to TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus control group. #: p< 0.05 versus
TNF-α alone treatment group.
4.4 AM protein expression
Figure 4.14: Effect of UBC on TNF-α-induced ICAM-1 gene expression in HUVEC cells.
Effect of different doses of UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) on HUVEC
cells. Cells were collected after 2, 6 and 24 h and the relevant, specific mRNAs were analyzed by
Real Time RT-PCR. Results are expressed as mean percentage of folds of expression (%), related
to TNF-α (20 ng/mL) alone treatment, ±SD. *: p< 0.05 versus control group. #: p< 0.05 versus
TNF-α alone treatment group.
Figure 4.15: TNF-α induces E-selectin protein expression in H5 V cells. Cells were treated
with TNF-α (20 ng/mL) for the indicated time periods. Total cell lysates were analyzed by Western
blot with specify antibody as described in Materials and Methods. Western blot analysis shown
are normalized by Actin. The density of the specific band was scanned and quantified with an
imaging analyzer. Results indicate the fold of increase of one representative of three reproducible
experiments per treatment group.
4.4 AM protein expression
Figure 4.16: TNF-α induces Vcam-1 protein expression in H5 V cells. Cells were treated with
TNF-α (20 ng/mL) for the indicated time periods. Total cell lysates were analyzed by Western
blot with specify antibody as described in Materials and Methods. Western blot analysis shown
are normalized by Actin. The density of the specific band was scanned and quantified with an
imaging analyzer. Results indicate the fold of increase of one representative of three reproducible
experiments per treatment group.
Figure 4.17: TNF-α induces Icam-1 protein expression in H5 V cells. Cells were treated with
TNF-α (20 ng/mL) for the indicated time periods. Total cell lysates were analyzed by Western
blot with specify antibody as described in Materials and Methods. Western blot analysis shown
are normalized by Actin. The density of the specific band was scanned and quantified with an
imaging analyzer. Results indicate the fold of increase of one representative of three reproducible
experiments per treatment group.
4.4 AM protein expression
Figure 4.18: Effect of UBC on protein expression of three AM in H5 V cells treated with
TNF-α. Bilirubin reduces the TNF-α protein induction of E-selectin (A), Vcam-1 (B) and Icam-1
(C) at 24 hours. Western blot analysis shown are normalized by Actin. The density of the specific
band was scanned and quantified with an imaging analyzer. Results indicate the fold of increase
of one representative of three reproducible experiments per treatment group.
UCB effects via NF-κB pathway
UCB and PDTC inhibit gene over-expression in an addictive pattern
H5 V cells from all experimental groups were pre-treated with PDTC to specifically inhibit the NF-κB pathway. The AM and iNOS mRNAs were measured
by Real Time RT-PCR after 2 hours of treatment, as described in Materials and
PDTC effects seams not to be dose related mediated (Figure 4.19). The addition of PDTC (10 µM) caused a significant reduction in gene over-expression
induced by TNF-α for iNOS (46%), E-selectin (73%), Vcam-1 (80%) and Icam-1
(24%) mRNA (respect TNF-α alone considered as 100%, p< 0.05 Figure 4.20).
An additive inhibition of the expression of E-selectin gene, was seen upon
addition of UCB at either 15 or 30 nM Bf (55%, 46%, p< 0.05). The additive
inhibition by UCB of Vcam-1 gene expression was significant only at a Bf of 30
nM (56%, p< 0.05). Indeed, the additive inhibition on iNOS gene expression was
significant only at a Bf of 15 nM (40%, p< 0.05) (Figure 4.20).
The definition of an “additive effect” was concluded only when the sum of
the individual inhibitions by UCB and PDTC did not differ statistically from the
experimentally-measured inhibition obtained by combined treatment with UCB
and PDTC (Kuldo et al., 2005). The combinatory addictive theorically expected
effect was equal to the observed addictive effect on iNOS, E-selectin and Vcam-1
gene expression at Bf 30 nM and for also at Bf 15 nM for E-selectin. Icam-1
gene induction by TNF-α was not further inhibited by the addition of UCB to the
treatment PDTC.
CREB phosphorylation is not influenced by UCB
To investigate whether CREB is inactivated by UCB, western blot analysis was
performed by using an antibody that recognizes the phosphorylated form of CREB
4.5 UCB effects via NF-κB pathway
Figure 4.19: PDTC inhibits the TNF-α AM and iNOS mRNA gene over-expression. H5 V
cells were incubated with different doses of PDTC (250, 100, 50, 10 and 5 µM) with or without
TNF-α (20 ng/mL). E-selectin, Vcam-1, Icam-1 and iNOS specific mRNAs were evaluated after 2
hours of treatment. Bars represent fold of expression, obtained by Real Time RT-PCR, compared
to control cells and normalized to β-actin as housekeeping gene. Results are representative of two
independent experiments. Values are mean±SD.
