: microparticles (EMPs) [3]. EMPs are ... population of submicronic vesicles that are released in response

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Apoptotic Microparticles to Progenitor
Mononuclear Cells Ratio in Heart Failure:
Relevance of Clinical Status and Outcomes
Alexander E. Berezin, MD, PhD*, Alexander A. Kremzer, MD,
Tatyana A. Samura MD, and Yulia V. Martovitskaya MD
Abstract
microparticles (EMPs) [3]. EMPs are a heterogeneous
population of submicronic vesicles that are released in response
to cell activation or apoptosis [4]. It has been investigated that
EMPs represent an intercellular communication and delivery
mechanism for the efficient and effective transfer of biological
information, which selectively packaged as intracellular
material included bioactive lipids, integrins, cytokines,
enzymes, mRNA and micro-RNA that lead to reprogramming
recipient cells; proatherogenic and prothrombotic effects; as
well as modulating inflammatory response [5, 6]. Increase in
circulating EMPs is detectable in several cardiovascular
diseases, such as acute coronary syndrome, atherosclerosis,
dyslipidaemia, hypertension, stroke, atrial fibrillation, as well
as sepsis, cancer, lupus erythematosus, chronic kidney disease,
type two diabetes mellitus, obesity [7-9].
Circulating mononuclear progenitor cells (MPCs) ensure
reparative processes including endotelization of fragments of
vascular lesion, as well as remodeling of extracellular matrix
and neoangiogenesis [10, 11]. Mobilization of bone-marrow
MPCs is regulated by a wide range of pro-inflammatory
cytokines, signal molecules, including micro-RNA,
ischemia-induced factors, neurohormones, glycopeptides and
products of oxidative stress [12]. At the same time, mature
endothelial cells also can differentiate from activated
mononuclears mobilized from peripheral tissues [13, 14]. As
well as EMPs, circulating proangiogenic MPCs are involved in
the process of vasculogenesis repairing damaged and
dysfunctional endothelium [15]. Because no association
between CD34+CD45- phenotyped circulating MPCs and
survival of the CHF patients was found, other subpopulations
of MPCs co-expressing CD34+ antigen and VEGFR-2+
vascular growth ligands (Vascular Endothelial Growth Factor
Rreceptor-2), CD133+, CD14+, and Tie2+ (tyrosine kinase
ligand) were tested as predictors of nature evolution of
ischemic CHF [16]. While circulating apoptotic EMPs are
powerful sensitivity markers of endothelial dysfunction and
tissue remodeling [3, 7], EMPs to MPCs ratio is considered
more tremendous indicator of an imbalance between
pro-angiogenic and apoptotic responses with possible relation
to cardiovascular outcomes [17, 18]. We postulated that EMPs
to MPCs ratio might be discussed as prognostic factors in
ischemic CHF, but it predictive value has not been defined. The
aim of this study was to evaluate the potential prognostic value
The aim of this study was to evaluate the predictive value
of circulating endothelial-derived apoptotic microparticles
(EMPs) to proangiogenic mononuclear cells (MPCs) ratio
for cumulative survival in patients with ischemic chronic
heart failure (CHF). A total of 154 patients with
moderate-to-severe CHF were enrolled. Flow cytometry
analysis for quantifying the number of EMPs and
proangiogenic MPCs was applied. Calculated EMPs to
MPCs ratios in survived and died patient cohort were 8.4
(95% CI = 7.6 – 9.2) and 78.9 (95% CI = 53.0 – 116.6)
respectively (P=0.001). The values of EMPs to MPCs ratio
for entire patient population included in the study obtained
by calculation were further distributed by quartiles (Q).
Using Kaplan-Meier survival analysis we found a
significantly divergence of survival curves in patients with
top quartile (Q4) of EMPs to MPCs ratio when compared
with low quartiles (Q1-Q3). Top quartile of EMPs to MPCs
ratio associates with increased 3-year CHF-related death,
all-cause mortality, and risk for recurrent hospitalization
due to CHF.
Keywords — heart failure, endothelial-derived apoptotic
microparticles, hospitalization, proangiogenic mononuclear cells;
prognosis, survival.
Cite this article as: Berezin AE, Kremzer AA, Samura AA and
Martovitskaya YV. Apoptotic microparticles to progenitor
mononuclear cells ratio in heart failure: relevance of clinical
status and outcomes. JCvD 2014;2(2):50-57.
I. INTRODUCTION
C
hronic heart failure (CHF) represents the leading cause of
cardiovascular morbidity and mortality worldwide [1].
