Accepted Manuscript Circulating endothelial progenitor cells as markers for severity of ischemic chronic heart failure Alexander E. Berezin, Alexander A. Kremzer PII:

S1071-9164(14)00080-3

DOI:

10.1016/j.cardfail.2014.02.009

Reference:

YJCAF 3266

To appear in:

Journal of Cardiac Failure

Received Date: 19 July 2013 Revised Date:

21 February 2014

Accepted Date: 24 February 2014

Please cite this article as: Berezin AE, Kremzer AA, Circulating endothelial progenitor cells as markers for severity of ischemic chronic heart failure, Journal of Cardiac Failure (2014), doi: 10.1016/ j.cardfail.2014.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title page

Circulating endothelial progenitor cells as markers for severity of ischemic chronic heart failure Alexander E. Berezin (1), Alexander A. Kremzer (2) (1) State Medical University, Internal Medicine Department, Zaporozhye, Ukraine

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(2) State Medical University, Clinical Pharmacology Department, Zaporozhye, Ukraine The authors have contributed equally to this paper.

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The study results were presented for the first time at Heart Failure Congress of European Society of Cardiology on 25-28 May, 2013, in Lisbon, Portugal. The manuscript was never been

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published.

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Corresponding author:

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Short title of the article: Endothelial progenitor cells in heart failure

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Alexander E. Berezin, Professor, MD, PhD

Internal medicine Department, State Medical University, 26, Mayakovsky Av., Zaporozhye,

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Postcode 69035, Ukraine.

Address for correspondence: # 28 Chuykov Str., Apt. 137, Box 6323, Zaporozhye, Post code: 69121, Ukraine Tel.: +38 061 2729607 Fax: +38 061 2729607 E-mail: [email protected] Manuscript Type: original article

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Introduction. Despite a high potential of endothelial progenitor cells (EPC) for diagnostic purposes, the EPC role in developing ischemic chronic heart failure (CHF) has not been determined obviously.

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The objective of the study is to assess the counts of CD45+CD34+, CD45-CD34+, CD14+CD309+, and CD14+CD309+Tie2+ phenotyped circulating EPC of various subpopulations in patients with ischemic chronic heart failure (CHF).

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Methods and Results. The study involved 153 patients (86 males), aged 48 to 62 years, with angiografically proven coronary artery disease (CAD) and 25 healthy volunteers. CHF was

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diagnosed in 109 patients (71.2%). Mononuclear cells populations were phenotyped by flow cytofluorimetry. Cardiovascular risk factors, such as type 2 diabetes mellitus, hyperlipidemia, arterial hypertension, and adherence to smoking, may have a negative effect on circulating EPC counts in CAD patients regardless the presence of CHF. The depletion of the CD14+CD309+- and CD14+CD309+Tie-2+-phenotyped circulating EPC counts is associated with the severity of left

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ventricular dysfunction, while the CD45+CD34+- and CD45-CD34+- mononuclears counts are more representative for the severity of atherosclerotic coronary arteries lesion.

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Conclusion. The authors found that NYHA functional class of CHF, LVEF less than 42%, the NT-pro-BNP level over 554 pg/mL, and the Е/Еm ratio over 15 U had the highest predictive

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value for the depletion of the EPC count in CAD patients.

Key words: coronary artery disease; mononuclears; predictive value.

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Introduction

A number of previous studies have shown the role of circulating endothelial progenirot cells (EPCs) of hematopoietic origin in the pathogenesis of cardiovascular diseases [1, 2]. For instance, it has been found that the CD34+CD45-phenotyped EPC count may increase in patients

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with myocardial infarction, unstable angina pectoris and acute coronary syndrome [3, 4]; and it may decrease in patients with subclinical atherosclerosis, chronic heart failure (CHF) with the total contractile myocardial dysfunction [5, 6]. Circulating EPCs ensure reparative processes,

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including endothelization of the fragments of vascular lesion, as well as remodeling of extracellular matrix and neoangiogenesis [7, 8]. Mobilization of endothelial progenitor cells,

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which possess a potential for angiopoiesis and organ protection, 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 [3]. Mature endothelial cells also may differentiate from activated mononuclears mobilized from peripheral tissues [9, 10]. Circulating EPCs of nonhematopoietic origin, which express CD34+CD45-, are phenotypically

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identical with primitive progenitor cells of other origin; and they are functionally different from primitive progenitor cells of other origin only due to their colony-formation ability when