Figure 4.20: UCB and PDTC inhibit, in an addictive pattern, the gene over-expression of
AM and iNOS induced by TNF-α. Effect of different doses of UCB (Bf 15 and 30 nM) with or
without TNF-α (20 ng/mL) and PDTC (10 µM) on H5 V cells. Cells were collected after 2 hours
of treatment and the relevant, specific mRNAs were analyzed by Real Time RT-PCR. Results are
expressed as mean of folds of expression, related to TNF-α (20 ng/ml) alone treatment, ±SD. *:
p< 0.05 versus TNF-α alone treatment group. #: p<0.05 versus TNF-α and PDTC treatment
4.5 UCB effects via NF-κB pathway
Figure 4.21: Time dependent induction of CREB phosphorylation by TNF-α in H5 V cells.
CREB was detected by Western blot analysis using a phospho-specific and total CREB antibody.
Cells were incubated with TNF-α(20 ng/mL) for 0, 5, 15, 30, 60, 120 and 240 minutes. The
density of the specific band was scanned and quantified with an imaging analyzer. The ratio
of phosphorylated CREB to total CREB in TNF-α stimulated cells is shown as the relative fold
increase compared with that in un-stimulated cells. Results indicate the fold of increase of one
representative of two reproducible experiments per treatment group.
at Ser 133 and an antibody that recognizes both forms of CREB. Phosphorylation of CREB was significantly increased in a time dependent manner by TNF-α
alone, with a maximum increase reached after 15 min (Figure 4.21). UCB did not
affected CREB phosphorylation whether cells were treated with UCB alone or in
co-treatment with TNF-α (Figure 4.22).
NF-κB nuclear translocation is inhibited by UCB
It was also investigated whether the NF-κB translocation to the nucleus was inhibited by UCB (Bf 15 and 30 nM). Cytoplasmic and nuclear localization of the p65
NF-κB subunit were evaluated by Western blot. As reported, TNF-α stimulated
p65 NF-κB nuclear translocation (Baeuerle, 1998a; May & Ghosh, 1998). UCB
alone did not affected p65 translocation (data not shown). On the contrary, when
cells were co-treated with UCB and TNF-α, the p65 nuclear translocation induced
by TNF-α was prevented by UCB in a dose dependent manner. Furthermore, the
co-treatment with UCB and TNF-α caused an increase of the cytoplasmic fraction
of p65 compared to control and to treatment with TNF-α alone (Figure 4.23).
Figure 4.22: UBC does not affect CREB phosphorylation in H5 V cells. CREB was detected
by Western blot analysis using a phospho-specific and total CREB antibody. Cells were incubated
with UCB (Bf 15 and 30 nM) with or without TNF-α (20 ng/mL) for 15 minutes. The density of the
specific band was scanned and quantified with an imaging analyzer. The ratio of phosphorylated
CREB to total CREB in TNF-α stimulated cells is shown as the relative fold increase compared
with that in un-stimulated cells. Results indicate the fold of increase of one representative of two
reproducible experiments per treatment group.
4.5 UCB effects via NF-κB pathway
Figure 4.23: UCB inhibits TNF-α-induced nuclear translocation of NF-κB in H5 V cells.
Panel A) TNF-α stimulates translocation of NF-κB from cytoplasm to nucleus, which is inhibited
by UCB. NF-κB was detected by Western blot analysis using a p65 NF-κB antibody after 30
minutes of incubation with TNF-α and/or UCB. Western blot analysis shown are normalized by
Actin. The purity of the cytoplasmic fraction was confirmed by αP84 antibody. Panel B) The
density of the specific band was scanned and quantified with an imaging analyzer. Results indicate
the fold of increase compared with unstimulated cells in cytoplasmic and nuclear fraction of one
representative of three reproducible experiments.
Chapter 5
For a long time bilirubin was considered to be simply a waste end product of heme
metabolism. More recently strong evidence has emerged pointing to bilirubin as
an independent factor in the prevention of atherosclerotic disease (Djouss et al.,
2001). In particular, mildly elevated serum bilirubin levels were associated with a
lower incidence of ischemic cardiovascular effects (Vitek et al., 2002) raising the
idea that bilirubin can interfere with the mechanisms involved in the development
of atherosclerosis. Based on the antioxidant properties of bilirubin, the hypothesis
was formulated that bilirubin can act as a ROS scavenger was formulated (Stocker
& Keaney, 2004; Baranano et al., 2002).
More recently bilirubin was demonstrated to inhibit the proliferation of vascular smooth muscle cells (Ollinger et al., 2005) and the endothelial migration of
monocytes (Keshavan et al., 2005). Furthermore, heme oxigenase-1, the widely
distributed enzyme that converts hemes into bilirubin, CO and Fe+2 formation, was
demonstrated to inhibit the over-expression of vascular adhesion molecules induced by TNF-α (Blankenberg et al., 2003).
Based on previous data, we postulated that bilirubin, by itself, and in particularly its active free form (unbound unconjugated bilirubin) can interfere with the
expression of adhesion molecules on endothelial cells. We created an in vitro
model of endothelial dysfunction, the earliest step in atherosclerotic disease, in
which over-expression of ICAM-1, VCAM-1 and E-selectin was induced by treatment of endothelial cells with TNF-α.