Endothelial dysfunction plays a critical role in the clinical
manifestations of CHF [2]. Recent studies suggested that lesion
of endothelial monolayer due to any reasons leads to dramatic
increase of circulating level of endothelial-derived apoptotic
Conflict of Interest: none.
From the Departments of Internal Medicine (AEB), Clinical Pharmacology
(AAK, TAS), and Pathology (YVM), State Medical University, 26,
Mayakovsky av., 69035 Zaporozhye, Ukraine.
*Correspondence to Alexander E. Berezin: [email protected]
50
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SIEMENS, Germany, in В-mode regimen and tissue Doppler
echocardiography regimen from parasternal, subcostal, and
apical positions over the short and long axis with sensor Р of 5
МHz. Left ventricular end-diastolic and end-systolic volumes
were measured by modified Simpson’s planimetric method;
and they were measured by cylinder method if severe failure of
local myocardial contractility was verified. Left ventricular
ejection fraction (LVEF) was assessed in compliance with the
requirements of American Society of Echocardiography [21].
Tissue Doppler echocardiography was carried out in 4-, 3- and
2-chamber projections in each of 16 segments of the left
ventricle and in 4 spots of the mitral annulus: at the base of
posterior septal, lateral, inferior, and anterior left ventricular
walls [22]. Peak systolic (Sm), early diastolic (Em), and late
diastolic (Аm) myocardial velocities were measured in the
mitral annulus area, followed by calculating velocity of early
diastolic left ventricular filling (E) to Аm (Е/Аm) ratio and to Em
(Е/Em) ratio.
of circulating EMPs to MPCs ratio for cumulative survival in
patients with ischemic CHF.
II. METHODS
Patient population under study
The study evolved 154 patients (86 males) aged 48 to 62
years with ischemic symptomatic moderate-to-severe CHF.
Chronic heart failure (CHF) was diagnosed according to
current clinical guidelines [19]. Table 1 shows characteristic of
the patients participated in the study. All the patients have given
their written informed consent for participation in the study.
The following are exclusion criteria: Q-wave and non-Q-wave
MI within 3 months before study entry; severe kidney and liver
diseases that may affect clinical outcomes; malignancy;
creatinin plasma level above 440 μmol/L; estimated GFR index
< 35 ml/min/м2; brain injury within 3 months before the
enrollment; body mass index above 30 kg/m2 and less 15
kg/m2; pulmonary edema; tachyarrhythmia; valvular heart
disease; thyrotoxicosis; ischemic stroke; intracranial
hemorrhage; acute infections; surgery; trauma; all the ischemic
events within 3 previous months; inflammations within a
previous month; neoplasm; pregnancy; implanted pacemaker,
any disorder that may discontinue patient’s participation in the
study according to investigators; and patient’s refusal to
participate in the study or to give his consent for it. Observation
period was up to 3 years. We analyzed cumulative survival
related to CHF, and additionally all-cause mortality was
examined.
Calculation of glomerular filtration rate
Calculation of glomerular filtration rate (GFR) was carried
out using MDRD-6 formula [23].
Measurement of NT-pro-BNP, total cholesterol and its
fractions
To determine N-terminal pro-brain natriuretic peptide
(NT-pro-BNP), total cholesterol and cholesterol fractions,
blood samples were drawn in the morning (at 7-8 a.m.) into
cooled silicone test tubes. Samples were processed according to
the recommendations of the manufacturer of the analytical
technique used. They were centrifuged upon permanent cooling
at 6,000 rpm for 3 minutes. Then, plasma was refrigerated
immediately to be stored at a temperature not higher than -35оС.
Circulating NT-pro-BNP level was measured by
immunoelectrochemoluminescent assay using sets by R&D
Systems (USA) on Elecsys 1010 analyzer (Roche, Mannheim,
Germany). Concentrations of total cholesterol (TC) and
cholesterol of high-density lipoproteins (HDLP) were
measured by fermentation method. Concentration of
cholesterol of low-density lipoproteins (LDL-C) was calculated
according to the Friedewald formula (1972).
Methods for visualization of coronary arteries
Multispiral computed tomography angiography and/or
angiographic study have been carried out to verify the ischemic
nature of the disease in patients. Multispiral computed
tomography angiography has been carried out for all the
patients prior to their inclusion in the study. When
atherosclerotic lesions of the coronary arteries were verified,
patients were subjected to conventional angiographic
examination provided indications for revascularization were
available. CAD was considered to be diagnosed upon
availability of previous angiographic examinations carried out
not later than 6 months ago provided no new cardiovascular
events occurred for this period, and the procedure are available
for assay. The coronary artery wall structure was measured by
means of contrast spiral computed tomography angiography
[20] on Somatom Volum Zoom scanner (Siemens, Erlangen,
Germany) with two detector rows when holding patients
breathe at the end of breathing in. After preliminary native
scanning, non-ionic contrast Omnipak (Amersham Health,
Ireland) was administered for the optimal image of the coronary
arteries. To reconstruct the image, 0.6-mm-width axial
tomographic slices were used.