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cultivated [11, 12]. This raises difficulties when identifying the EPC production source if expression of CD34 antigen in CD45-negative mononuclears was verified. Moreover, no

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association has been verified between the CD34+CD45- phenotyped circulating cells count, on the one hand, and the severity of coronary atherosclerosis and patients’ survival rate, on the other hand [13]. It has caused attempts to verify other subpopulation of EPC coexpressing CD34+ antigen and VEGFR-2+ vascular growth ligands (Vascular Endothelial Growth Factor Receptor2), CD133+, CD14+ and Tie2+ (tyrosine kinase ligand). CD34+ granulocytes, which express Tie2+ and VEGFR-2+, are supposed to include activated mononuclears of non-hematopoietic origin, which have phenotypic differences manifested in additional expression of CD14 antigen, show their pluripotency, and are a potential source of endotheliocytes [14]. For EPC of

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CD14+CD309+ and CD14+CD309+Tie2+ phenotypes, association has been found with the atherosclerosis incidence and survival of patients with acute coronary syndrome and myocardial infarction [15]. However, despite significant steps forward in defining EPC potential for diagnostic purposes, the role of EPCs of hematopoietic and nonhematopoietic origin in

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developing ischemic CHF has not been determined obviously [16]. The objective of this study was to assess counts of CD45+CD34+, CD45-CD34+, CD14+CD309+, and CD14+CD309+Tie2+ phenotyped circulating endothelial progenitor cells of various

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subpopulations in patients with ischemic chronic heart failure. Methods

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The study involved 153 patients (86 males) aged 48 to 62 years with angiografically proven coronary artery disease (CAD) with stenotic lesion of at least one coronary artery > 50%, and 25 healthy volunteers. Chronic heart failure (CHF) was diagnosed in 109 patients (71.2%) with angiografically proven CAD by means of conventional criteria according to current clinical guidelines [17]. Table 1 shows characteristic of the patients participated in the study. All the

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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 prior to study enrollment;

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severe kidney and liver diseases that may affect clinical outcomes; malignancy; creatinin plasma level above 440 µmol/L; estimated glomerular filtration rate (GFR) < 35 ml/min/м2; brain injury

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within 3 months prior to study enrollment; body mass index (BMI) over 30 kg/m2 and less than 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, according to investigators, might discontinue patient’s participation in the study; and patient’s refusal to participate in the study or to give his consent for it. Methods for visualization of coronary arteries

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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 asymptomatic patients at high risk of CAD prior to study enrollment. When atherosclerotic lesions of the coronary arteries were verified, patients subjected

to

conventional

angiographic

examination

provided

indications

for

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were

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

Multispiral computed tomography angiography

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cardiovascular events occurred for this period, and the procedures are available for assay.

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The coronary artery wall structure, as well as atheroma geometries and compositions, were measured by means of contrast spiral computed tomography angiography [18] on Somatom Volum Zoom scanner (Siemens, Erlangen, Germany) with two detector rows when holding breath at the end of breathing in. After preliminary native scanning, Omnipak non-ionic contrast (Amersham Health, Ireland) was administered for the optimal image of the coronary arteries. To

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reconstruct the image, 0.6-mm-width axial tomographic slices were used. The coronary arteries calcification was assessed by calculating Agatston score index and by measuring the

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calcification mass [19]. The authors determined the presence of calcified atheromas (CAT), high density noncalcified atherosclerotic plaques (HD-NCP) and low density noncalcified

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atherosclerotic plaques (LD-NCP). The presence of calcified atheromas was supposed at the computed tomography density equal to or higher than +150 Hounsfield unit (HU); the presence of HD-NCP was supposed at the density level within the range of +30 to +149 HU; and the presence of LD-NCP was supposed at the density level within the range of -100 to +30 HU [19, 20]. Assessment of intracardiac hemodynamics Intracardiac hemodynamics was assessed by means of transthoracic ultrasonic cardiography according to a conventional procedure on ACUSON apparatus, SIEMENS, Germany, in В

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ultrasonography regimen and tissue Doppler echocardiography regimen from parasternal, subcostal, and apical positions over the short and long axis with Р sensor 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

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myocardial contractility was verified. The 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

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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),

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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 the Аm (Е/Аm) ratio and to the Em (Е/Em) ratio.

Calculation of glomerular filtration rate

Calculation of glomerular filtration rate (GFR) was carried out using MDRD-6 formula [23].