Two endothelial cell lines were studied: immortalized H5 V cells from mice
and HUVEC cells, derived from the human umbilical cord. These cells were used
to study the nitric oxide metabolism, and the gene and protein expression of three
adhesion molecules after induction by TNF-α and/or treatment with low doses of
UCB. Our experiments utilized two concentrations of unbound bilirubin (Bf) of
15 and 30 nM, in order to mimic as closely as possible the plasma Bf levels found
in humans with mild unconjugated hyperbilirubinemia (Jacobsen & Wennberg,
1974; Nelson et al., 1974). In this in vitro system, the Bf in the medium is the
equivalent of plasma Bf, since the endothelial cells are directly bathed by the fluid
in both cases. It thus differs from others studies of central nervous system cells,
since the blood brain barrier and choroid plexus intervene between the plasma and
the neurons and astrocytes.
Viability and UCB
It is well recognized that infants who died from sever hyperbilirubinemia not only
demonstrate yellow staining of the brains but also yellow coloration of tissue
and organs. Although the occurrence of central nervous system sequelae is the
most constant clinical feature, other functional abnormalities such as diarrhoea
and urine concentration defect have also been described (Ostrow, 1986).
There have been a handful on in vitro studies demonstrating cytotoxic effects
of UCB depending on the cell type. Although this difference in most of the in vitro
cells models UCB has been found to be toxic, including fibroblast (Nelson et al.,
1974; Chuniaud et al., 1996; Ngai et al., 2000), astrocytes (Chuniaud et al., 1996),
oligodendrocytes (Genc et al., 2003), hepatocytes, erythrocytes, leukocytes, liver
(Czernobilsky & Dubin, 1965), HeLa cells (Shimabuku & Nakamura, 1983; Ngai
et al., 2000), platelets (Amit et al., 1992), neuroblastoma (Schiff et al., 1985), hepatoma (Thaler, 1971; Seubert et al., 2002), glioblastoma, Chang Liver (Ngai et al.,
2000) and fibrosarcoma (Cowger, 1971). Even thought there is scant information
regarding the molecular mechanism underlying these effects; it has been recently
5.1 Viability and UCB
demonstrated that Bf and not the total UCB elicits the toxic effect (Calligaris et al.,
The results obtained in H5 V cells clearly demonstrated a reduction on the cell
viability. Interestingly, HUVEC cells seams to be more sensitive to UCB than
H5 V, at same levels of Bf and at shorter incubation times (Figure 4.1). On the
other hand, as it was shown UCB did not cause membrane permeability, leading
to the release of LDH (Table 4.1), this effect may reflect the absence of necrosis at
the Bf studied. Cellular release of LDH is generally considered to be a hallmark
of necrotic cell death (O’Brien et al., 2000).
The fact that mitochondrial activity was impaired when no lysis was detectable
is in line with the hypothesis proposed by Mustapha et al. (Mustafa et al., 1969)
where mitochondrial may be the primary target for Bf. In line with this observation was the demonstration that, in astrocytes and fibroblast, the Bf level is responsible of toxic effects, mainly correlated with alteration on mitochondrial activity
instead of cell lysis. Indeed, it was proposed that the aggregation of higher levels
of Bf molecules inside the cell may result in the breaking of the membrane architecture and cytolisis (Chuniaud et al., 1996).
Indeed, toxicity of higher doses of UCB (86 µM, at a UCB/albumin ratio of
3:1) in immature cells neurons is typically characterized by a perturbation of the
mitochondrial membrane, with alteration of polarity, fluidity and increasing the
permeability, leading to the release of cytochrome c (Rodrigues et al., 2002b; Rodrigues et al., 2002a), critical events associated with the initiation of the cell death
by apoptotic pathways.
Another study of the mitochondrial functionality clearly demonstrated that
bilirubin at high non physiological doses (25–50 µM) stimulates apoptosis of
colon adenocarcinoma cells in vitro through activation of the mitochondrial pathway, by directly dissipating mitochondrial membrane potential. As this effect is
triggered at concentrations normally present in the intestinal lumen, it was postulated a physiologic role for bilirubin in modulating colon tumorigenesis (Keshavan
et al., 2004).
Furthermore, it was demonstrated that bovine brain microvascular endothelial
cells may undergo apoptosis after exposure to higher doses of bilirubin (10-100
µM). These effect appeared to be time-dependent but not clearly concentrationdependent. Indeed, biochemical markers for apoptosis such as DNA fragmentation and PARP cleavage were induced by bilirubin (Akin et al., 2002).
It was recently reported that a given Bf concentration may or may not cause
cell lysis, depending on the ratio UCB/albumin (Calligaris et al., 2007). Furthermore, different results have been obtained depending on the cell type and the assays used but as mentioned previously the level of Bf used. Amplification of UCB
cytotoxicity by TNF-α and LPS was observed in fibroblast cells at a UCB/HSA
molar ratio of 1.0 (Ngai & Yeung, 1999).