Assay of circulating CD31+/Annexin V+ endothelialderived apoptotic microparticles
Circulating EMPs were isolated from 5 ml of venous citrated
blood drawn from the fistula-free arm. Platelet-free plasma
(PFP) was separated from whole blood and then was
centrifuged at 20,500 × rpm for 30 min. EMPs pellets were
washed with DMEM (supplemented with 10 μg/ml polymyxin
B, 100 UI of streptomycin, and 100 U/ml penicillin) and
centrifuged again (20,500 rpm for 30 min). The obtained
supernatant was extracted, and pellets were resuspended into
the remaining 200 μl of supernatant. PFP, EMPs, pellet, and
supernatant were diluted five-, 10-, and five-fold in PBS,
respectively. Endothelial-derived apoptotic microparticles
Assessment of hemodynamics
Transthoracic ultrasonic echocardiography was performed
according to a conventional procedure on ACUSON apparatus,
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transversal directions in the flowcytofluorimeter, the
scattergram results were analyzed by using Boolean principles
for double or triple positive events.
Fig. 1. Results of Kaplan-Meier survival analysis: The cumulative survival in
four cohorts’ patients dependent on quartiles of EMPs to MPCs ratio. Each
Quartile is enumerated as follow: 1- Quartile I; 2- Quartile II; 3 – Quartile III, and
4 – Quartile IV. Censored data are marked on the curves suitable for appropriate
Quartiles
were phenotyped by flow cytofluorimetry by phycoerythrin
(PE)-conjugated monoclonal antibody against CD31 (BD
Biosciences, USA) followed by incubation with fluorescein
isothiocyanate (FITC)-conjugated annexin V (BD Biosciences,
USA) per HD-FACS (High-Definition Fluorescence Activated
Cell Sorter) methodology independently after supernatant
diluted without freeze. The samples were incubated in the dark
for 15 min at room temperature according to the manufacturer’s
instructions. The samples were then analyzed on a FC500 flow
cytometer (Beckman Coulter) after 400 𝜇L annexin-V binding
buffer was added. For each sample, 500 thousand events have
been analyzed. EMPs gate was defined by size, using 0.8 and
1.1 mm beads (Sigma, St Louis, MO, USA). CD31+/annexin
V+ microparticles were defined as EMPs positively labeled for
CD31 and annexin V (CD31+/annexin V+) [24].
Statistical Analysis
Statistical analysis of the results obtained was carried out in
SPSS system for Windows, Version 22 (SPSS Inc, Chicago, IL,
USA). The data were presented as mean (М) and error of mean
(±m) or 95% confidence interval (CI); median (Ме) and
interquartile range. To compare the main parameters of
patients’ groups (subject to the type of distribution of the
parameters analyzed), one-tailed Student t-test or
Shapiro–Wilk U-test were used. To compare categorical
variables between groups, Chi2 test (χ2) and Fisher F exact test
were used. The circulating EMPs, MPCs and NT-pro-BNP
level in the blood failed to have a normal distribution, while
distribution of the total cholesterol and cholesterol fractions
had a normal character (estimated by means of
Kolmogorov-Smirnov test) and was not subjected to any
mathematical transformation. The factors, which could be
associated potentially with EMPs to MPCs ratio, were
determined by logistic regression analysis. Odds ratio (OR)
and 95% confidence interval (95% CI) were calculated for all
the independent predictors of survival of the patients. A
calculated difference of P<0.05 was considered significant.
III. RESULTS
Identifying fractions of mononuclear and endothelial
progenitor cells
Mononuclear cells populations were phenotyped by flow
cytofluorimetry by means of monoclonal antibodies labeled
with FITC fluorochromes (fluorescein isothiocyanate) or
double-labeled with FITC/PE (phycoerythrin) (BD Biosciences,
USA) to CD45, CD34, CD14, Tie-2, and СD309(VEGFR2)
antigens as perHD-FACS (High-Definition Fluorescence
Activated Cell Sorter) methodology, with red blood cells
removed obligatory with lysing buffer according to gating
strategy of International Society of Hematotherapy and Graft
Engineering sequential (ISHAGE protocol of gating strategy)
[25]. For each sample, 500 thousand events have been analyzed.
Circulating MPCs have been identified as CD45-CD34+ cells.
Proangiogenic phenotype for endothelial MPCs was
determined as CD14+СD309 (VEGFR2)+Tie-2+ antigens.