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Measurement of highly sensitive C-reactive protein, NT-pro-BNP, total cholesterol and its fractions

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To determine highly sensitive C-reactive protein (hs-CRP), N-terminal pro-brain natriuretic peptide (NT-pro-BNP), total cholesterol and cholesterol fractions, blood samples were drawn in

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the morning (at 7-8 a.m.) into cooled silicone test tubes wherein 2 mL of 5% Trilon B solution were added; then 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. NT-pro-BNP content 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. The concentration of cholesterol of low-density lipoproteins (LDL-C) was calculated according to the Friedewald formula (1972).

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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 doublelabeled with FITC/PE (phycoerythrin) (BD Biosciences, USA) to CD45, CD34, CD14, Tie-2,

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and СD309(VEGFR2) antigens as per HD-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) [24]. For each sample, 500 thousand events have been

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analyzed. Circulating EPC have been identified as CD45-CD34+. СD309(VEGFR2) and Tie-2

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antigens were also determined to identify subpopulations of EPC coexpressing CD14 antigen. Obtained when laser beam is scattered in longitudinal and transversal directions in the flow cytofluorimeter, the scattergram results were analyzed by using Boolean principles for double or triple positive events. The pool of the cells identified was standardized with regard to CD45+ circulating mononuclear count.

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Study design: open cohort study. Ethical principles

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The investigators followed strictly all the requirements to clinical trials in conformity with the World Medical Association (WMA) Declaration of Helsinki, 1964, Good Clinical Practice

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proveided by International Conference on Harmonization (GCP-ICH), Council of Europe Convention for the Protection of Human Rights and Dignity of the Human Being in view of using achievements in biology and medicine, Convention on Human Rights and Biomedicine, including

Additional

Protocol

to

the

Convention

on

Human

Rights

and

Biomedicine, concerning Biomedical Research, and legislation of Ukraine. Statistical Analysis Statistical analysis of the results obtained was carried out in SPSS system for Windows, Version 20 (SPSS Inc, Chicago, IL, USA). The data were presented as mean (М) and error of mean (±m)

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or a 95% confidence interval (CI); the median (Ме) and the interquartile range. The hypothesis of normal distribution of the parameters analyzed was checked by means of Shapiro–Wilk test and Kolmogorov-Smirnov test. 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-

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test were used. The two-tailed version of Wilcoxon test was used for paired comparison of parameter values inside the group. To compare categorical variables between groups, Chi2 test (χ2) and Fisher F exact test were used. The circulating EPC count and NT-pro-BNP level in the

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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)

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and was not subjected to any mathematical transformation. The factors, which could be associated potentially with circulating EPC count, were determined by means of univariate analysis of variance (ANOVA); and then, the identified factors with Р < 0.1 also were studied by means of miltivariate analysis of variance. Receive Operation Curve (ROC) analysis was carried out to identify the optimal cutoff points of the left ventricular ejection fraction value, the Е/Еm

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ratio, and NT-pro-BNP level in the blood, which have the most substantial effect on the depletion of the circulating EPC count. The odds ratio (OR) and a 95% confidence interval (95%

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CI) were calculated for all the independent predictors of depletion of the circulating endothelial progenitor cells count. The calculated difference of P 554 pg/mL (OR=2.06; 95% CI=1.80-2.54; P=0.002), the Е/Еm ratio > 15 U (OR=1.80; 95% CI=1.34 – 2.02; Р=0.001), intense coronary

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arteries calcification assessed by Agatston score index (OR=1.20; 95% CI=1.09–1.31; Р=0.001). Moreover, the authors have found that the following are the most important independent

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predictors of the depletion of the CD14+CD309+Tie2+ phenotyped circulating EPC count: the CHF functional class (OR=1.65; 95% CI=1.44-1.94; P=0.001), the left ventricular ejection fraction < 42% (OR=1.50; 95% CI=1.40–1.80; P=0.001), the NT-pro-BNP level in the blood > 554 pg/mL (OR=2.13; 95% CI=1.94–2.48; P=0.001), hyperlipidemia (OR=1.12; 95% CI=1.05– 1.23; P=0.005), the Е/Еm ratio > 15 U (OR=2.00; 95% CI=1.75–2.26; P=0.001), as well as a

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multivessel lesion of the coronary arteries (OR=1.30; 95% CI=1.28–1.44; P=0.001). Thus, the following have the highest prognostic value for the depletion of the circulating EPC count: the

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CHF functional class, a decrease in the left ventricular ejection fraction < 42%, an increase in the NT-pro-BNP level in the blood > 554 pg/mL, an increase in the Е/Еm ratio > 15 U. In this case,