It is well known that TNF-α exhibits its cytotoxic effect through binding to the
cell receptor (Fiers, 1991). The present results confirm that TNF-α was able to induce cytotoxicity (Figure 4.1). Interestingly, cytotoxicity effect was more evident
on HUVEC cells. I agreement with previously studies this HUVEC seems to be
more sensitive to the absence of serum and the treatments with TNF-α or others
growth factors (Emmanuel et al., 2002). However, no further combinatory effects
were seen in co-treatment with different physiological doses of UCB in both cell
model studied. Moreover, the lack of further cytotoxic effects in co-treatments
UCB and TNF-α seems to reenforce the hypothesis of antioxidant properties of
On the other hand, Harlan et al. (Harlan et al., 1983) reported that LPS in
concentration up to 10 µg/mL did not induce detectable cytotoxicity in human endothelial cells derived from umbilical vein, pulmonary artery, or pulmonary vein.
In contrast, significant cytotoxicity was observed in bovine aortic endothelial cells
exposed to LPS as low as 0.01 µg/mL. Indeed, these data demonstrated an important direct LPS-mediated cytotoxic effect, and that this toxic effect depends on the
species from which the endothelial cells are derived.
5.1 Viability and UCB
Barañano et al. (Baranano et al., 2002) introduced the hypothesis of UCB
actin as antioxidant compound like others known antioxidants such as glutathione
and α-tocopherol. It was demonstrated that the potent physiologic antioxidant actions of bilirubin reflects an amplification cycle whereby bilirubin, acting as an
antioxidant, is itself oxidized to biliverdin and then recycled by biliverdin reductase back to bilirubin. This redox cycle may constitute the principal physiologic
function of bilirubin. However, most of the previous studies that evaluated antioxidant actions of bilirubin, were restricted to in vitro experiments measuring
the antioxidant potential of bilirubin, or examined protection conferred by exogenous bilirubin. In line with this idea, the cytoprotective UCB effects have been
then confirmed in vivo and in vitro by inhibiting ROS. UCB and α-tocopherol protected oligondendrocytes from H2 O2 (100 µM). Interestingly, bilirubin seems to
be more effective than α-tocopherol at the same concentration (50 nM). However,
the cytoprotection of bilirubin diminished at higher concentration (100 µM) presumably because higher levels of UCB are themselves cytotoxic (Liu et al., 2003).
Another study conducted in erythrocytes derived from cord blood demonstrated antioxidant properties of bilirubin. It was concluded that bilirubin, at physiologic concentrations, protects neonatal red blood cells against oxidative stress.
However, bilirubin at concentrations equal or exceeding 30 mg/dL and a bilirubin/BSA ratio of greater than one, was associated with significant cytotoxicity.
Additionally, cytotoxicity was evaluated by increased protein oxidation, decreased
erythrocyte glucose-6 phosphate dehydrogenase and adenosine triphosphatase activity, and altered cell membrane integrity (Mireles et al., 1999).
Moreover, bilirubin physiologic role relates to cytoprotection generated endogenous by Heme oxygenase-1, the rate limiting enzyme of heme degradation,
was confirmed in several studies directly related to inflammatory stress and endothelial dysfunction that will discuss latter (Kawamura et al., 2005; Taille et al.,
Although it is believed that the action of UCB is basically related to its toxic
and antioxidant effects in respect of its concentration (Ostrow & Tiribelli, 2003),
the aim of the present data is to demonstrate that UCB may have some other cellular functions. Moreover, the previous findings outline the idea that may be difficult
to formulate a unifying concept of UCB effects. This conclusion is supported by
several reports in neural cells that demonstrated different sensitivities to bilirubin
cytotoxicity (Schiff et al., 1985; Notter & Kendig, 1986; Calligaris et al., 2007).
The contradictory observations in the cellular response in several studies may be
the result of non-physiologic concentration of bilirubin used or in alternative, the
presence of different mechanism of action in different cell lines (Calligaris et al.,
Nitric oxide and UCB
Accumulating evidence suggested that increased vascular oxidant stress represent
a major cause of reduced endothelial NO bio-availability in experimental and clinical cardiovascular disease. Different mechanism may explain why changes of the
endothelial redox state have a profound impact on endothelial NO availability
(Boulden et al., 2006):
• a direct inactivation of NO by superoxide O2– ;
• a reduced NOS activity, by increasing endogenous inhibitors;
• an increased oxidation of critical cofactors, such as BH4 by changes of the
endothelial redox state .
All these mechanisms may contribute to explain the NO levels in endothelial
cells which are important for endothelium dependent vasodilation, suppression of
thrombosis, vascular inflammation and thrombosis. This concept has been supported by several clinical studies, where endothelium dependent vasomotion is
therefore to represent a surrogate marker for cardiovascular events (Landmesser
et al., 2006).
The current study provides several evidence about NO regulation in two models of endothelial cells (H5 V and HUVEC). The regulation of NO metabolism and
5.2 Nitric oxide and UCB
the enzymes involved the synthesis (eNOS and iNOS) during the pro-inflammatory
state seems to be controversial (Wever et al., 1998). Cytokines are believed to induce the production of substantial amounts of NO by increasing iNOS expression
and activity during the pro-inflammatory state (Nathan, 1992). However, eNOS
down-regulation by TNF-α, and the decreased bio-availability of NO on the development of the endothelial dysfunction was also reported (Lai et al., 2003; Govers
& Rabelink, 2001).