Obtained when laser beam is scattered in longitudinal and
52
General characteristics of study patient population
During a median follow-up of 2.18 years, 21 participants died
and CHF-related death was defined in 18 patients. Additionally,
106 subjects were hospitalized repetitively due to advance CHF
(17 cases in died cohort and 89 cases in survival cohort). Table
1 shows a general characteristic of the patients included in the
study. As one can see from Table 1, no substantial age and
gender differences were found among persons who died and
survived, as well as differences in body mass index (BMI),
glomerular filtration rate (GFR), HbA1c, fasting blood glucose
level, blood creatinine level, total cholesterol (TC), low-density
lipoprotein cholesterol (LDL-C) and high-density lipoprotein
cholesterol (HDL-C), numerous of coronary vessels damaged.
No difference was found between the two cohorts in systemic
office blood pressure (BP) and heart rate (HR). Documented
incidence of type 2 diabetes mellitus in patients of the two
cohorts was 38.1% and 33.8% (P=0.06). Note that there was not
a statistically significant change in Е/Аm and Е/Em between the
two cohorts, while decrease in the left ventricular ejection
fraction value was quite anticipated in the setting in died
patients.
At the same time, the level of circulating NT-pro-BNP was
statistically significantly higher in died patients than in
survived persons. When analyzing details of pharmacotherapy,
no substantial differences were found between the two cohorts
with regard to administration of the majority of drugs.
Circulating EMPs and MPCs level in survival and died
patients
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Heart Association, BNP – brain natriuretic peptide, LVEF - Left ventricular
ejection fraction, U – unit, Em - early diastolic myocardial velocity, Аm - late
diastolic myocardial velocity, E – peak velocity of early diastolic left
ventricular filling, ACEI – angiotensin-converting enzyme inhibitor, ARAs –
angiotensin-2 receptors antagonists.
Medians of circulating levels of EMPs in survived and died
patient cohort were 0.286 n/mL (95% confidence interval [CI]
= 0.271-0.309 n/mL) and 0.673 n/mL (95% CI = 0.65-0.74
n/mL) (P<0.001).
Variable
Age, years
Males, n (%)
Hypertension, n (%)
Dyslipidemia, n (%)
T2DM, n (%)
Smoking, n (%)
NYHA II Class
NYHA III Class
NYHA IV Class
BMI, kg/m2
eGFR, mL/min/1.73
m2
HbA1c, %
Fasting blood glucose,
mmol/L
Creatinine, μmol/L
TC, mmol/L
LDL-C, mmol/L
HDL-C, mmol/L
NT-pro-BNP, pg /mL
Died subjects
(n=21)
Survived subjects
(n=133)
57.20±6.70
12 (57.1%)
12 (57.1%)
9 (42.8%)
8 (38.1%)
7 (33.3%)
6 (28.6%)
9 (42.8%)
6 (28.6%)
23.7 (95%
CI=22.5–27.3)
82.1 (95%
CI=69.9–93.1)
6.3 (95%
CI=4.4-9.0)
4.80 (95%
CI=3.6-8.5)
70.5 (95%
CI=59.6–88.3)
5.3 (95%
CI=4.6-6.0)
3.60 (95% CI
=3.20–4.18)
0.94 (95% CI =
0.92–1.06)
1533.6 (95% CI
644.5 – 2560.6)
129±4
76±6
59.50±7.30
67 (50.3%)
61 (45.9%)
52 (39.1%)
45 (33.8%)
24 (29.3%)
35 (26.3%)
65 (48.9%)
33 (24.8%)
24.2 (95%
CI=22.0–27.9)
85.2 (95%
CI=70.3–112.5)
7.0 (95%
CI=4.3-9.2)
5.40 (95%
CI=3.4-9.1)
74.9 (95%
CI=65.1–90.3)
5.0 (95%
CI=4.2-5.8)
3.02 (95%
CI=2.80–3.90)
0.88 (95% CI =
0.82–0.97)
1031.2 (95% CI
704.8 – 1560.7)*
135±5
68±3
The data suggested that EMPs number in plasma were directly
related to NYHA functional class of CHF (r = 0.514, P = 0.001),
NT-pro-BNP (r = 0.416, P = 0.001), T2DM (r = 0.402, P =
0.003), multi-vessel lesion of coronary arteries (r = 0.362, P =
0.001), Е/Аm (r = 0.360, P = 0.001), Е/Em (r = 0.344, P = 0.001),
gender (r = 0.318, P < 0.001 for male), TC (r = 0.313, P =
0.001), age (r = 0.275, P = 0.001), smoking (r = 0.212, P =
0.001) and inversely to LVEF (r = -0.496, P = 0.001) and
estimated GFR (r = -0.408, P = 0.003).