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the impact of conventional risk factors, such as arterial hypertension (HT), hyperlipidemia, and type 2 diabetes mellitus, is preserved upon a long-term follow-up in terms of the depletion of the circulating EPC count predominantly for CD45+CD34+ and CD45-CD34+ phenotyped mononuclears. The pool of endothelial progenitor cells (EPC), which coexpress universal receptors for tyrosine kinase (Tie2) and vascular endothelial growth factor (CD309), is associated more with the NT-pro-BNP level in the blood, the severity of contractile and relaxation dysfunction, as well as with the severity of CHF clinical manifestations. Discussion

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Previous studies have found that endothelial progenitor cells are the result of the mobilization of unipotent stem cells lines, and they play an important role in tissue reparation, neovascularization and angiogenesis [4, 25]. It has been considered that EPC number and functionality are assumed to reflect the endogenous vascular repair capacity, with the EPC pool

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declining in older age and being exhausted by unfavorable life-style and risk factors [26]. The positive association between the EPC number and several vascular risk factors emerged in general population was found in several studies [26, 27], while other investigators found a

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negative association between various cardiovascular risk factors and the circulating EPC count [28, 29]. Following the results of this study, the authors consider that a high sympathetic

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overload status in CHF patients alters probably the proliferation, mobilization and differentiation of various types of hematopoietic and nonhematopoietic cell populations. It was emphasized that, based on consideration of conventional risk factors, the estimation of an individual risk value remained nonoptimal [14, 28]. The authors agree with the opinion expressed by Padfield GJ. et al. (2013) [15] that the CD45-CD34+ cells count may reflect indirectly the prevalence rate

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of atherosclerosis, and it may correlate with the number of atheromas that pose a threat. Indeed, being a hematopoietic EPC marker, an Е-selectin ligand, and a cytoadherence indicator at the

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same time, CD34 antigen may reflect intension of reendothelialization processes in case of atherothrombosis of various localizations [30]. In this regard, the CD34+CD45- identified

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circulating EPC count in patients with stable CAD may be essentially similar to that in healthy individuals; and it may show decrease trends in patients with CHF. Nevertheless, one may not rule out the following particularities of pharmacotherapy in patients participating in the study: over 70% of patients received statins, while 25% of patients received antagonists of mineralocorticoid receptors. Currently, the role of the two drug classes in mobilizing EPC is being investigated extensively [31, 32]. It makes an opportunity to suppose the presence of an association between the administration of these drugs and relatively negligible alteration of the CD34+CD45- phenotyped EPC pool in the cohort of patients with CAD without CHF. On the

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other hand, with no clinically significant pro-inflammatory activation mediated, in particular, by deep microcirculation disorder common in patients with manifest CHF predominantly with the diminished total contractile function, the CD34+CD45- phenotyped circulating EPC count may reflect the disease progress risk not in full [33, 34]. One may suppose that, involved in

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neoangiogenesis, tissue reparation, and cardiovascular remodeling, endothelial progenitor cells coexpressing receptors for VEGF and tyrosine kinase contribute greatly to tension of adaptive alteration that hampers manifestation of CHF as a brand new morbid condition [35]. Moreover,

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the deficit of CD14+CD309+ and CD14+CD309+Tie-2+ phenotyped angiopoetic EPC may be considered an indicator of devolution of ischemia-induced CHF even in case when the NT-pro-

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BNP level in the blood is within the grey zone and has a limited prognostic value [17]. Indeed, the results of our study substantiate the hypothesis that CD14+CD309+ and CD14+CD309+Tie-2+ phenotyped circulating EPC counts are statistically significantly lower in the cohort of patients with angiographically proven CAD then in healthy volunteers; and they depend more on the presence of CHF and the number of cardiovascular risk factors than on the severity of coronary

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atherosclerosis. With this in mind, one may not rule out that the depletion of the pool of CD14+CD309+ and CD14+CD309+Tie-2+ phenotyped circulating endothelial progenitor cells

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with angiopoetic potential and their dysfunction being formed may negatively affect the CHF progression because of EPC involvement in cardiovascular remodeling mechanisms [5], while

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the CD34+CD45- phenotyped EPC count does reflect more the severity of atherosclerotic lesion of the coronary arteries and atherothrombosis [9, 26]. Indeed, despite an obvious depletion of the CD45+CD34+, CD45-CD34+, CD14+CD309+ and CD14+CD309+Tie2+ phenotyped circulating EPC pool with an increase in the CHF functional class, the multivariant analysis has made it possible to found that conventional cardiovascular risk factors contribute to the dynamics of CD45+CD34+ and CD45-CD34+ phenotyped circulating EPC counts. On the contrary, CD14+CD309+ and CD14+CD309+Tie2+ phenotyped mononuclerars counts correlate more with the intensity of left ventricular contractile and relaxation myocardial dysfunction, as well as with