Our data demonstrated a redaction of NO levels by treatments with the proinflammatory cytokine TNF-α in time (Table 4.3) and in dose-dependent manner
(Table 4.2). Moreover, the increase of the NO content in control conditions seems
to be time dependent (Table 4.3). Even though these results were further confirmed by using an ion chromatography with suppressed conductivity detection,
the interferences of the Griess’ method can not be exclude (Nithipatikom et al.,
1996). However, it was demonstrated in rabbit corneal cells that a mixture of
cytokines, TNF-α, IL-1β and INF-γ are required to induce significant nitrite accumulation and iNOS expression. Indeed in absence of INF-γ, little or no nitrite
accumulation by TNF-α was reported (O’Brien et al., 2001).
It is well known that the activity of NO is not restricted to the site of production. As un-charged gas, NO radicals are highly diffusible. Indeed, the generation
of s-nitrosothiols, s-nitrosylated proteins, and s-nitrosyl-metal complex can mediate its functions for instances to long distances. Moreover, the imbalance of proand anti-apoptotic effects can thus be best understood in terms of the specific cyscontaining proteins that are targets of NO in the context of cell type and stimulus
(Gaston et al., 2006).
On the other hand, during the endothelial dysfunction state a reduction of
NO levels were reported and explained by different mechanism, increasing of
ROS production, among others. ECs have been shown to generate significant
amounts of ROS (Stroes et al., 1998) and to express enzymes (e.g. eNOS, NADPH
oxidase, CYP, COX) that can produce ROS in response to receptor activation
or other cellular events that elevate intracellular calcium. NO is the principal
endothelium-derived dilator operating in the vasculature and its activity can be
governed by the amount of ROS in the vascular milieu, whereby superoxide anions
can rapidly scavenge NO at a diffusion-controlled rate (Endemann & Schiffrin,
2004; Madamanchi et al., 2005). NO displays high affinity for heme groups and
many enzymes, including those noted above (NOS, COX, CYP etc) have heme
groups. Thus NO itself may inhibit the enzymatic production of superoxide and
H2 O2 (Griscavage et al., 1994). The relative contributions of NO and ROS to
vascular tone are inversely proportional to each other and the appearance of one
could likely compensate for the absence of the other.
Pathophysiological conditions such as diabetes and atherosclerosis display
signs of oxidative stress and dysfunctions in the NO pathway, thus it may be valid
to argue that endothelial ROS production could be compensating for impairments
to normal relaxant mechanisms (Wever et al., 1998). If this hypothesis is correct then there should be an increased contribution of H2 O2 in pathophysiological
states where the normal production of NO is compromised.
Boulden et al. (Boulden et al., 2006) demonstrated that endothelial dysfunction can be induced by H2 O2 and may be mediated by the NADPH oxidase and
its product, O2– . The activation of the NADPH oxidase results in increased O2–
with effects on NO production. This implies that ROS may be a downstream effector of NADPH oxidase activation in order to decreased NO levels and mediate
endothelial dysfunction.
Furthermore, increased NADPH oxidase activity is associated with hypertension and progression of atherosclerosis, suggesting that this enzyme may be part
of the pathogenic cascade leading to uncompensated oxidative stress (Cai et al.,
In agreement with these findings, several reviews indicated the role of TNF-α
in ROS production. TNF-α induces oxidative stress by activating the NADPH
oxidase complex, the major source of endothelial reactive oxygen species production (Li et al., 2002).
5.2 Nitric oxide and UCB
Jiang et al. (Jiang et al., 2006) demonstrated in human microvascular endothelial cells that NO donors strongly induce expression of heme oxygenase-1.
This was associated with a reduction of the superoxide-generating capacity of
NADPH oxidase, an effect that depends on de novo gene transcription and heme
oxygenase-1 activity. Activation of NADPH oxidase by TNF-α increased generation of reactive oxygen species, specially when heme oxygenase-1 expression was
blocked with specific small-interfering RNA. Interestingly, these results demonstrated that bilirubin (1-100 nM) suppressed TNF-α induced ROS formation by
inhibiting NADPH oxidase activity.
Moreover, it was reported in fibroblast that high UCB levels inhibit protein
kinase C phosphorylation. Then UCB may regulate the activation of NADPH oxidase by changing the phosphorylation state, crucial for NADPH oxidase activity,
of the p47 phox subunit (Amit & Boneh, 1993).
In the present study, TNF-α was able to induce iNOS expression in a time
dependent manner without modifying eNOS mRNA (Figure 4.3), in line with previews reports (Bruch-Gerharz et al., 1998). However, a discrepancy between the
induction of iNOS expression and the NO levels was observed. Probably, a reduction of NO levels or a generation of other forms of nitrosative reactive species
generated by ROS can occurred. The ROS hypothesis was then verified by using NAC, a well known antioxidant. The present data demonstrate that NO basal
levels were reduced by treatment with NAC alone (Figure 4.6). These data may
partially demonstrate the hypothesis of ROS generation in H5 V cell model. Moreover when NAC and TNF-α where added together, a further reduction on NO
levels was observed. This reduction may be the result of the reduction of ROS
generated by NAC as antioxidant but also by a reduction on iNOS expression, as
shown in Figure 4.5 and Figure 4.6. Interestingly, co-treatment with TNF-α and
NAC was able to inhibit NO production. Furthermore, as shown in Figure 4.6,
iNOS expression induced by TNF-α was blunted by treatment with NAC. These
results clearly demonstrated a complex and multi-step regulatory mechanism in
the synthesis of NO, iNOS expression, and consumption, ROS.