Medians of circulating levels of MPCs in both patient cohorts
were 0.28 n/µL (95% CI = 0.26 – 0.30 n/µL) for survived
persons and 0.12 n/µL (95% CI = 0.098 – 0.14 n/µL) for
subjects who died (P=0.001).
There was a positive association of MPCs count with left
ventricular ejection fraction (r=0.639; P=0.001), Е/Е m ratio
(r=0.52; P=0.001), eGFR value (r=0.486; P=0.002); and a
negative association with CHF functional class (r=-0.657;
P=0.001), with the presence of T2DM (r=-0.610; P=0.001), age
(r=-0.398; P=0.001), creatinine (r=-0.394; P=0.001),
NT-pro-BNP level in the blood (r=-0.473; P=0.001), LDL
cholesterol (r=-0.354; P=0.001), total cholesterol level
(r=-0.258; P=0.043), adherence to smoking (r=-0.285;
P=0.042), body mass index (r=-0.272; P=0.046).
Calculated EMPs to MPCs ratios in survived and died patient
cohort were 8.4 (95% CI = 7.6 – 9.2) and 78.9 (95% CI = 53.0 –
116.6) respectively (P=0.001). EMPs to MPCs ratio was
directly related to NYHA functional class of CHF (r = 0.62, P =
0.001), NT-pro-BNP (r = 0.513, P = 0.001), T2DM (r = 0.422,
P = 0.001), multi-vessel lesion of coronary arteries (r = 0.432, P
= 0.001), Е/Аm (r = 0.387, P = 0.002), Е/Em (r = 0.356, P =
0.002), gender (r = 0.396, P < 0.001 for male), TC (r = 0.322, P
= 0.001), age (r = 0.301, P = 0.001), smoking (r = 0.287, P =
0.001) and inversely to LVEF (r = -0.506, P = 0.001) and
estimated GFR value (r = -0.502, P = 0.001). No significant
association between the EMPs to MPCs ratio with fasting
plasma glucose, HbA1c, means systolic and diastolic BP,
premature CAD in family anamnesis, and medications for both
cohorts of the patients was found. For further analysis the
values of EMPs to MPCs ratio for entire patient population
included in the study obtained by calculation were distributed
by quartiles (Q): Q1 (median = 9.7; 95% CI = 5.8 – 10.6); Q2
(median = 18.2; 95% CI =11.0 – 22.2); Q3 (median = 41.4;
95% CI =22.5 – 57.8); Q4 (median = 73.0; 95% CI =58.9 –
96.6).
Systolic BP, mm Hg
Heart rate, beats per 1
min.
42.80±0.76
55.40±0,80*
LVEF, %
16.6±0.94
16.5±1.20
Е/Аm, U
16.6±1.00
16.6±0.84
Е/Em, U
5 (23.8%)
24 (18.0%)
One-vessel coronary
arteries lesion, n (%)
8 (38.1%)
54 (40.1%)
Two-vessel coronary
arteries lesion, n (%)
8 (38.1%)
55 (41.4%)
Multivessel lesion, n
(%)
21 (100%)
133 (100%)
ACEI / ARAs, n (%)
19 (90.5%)
121 (91.0%)
Acetylsalicylic acid, n
(%)
2 (9.5%)
12 (9.0%)
Other antiaggregants,
n (%)
14 (66.7%)
80 (60.2%)
Statins, n (%)
8 (38.1%)
45 (33.8%)
Metformin, n (%)
18 (85.7%)
121 (91.0%)
Diuretics, n (%)
9 (42.9%)
70 (52.6%)
Mineralcorticoid
receptors antagonists,
n (%)
Table 1 General characteristic of patient population
The predictive value of EMPs to MPCs ratio in study
patient population
Using Kaplan-Meier survival analysis we found a significantly
divergence of survival curves in patients with top quartile (Q4)
of EMPs to MPCs ratio when compared with low quartiles
(Q1-Q3) (Figure 1). The curves divergence of events
accumulation reached a statistical significance in 50 weeks of
Note: * - statistically differences between parameters in the two groups
(P<0.05); CI – confidence interval; CAD – coronary artery disease, T2DM –
type two diabetes mellitus, TC – total cholesterol, eGFR - estimated
Glomerular filtration rate, HbA1c – glycated hemoglobin, HDL-C high-density lipoprotein cholesterol, LDL-C - Low-density lipoprotein
cholesterol, BP – blood pressure, BMI - Body mass index, NYHA - New York
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observation period (P<0.001 for all cases). No statistically
significance differences between survival in patient cohorts
with Q1 and Q2, as well as Q2 and Q3 EMPs to MPCs ratio
Variable
were found. The divergence between two cohorts with EMPs to
MPCs ratio equal Q1 and Q3 was reached be able significance
at study end.
EMPs
All-cause mortality
OR
95% CI
1.58
1.20–1.88
P
0.001
CHF-related death
OR
95% CI
1.22
1.12–1.36
P
0.001
MPCs
1.21
1.06–1.43
0.002
1.15
1.03–1.28
0.001
EMPs/MPCs
ratio
NYHA class
1.69
1.40–1.92
0.001
1.38
1.20–1.54
0.001
1.12
1.01–1.24
0.05
1.18
1.05–1.30
0.001
NT-pro-BNP
LVEF
T2DM
three- and
multi-vessel
lesion
1.09
1.06
1.05
1.02
1.02–1.16
1.01–1.12
1.01–1.11
0.88–1.09
0.002
0.001
0.001
0.56
1.42
1.15
1.03
1.01
1.22–1.73
1.12–1.18
0.93–1.10
0.92–1.07
0.006
0.014
0.32
0.27
CHF-related re-hospitalization
OR
95% CI
P
1.20
1.11 –
0.001
1.32
1.06
1.02 –
0.001
1.09
1.32
1.18 –
0.001
1.66
1.12
1.07 –
0.001
1.22
1.44
1.28–1.67
0.002
1.22
1.07–1.45
0.016
1.04
0.97–1.06
0.42
1.14
1.03–1.26
0.012
Table 2. Variables Independently Related to 3-years all-cause mortality, CHF-related death, and CHF-related rehospitalisation, obtained by
Logistic Regression Analysis.
Note: OR – odds ratio, CI – confidence interval; LVEF – left ventricular ejection fraction; BNP – brain natriuretic peptide; T2DM – type two diabetes mellitus.
EPMs to MPCs ratio + NT-pro-BNP + NYHA class + LVEF + T2DM; Model 6:
EPMs to MPCs ratio + NT-pro-BNP + NYHA class + LVEF + T2DM + threeand multi-vessel lesion of coronary arteries.
Multivariate logistic regression was used to assess whether any
combination of assays was able to better discriminate between
survival and died patients. In the logistic regression analysis,
the main factors independently related with cumulative
mortality and CHF-related re-hospitalizations were EPMs,
MPCs, EPMs to MPCs ratio, NT-pro-BNP, NYHA class,
LVEF, T2DM, and three- and multi-vessel lesion. Noteworthy,
EPMs to MPCs ratio had the most independently predicted
potential for all-cause mortality (OR = 1.69; 95% CI =
1.40–1.92; P = 0.001), CHF-related death (OR = 1.38; 95% CI
1.20–1.54; P = 0.001), and also CHF-related re-hospitalization
(OR = 1.32; 95% CI = 1.18 – 1.66; P=0.001) when compared
with other variables (Table 2).
MPCs, EMPs, NYHA class, NT-pro-BNP and LVEF remained
statistically significant for all categories: all-cause mortality,
CHF-related death, and CHF-related re-hospitalizations,
whereas T2DM and three- and multi-vessel lesion for all
variables did not. Using a stepwise model selection method for
multivariable prediction model we have been investigated the
summary effect of any combinations of EPMs to MPCs ratio,
NT-pro-BNP, LVEF on all-cause mortality, CHF-related death,
and CHF-related re-hospitalizations (Table 3).
We found that EPMs to MPCs ratio remained statistically
significant predictors for combined end-point included
all-cause mortality and CHF-related re-hospitalizations. A
stepwise model selection method has been demonstrated that
NT-pro-BNP, NYHA class, LVEF, T2DM and three- and
multi-vessel lesion of coronary arteries added to EPMs to
MPCs ratio do not offer any additional information to
discriminate between survived and died patients with
symptomatic ischemic CHF.
IV. DISCUSSION
OR
95% CI
P value
1.62
1.12 – 1.92
0.001
Model 1
1.56
1.09 – 1.77
0.003
Model 2
1.42
1.12 – 1.61
0.001
Model 3
1.25
1.07 – 1.62
0.003
Model 4
1.09
1.02 – 1.17
0.002
Model 5
1.11
1.01 – 1.19
0.001
Model 6
Table 3. Predictive value of EPMs to MPCs ratio for combined
end-point (all-cause mortality and CHF-related re-hospitalisations).
Multivariable prediction model shows lack of additional information
to discriminate between survived and died patients with symptomatic
ischemic CHF when NT-pro-BNP, NYHA class, LVEF, T2DM and
three- and multi-vessel lesion of coronary arteries were added to EPMs
to MPCs ratio.