The present data also clearly demonstrate a role of UCB on the modulation of
NO metabolism. However, the molecular events on NO levels by UCB may be
difficult to explain for several reasons. First, NO levels are the result of a very
complex regulation and UCB may be involved in different steps. At 48 hours NO
levels were reversed by UCB even at upper-normal physiological (15 nM) and
mildly elevated (30 nM) Bf (Figure 4.2). These results may be explained by an
up-regulation of iNOS induced by UCB at 24 hours (Figure 4.4). However, after 2 hours of treatment UCB significantly inhibits iNOS expression (Figure 4.4).
Interestingly, at 2 hours NO levels were not detectable. This complex biphasic
regulation of UCB on iNOS expression may be explained by a modulation the
NO levels. It was reported that NO levels are responsible of iNOS regulation itself (Schwartz et al., 1997). In this case as NO levels were not detectable, UCB
may prevent NO induction. On the other hand, when NO levels were dismissed
(at 48 hours), UCB may compensate these effects by a synergistic effect on iNOS
TNF-α induction (24 hours). However, further results need to be obtained in order
to prove these hypothesis. The additive effect observed between UCB and NAC
reinforced the hypothesis of the redox state (Figure 4.7).
It was demonstrated both in vivo and in vitro, that UCB limits the increase
hepatic levels of TNF-α, nitric oxide (NO) and iNOS caused by treatment with
endotoxin (Wang et al., 2004). These results, in agreement with our data, suggests
a role for bilirubin in the prevention of the tissue injury in response to inflammatory stimuli.
NO levels in freshly isolatedHUVEC cells were undetectable, in line with several in vitro studies. However, HUVEC cells freshly isolated and treated with
TNF-α seems to increase NO production together with an increase in the iNOS
expression (Orpana et al., 1997). Conversely, iNOS induction could not be further
detected in HUVEC subcultures passed once from cells presenting maximal levels
of iNOS expression in the primary culture (de Assis et al., 2002). No changes in
eNOS expression were seen in the present study by treatment with TNF-α at all
times studied (Table 4.4). Moreover, iNOS mRNA expression was undetectable.
5.3 Adhesion molecules and UCB
While accumulating evidence on different sources of endothelial cells demonstrated ROS effects, the TNF-α effects on NO metabolism are still not fully
elucidated (Govers & Rabelink, 2001; Yang & Rizzo, 2007). However our data,
accordingly with recent studies on heme oxygenase-1 (Jiang et al., 2006), pointed
out UCB as a potential modulator of of the oxidant stress and the cardiovascular
protective actions.
Adhesion molecules and UCB
Our results demonstrated, for the first time, that in both the mouse and human
endothelial cell lines UCB, at Bf that did not affect the expression of the three adhesion molecules, blunts the over-expression of E-selectin and VCAM-1 induced
by a pro-inflammatory cytokine such as TNF-α, indicating the lack of species
specific effect (Figure 4.9, 4.12, 4.10, 4.13). By contrast, the enhanced gene expression of ICAM-1 induced by TNF-α was blunted by UCB only in the human
(HUVEC) cell line (Figure 4.11, 4.14). In both cell lines the inhibitory effect of
UCB was usually modest (20-30%), detected and 2 and/or 6 hours, but vanished
by 24 hours.
E-selectin, ICAM-1 and VCAM-1 are known to share many common regulatory mechanisms but only partially shared in the NF-κB pathway (Marui et al.,
1993; Zerfaoui et al., 2008). Thus, in endothelial cells the treatment with hemeoxygenase, a precursor of bilirubin formation, inhibited only the TNF-α induced
over-expression of VCAM-1 and E-selectin but not ICAM-1, indicating that different regulatory mechanism are involved (Soares et al., 2004).
Interestingly, in both cell models E-selectin was the adhesion molecule whose
gene expression was the earliest to be influenced by UCB. The variations were
observed just 2 hours after exposure to the pigment. This data is consistent with
the well established observation that E-selectin is the first adhesion molecule to
be involved, in a time dependent manner, in the leukocyte recruitment by rolling
and tethering (Blankenberg et al., 2003).
On the other hand, in H5 V cells, UCB blunted the protein over-expression of
all three adhesion molecules induced by TNF-α (Figure 4.18). These findings
suggests a post-transcriptional influence of bilirubin, yet to be demonstrated.