Note: Model 1: EPMs to MPCs ratio; Model 2: EPMs to MPCs ratio +
NT-pro-BNP; Model 3: EPMs to MPCs ratio + NT-pro-BNP + NYHA class;
Model 4: EPMs to MPCs ratio + NT-pro-BNP + NYHA class + LVEF; Model 5:
54
Circulating EMPs play a pro-inflammatory and procoagulant
detrimental role in the vascular dysfunction that is a key
mechanism in the development and progression of a wide range
of cardiovascular diseases [26]. Recent studies revealed that
EMPs may trigger endothelial dysfunction by disrupting
production of nitric oxide release from vascular endothelial
cells and subsequently modifying vascular tone [3, 7].
Circulating EMPs affect both pro-inflammatory and
pro-atherosclerotic processes, promote coagulation and
inflammation, and also modulate angiogenesis and apoptosis in
endothelial cells [27-29]. Because endothelium is one of the
primary targets of circulating microvesicles, EMPs have been
considered biomarkers of vascular injury, inflammation and
stage of progression of cardiovascular diseases. Recent study
revealed an association between circulating EMPs labelled as
CD31+ / annexin V+ cells and MPCs with different nature with
cardiovascular outcomes [9, 10, 12]. However, no previous
study has mentioned the possible predicted role of circulating
EMPs levels and MPCs count in the CHF. In our investigation
we found a significantly increase of EMPs to MPCs ratio in
circulation in ischemic CHF patients who were died when
compared with those who survived. Quartile distribution of
EMPs to MPCs ratio with further cumulative survival analysis
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APRIL 2014
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with Kaplan-Meier has been shown a significant divergence
between curves in Q4 EMPs and other quartiles. Using
stepwise predictive methods we found that increased EMPs to
MPCs ratio independently predicted combined end-point
(all-cause mortality and CHF-related re-hospitalization) within
observation period. Multivariable prediction model has been
shown a high decremented potential of EMPs to MPCs ratio
alone, when compared with other markers of unfavorable of
clinical outcomes suitable for CHF, such as NT-pro-BNP and
LVEF. These findings suggest that increased EMPs to MPCs
ratio might improve the predictive value of contemporary
model in CHF based on clinical performances and
NT-pro-BNP measurements. Although the cellular mechanism
of action of EMPs largely remains unclear, we believe that
increased EMPs to MPCs ratio in CHF may reflect a reduced
vascular repair capacity and severity of endothelial dysfunction,
that is, probably, considered staging disease. In this study,
EMPs to MPCs ratio was sufficient to predict long-term
changes significant as independent factors in cumulative
survival, re-hospitalization due to CHF, and CHF-related death.
It should emphases that while EMPs and MPCs have large
diagnostic potential as biomarkers in cardiovascular diseases
and cancer; however, due to current technological limitations in
purification of EMPs and an absence of standardized methods
of detection, the role of EMPs became controversial [30-32].
Noteworthy, the High-Definition FACS method that was used
in the study to be determined types of cells, is required a
correction for spectral overlap between two or more
fluorochromes used in the same staining combination [25].
Probably, some obstacles related with FACS methodology,
might be an explanation why not for cases a tremendous
relation between circulating EMPs and MPCs were found.
However, there is a controversial in results of recently clinical
studies that reflect a role of circulating different types MPCs in
cardiovascular diseases [16, 33, 34]. Alba AC et al. 92013) [16]
reported about reduced mortality in non-diabetic patients with
lower CD34+VEGFR2+ cells in comparison with T2DM
subjects. We have previously found that the reduction in
circulating CD14+CD309+ and CD14+CD309+Tei2+ MPCs is
related to a number of cardiovascular risk factors in
asymptomatic patients with CAD [35]. Probably, risk factors
contribute to CHF evolution through suppression of mobbing,
differentiation and migration of proangiogenic progenitor cells,
as well as increasing apoptosis of endothelial cells. Of note,
impaired kinase activity (Ca2+/calmodulin- dependent kinase
IV, AKT) of endothelial cells may be not only crucial in
endothelial dysfunction [36, 37], but plays a pivotal role in
functional disability of endothelial progenitor cells. Argued
around the important issue we predisposed that mishmash
between EMPs to MPCs in peripheral circulatory reflects an
interrelation of some genetic mechanisms with coexisting risk
factors and low intensity inflammation, which is suitable for
heart failure. Thus, calculation of EMPs to MPCs ratio looks
attractive for interpretation data of EMPs and MPCs
measurements that reflects tenderness of existing imbalance
between apoptosis of endothelial cells and repair of
endothelium [38]. Hsu CY et al (2013) [17] reported that
increased circulating EMPs to MPCs ratio is associated with
55
subsequent decline in GFR in hypertensive patients. However,
no more studies dedicated investigation of prognostic role of
such ratio in cardiovascular disease patients, although
dissociation between circulating apoptotic microparticles and
decreased circulating endothelial progenitor cell levels in
hypertensive patients with microalbuminuria has also described
[9]. Knowledge of the functional properties of EMPs and MPCs
will contribute to a better understanding of the pathological
mechanisms of communication between cells and CHF
progression, because EMPs to MPCs ratio may be an attractive
prognostic biomarker for CHF. Probably, new studies with
more statistical powerful are required to be improve the
understanding of this issue and attracted the attention of
specialists.
Study Limitations
This study has some restrictions. The authors believe that a
greater cohort of patients with more incidences detected is
desirable to improve the power of the study. It is necessary to
note that large pool of nanoparticles might be produced after
blood sampling due to destroy of platelets and blood cells.
Therefore, preparation of isolates of microparticles in samples
is the most sophisticated step for further examination. Venous
citrated blood drawn from the fistula-free arm was performed
obligatory. We believe that these risks are systemic, and to
minimize them, we refused to freeze the blood samples before
measurement of microparticles. Although High-Definition
FACS methodology is widely used, theoretically overlap
between two or more fluorochromes might be reflected some
obstacles for further interpretation of obtained results. However,
the authors suppose that these restrictions might have no
significant impact on the study data interpretation.
V. CONCLUSIONS
Among patients with symptoms of CHF, top quartile of
EMPs to MPCs ratio associates with increased 3-year
CHF-related death, all-cause mortality, and risk for recurrent
hospitalization due to CHF.
VI. ACKNOWLEDGMENTS
We thank all patients for their participation in the
investigation, staff of the Regional Zaporozhye Hospital
(Ukraine) and the doctors, nurses, and administrative staff in
City hospital # 6 (Zaporozhye, Ukraine), general practices, and
site-managed organizations that assisted with the study.
The study was approved by the local ethics committee of State
Medical University, Zaporozhye, Ukraine. The study was
carried out in conformity with the Declaration of Helsinki.
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Alexander E. Berezin received his MD
degree from State Medical University,
Zaporozhye, Ukraine, in 1992. PhD degree
in Heart Failure was earned in State
Medical University, Zaporozhye, Ukraine
in 1994. He is currently Professor of
Medicine, Chief of the Cardiology Unit of
Internal Medicine Department at State
Medical University, Zaporozhye, Ukraine. He became a
Member (M) the Ukrainian Cardiology Association in 1997; M
of the Ukrainian Heart Failure Association in 2000; M of Heart
Failure Association of the ESC in 2004; M of European Acute
Cardiovascular Care Association in 2010; M of W.G. on
Atherosclerosis and Vascular Biology of the ESC in 2010; M of
European Association of Percutaneous Cardiovascular
Interventions in 2012; M of European Association for
Cardiovascular Prevention and Rehabilitation in 2012, M of
W.G. on Hypertension & the Heart of the ESC in 2012.
From 2002 to 2012, he was Associate Professor at the Internal
Medicine Department at State Medical University, Ukraine.
He is currently Editor-in-Chief of Biological Markers and
Guided Therapy (since 2013); Member of the Editorial Board,
Ukranian Medical J (since 2000), J Cardiology and Therapy
(since 2013), Case Reports in Internal Medicine (since 2013);
reviewer for J Genetic Disorders and Genetic Reports; Obesity;
Eur. J Clinical Investigations; J Food Science and Engineering;
Case Reports in Internal Medicine; Diabetes, Metabolic
Syndrome and Obesity: Targets and Therapy.
His research interest includes the fundamental study of
biological markers of cardiovascular diseases, the
implementation of visualization procedures and percutaneous
cardiovascular
interventions,
the
development
of
cardiovascular prevention and rehabilitation, especially in the
field affected heart failure and coronary artery disease,
improving knowledge in vascular biology and regenerative
medicine.
Alexander A. Kremzer received his PhD
degree from Zaporozhye State Medical
University, Ukraine in 2000. He is currently
Associate Professor at the department of
clinical pharmacology, pharmacy and
pharmacotherapy in State Medical
University, Zaporozhye, Ukraine.
From 2003 to 2008, he was Assistant
Professor at the department of clinical pharmacology,
pharmacy and pharmacotherapy in State Medical University,
Zaporozhye, Ukraine. His research interest includes the
development of methods and approaches for predicting the
progressing of chronic heart failure using the modern
biochemical and immunological markers.
57