A recent study, in murine endothelial cells, demonstrated a different pattern of
expression of the three adhesion molecules studied in acute and chronic treatment
with TNF-α. E-selectin was strongly up regulated in acute but not in chronic inflammation. More over VCAM-1 reveled a similar patter in contrast with ICAM-1
(Rajashekhar et al., 2007). The authors proposed that the discrepancies found in
several studies can be explained as: 1) tissue culture conditions and immortalization procedures further contributing to the differences in response to TNF-α; 2)
mouse endothelial cells used respond differently from human endothelial cells. In
agrement with this hypothesis the presents results demonstrated that UCB effects,
at leat for the adhesion molecules expression, appears to be lack of species specific effect. Previous studies pointed out the potential role of UCB in modulating
the trans-endothelial migration Vcam-1 mediated in vivo (Keshavan et al., 2005).
Even though the culture conditions differ among the studies, specially for UCB
concentrations, these findings support a potential role for bilirubin as an endogenous immunomodulatory agent. However the molecular mechanisms underlying
this activation, or blunt in terms of UCB effect, are not fully understood, nor is it
known whether these genes are activated by common, or gene-specific, regulatory
Signalling pathways and UCB
The present data may rise the hypothesis that bilirubin can influence the inflammatory markers (NO and adhesion molecules) by interfering with the pathways
involved in the regulation endothelial dysfunction. However, the exact mechanism(s) responsible for bilirubin-mediated antioxidant o toxicity remains largely
5.4 Signalling pathways and UCB
unknown. An interesting question is: how bilirubin could mediate its effects?.
Several data demonstrated the UCB effects are limited to binding to nuclear receptors. It was shown that bilirubin might have a direct regulatory effect by binding
the aryl hydrocarbon receptor (Seubert et al., 2002) or indirectly by activation of
constitutive androstane receptor (Huang et al., 2004). Both receptors are associated with multiple cellular functions, cell cycle (Elizondo et al., 2000), apoptotic
response (Reiners & Clift, 1999), xenobiotics hepatic clearance (Wei et al., 2000),
indicating its interactions with signalling pathways.
Several signalling pathways are described to be involved in regulating the gene
expression of iNOS and adhesion molecules specially NF-κB (Baeuerle, 1998a;
Ghosh et al., 1998; Hanada & Yoshimura, 2002; Lin et al., 2007) and CREB (Gerritsen et al., 1997; Ono et al., 2006; Ciani et al., 2002).
In the present study it was demonstrated that PDTC, an IκB inhibitor that prevents the release of p65 (Schoonbroodt & Piette, 2000), has an additive inhibitory
effect on TNF-α induction of iNOS and the adhesion molecules indicating that
bilirubin may also act through an other signalling cascade (Figure 4.20).
When the extent of NF-κB nuclear translocation was evaluated after TNF-α
and UCB co-treatment in our H5 V cells, the TNF-α-stimulated nuclear translocation was inhibited by UCB (Figure 4.23). This result confirmed that UCB can
affect the NF-κB regulatory pathway, probably through an interaction with the
IKK proteins (Malek et al., 2001).
CREB is also involved in the up-regulation of Vcam-1 and E-selectin gene
expression induced by TNF-α (Gerritsen et al., 1997; Ono et al., 2006). It was
investigated the CREB cascade to look for a possible involvement of UCB on
this signalling cascade. However, no influence of UCB on the phosphorylation of
CREB (Figure 4.22) induced by TNF-α was observed (Figure 4.21). Thus CREB
does not mediate the influences of UCB on the expression of the adhesion molecules in H5 V cells.
Bilirubin is already known to be a modulator of the NF-κB signal transduction pathway in astrocytes. Furthermore it was demonstrated that high doses of
UCB (50 µM) induces p65 NF-κB subunit nuclear translocation after 4 hours of
treatment (Fernandes et al., 2006). On the other hand, it was recently reported that
biliverdin inhibits the transcriptional activity of NF-κB in HEK293A cells, by inhibiting TNF-α-induced DNA binding (Gibbs & Maines, 2007). The coimmunoprecipitation data showed that biliverdin reductase binds, under TNF-α stimulus,
to the p65 subunit of NF-κB. Indeed, an over-expression of biliverdin reductase
enhanced both the basal and TNF-α-mediated activation of NF-κB and the concomitant iNOS gene activation (Gibbs & Maines, 2007). These results do not fit
with the findings of the present study. These differences may be a further demonstration of the dual bilirubin effect, toxic at high concentration and protective at
low levels, modulating at least in part by NF-κB signalling pathway.
It is becoming clear that NO itself plays a pivotal role in the regulation of the
gene expression, specially by this regulatory activity may control iNOS gene induction.(Schwartz et al., 1997). Such mechanism would constitute a self-regulating
pathway by which NO production from this NOS could be fine-tuned (Schwartz
et al., 1997; Bogdan, 2001a). This biphasic activity of NO appears to play a central role in the time course of activation of these immune cells and, by inference,
in facilitating the initiation of a defense response against pathogenic stimuli and
in its termination to limit tissue damage. This mechanism, mainly due to the NFκB pathway, can also explain at least in part the reported ability of NO to act in
both a pro- and anti-inflammatory manner (Connelly et al., 2001). Interestingly,
the results described in the present data demonstrated that UCB may also have a
biphasic effects on iNOS regulation (Figure 4.4). Low concentrations of NO (such
as occur after 2 hours of treatment with TNF-α) activate NF-κB and up-regulated
iNOS while high concentrations of NO have the opposite effect. UCB may help
to regulate NO production preventing its overproduction and avoiding its reduction. As it was demonstrated previously UCB effects on iNOS expression may be
mediated, at least in part, by preventing the NF-κB nuclear translocation induced
by TNF-α (Figures 4.20 and 4.23). However, on the modulation of NO levels
by UCB the contribution of others pathways or post-transcriptional mechanisms
5.4 Signalling pathways and UCB
affecting NOS activity can not be excluded.
Other studies suggested that UCB would be able to modulate others signal
transduction pathways. In HeLa and mouse embryonic fibroblast, UCB at high
toxic concentrations (80 nM) induced oxidative stress, activated APE1/Ref-1, a
master redox signalling pathway regulator in eukaryotic cells and induced the activation of Egr-1 transcription factor by up regulation of PTEN tumor suppressor
(Cesaratto et al., 2007). In this way UCB may induce cell toxicity not only by
modulating NF-κB (Fernandes et al., 2006).
Adhesion molecules such as VCAM-1, E-selectin and ICAM-1 are highly regulates at transcriptional level by a large number of mediators. As it was mentioned
previously, NF-κB is believed to play a critical role in mediating inflammatory response in endothelium (Martin et al., 2000). However, the used of different doses
of PDTC, the specific inhibitor, could not complectly revert TNF-α stimulation
of iNOS and the three genes studied (Figure 4.19). These results suggest that an
overlapping distinct signalling pathways may serve to modulate pro-inflammatory
genes expression (Quinlan et al., 1999; Marui et al., 1993; Zerfaoui et al., 2008).
Several others pathways seams to be important by the modulation of the adhesion molecules. Among them, the NFAT family of transcription factors regulated
by calcium and calcineurin. NFAT proteins are phosphorylated and reside in the
cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocated to the nucleus to activate the transcription of a large number
of genes (Hogan et al., 2003). Moreover, the activation of endothelial cells by
thrombin involves an interplay between NFAT and NF-κB signaling pathways to
modulate cooperatively the VCAM-1 gene expression (Minami et al., 2006). It
was demonstrated that bilirubin is a modulator of calcium reservoirs increasing
intracellular calcium levels (Brito et al., 2004). These results according to the
present data may formulate the hypothesis that some other pathways are important to determine UCB effects, specially those relative to its protective functions.
Altogether, our data suggest that bilirubin may blunt the pro-inflammatory
state determined by the cytokine TNF-α by interacting, at least in part, with the
NF-κB transcription factor. However the contribution of other signalling pathways can not be excluded.
Chapter 6
The results obtained in the present study show that unconjugated bilirubin, even
at upper-normal physiological (15 nM) and mildly elevated (30 nM) Bf can modulate gene expression and endothelial cell function.
In this in vitro system UCB reduces the viability of endothelial cells in a dose
dependent manner. The cytotoxic is primary observed by a impaired mitochondrial function. This effect is more evident at high levels of bilirubin (100 nM).
Moreover, the accumulation inside the cell at higher Bf may induced a disruption on the cell membrane leading to necrosis and cell death. However, the lack
of combinatory further effects between UCB and the pro-inflammatory cytokine
TNF-α may reinforce the hypothesis of antioxidant properties od UCB.
This observation pointed out the important role of the Bf in order to compare
different results. Indeed, non-physiologic concentration of bilirubin used may reflect in different cellular responses.
The results described demonstrated in mouse endothelial cell line that UCB
(at normal and mildly elevated physiological levels) may have a biphasic effects
on iNOS regulation. This effect was described by NO itself, low concentrations
of NO (such as occur after 2 hours of treatment with TNF-α) may activate NF-κB
and up-regulated iNOS. High concentrations of NO could have the opposite effect. UCB may help to regulate NO production preventing its overproduction and
avoiding its reduction. As it was demonstrated previously, UCB effects on iNOS
expression may be mediated, at least in part, by preventing the NF-κB nuclear
translocation induced by TNF-α.
The results demonstrate also, for the first time, that in mouse and human endothelial cell lines UCB, at Bf that did not themselves affect the expression of
the three adhesion molecules, blunts the over-expression of E-selectin and Vcam1
induced by a pro-inflammatory cytokine such as TNF-α, indicating the lack of
species specific effect. By contrast, the enhanced gene expression of Icam-1 induced by TNF-α was blunted by UCB only in the human (HUVEC) cells line.
In both cell lines, the inhibitory effect of UCB was usually modest (20-30%) and
detected at 2 and/or 6 hours, but had worn off by 24 hours.
In summary, these data indicate that bilirubin may blunt the development of
endothelial dysfunction by modulating the adhesion molecules over-expression
and the NO metabolism in the pro-inflammatory state induced by the cytokine
TNF-α. Even though UCB alone does not alter these markers. UCB effects are
mediated in part by a modulation of the NF-κB transcription factor. These results
support the concept that modestly elevated concentrations of bilirubin may help
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