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the CHF severity and the NT-pro-BNP level in the blood. One may suppose that a negative effect of cardiovascular risk factors, including the extent and the severity of coronary atherosclerosis, on the manifestation of ischemia-induced CHF, is probably mediated by the deficit of CD45+CD34+ и CD45-CD34+ phenotyped hematopoietic circulating EPC. At the same time,

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mobilization of CD14+CD309+ and CD14+CD309+Tie2+ phenotyped nonhematopoietic circulating EPC from peripheral tissues is already substantially depleted at the stage when asymptomatic left ventricular myocardial dysfunction emerges; with the EPC mobilization being

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an important predictor of progression of manifest ischemia-induced CHF later on.

Conclusion: The authors found that the CHF functional class, LVEF decreased less than by

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42%, the NT-the pro-BNP level increased over 554 pg/mL, and the Е/Еm ratio increased over 15 U had the highest prognostic value for the depletion of the EPC count in CAD patients. Conflict of interests: not declared Figure legends:

Figure 1. The box plots show specific ratio of variously phenotyped circulating mononuclear

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counts in patients with coronary artery disease in comparison with healthy individuals Figure 2. Various circulating mononuclears populations counts in patients with various NYHA

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functional classes of chronic heart failure caused by coronary artery disease Supplementary figure: Samples of scattergrams obtained when scattering laser beam in the flow

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cytofluorimeter followed by phenotyping mononuclear cells population by means of monoclonal antibodies, which are labeled with FITC fluorochromes (fluorescein isothiocyanate) or doublelabeled with FITC/PE (phycoerythrin), to CD45, CD34, CD14, Tie-2 and СD309(VEGFR2) antigens by High-Definition Fluorescence Activated Cell Sorter methodology. Supplementary figure shows scattergrams with antibody controls for the group patients that are represented in Figure 1. Acknowledgement 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

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28. Bakogiannis C, Tousoulis D, Androulakis E, Briasoulis A, Papageorgiou N, Vogiatzi G, et al. Circulating endothelial progenitor cells as biomarkers for prediction of cardiovascular outcomes. Curr Med Chem. 2012; 19(16):2597-604. 29. Werner N, Nickenig G. Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy? Arterioscler. Thromb. Vasc. Biol. 2006; 26: 257–266. 30. Peichev M., Naiyer A.J., Pereira D, Zhu Z, Lane WJ, Williams M. et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood. 2000; 95, 952–958.

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number of cd34+, cd133+ and cd34+/cd133+ cells in peripheral blood of patients with congestive heart failure. Eur J Med Res. 2009; 14: 113–117. 35. Kissel CK, Lehmann R, Assmus B, Aicher A, Honold J, Fischer-Rasokat U, et al. Selective functional exhaustion of hematopoietic progenitor cells in the bone marrow of patients with

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postinfarction heart failure. J Am Coll Cardiol. 2007; 49: 2341–2349.

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Table 1. General characteristic of patients participating in study

Parameters (n=25)

In whole group (n=153)

Age, years

51.70±6.10

58.34±9.60

Males, n (%)

14 (56.0%)

86 (56.2%)

Arterial hypertension, n (%)

-

Hyperlipidemia, n (%) T2DM, n (%)

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Patients with angiographically proven coronary artery disease

Healthy individuals

With CHF (n=109)

57.20±6.70

59.50±7.30

29 (65.9%)

57 (52.3%)

99 (64.7%)*

32 (72.7%)

67 (61.5%)**

-

71 (46.4%)*

19 (43.2%)

52 (47.7%)**

-

54 (35.3%)*

17 (38.6%)

38 (34.5%)

CAD in family history, n (%)

2 (8.0%)

17 (11.1%)

5 (11.4%)

12 (11.0%)

Adherence to smoking, n (%)

6 (24.0%)

32 (20.9%)

8 (18.2%)

24 (22.0%)**

24.1 (95% CI=21.6–28.7)

23.7 (95% CI=22.5–27.3)

24.2 (95% CI=22.0–27.9)

82.3 (95% CI=68.7–102.6)

82.1 (95% CI=69.9–93.1)

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AC C

25.1)

EP

23.3 (95% CI=20.1– BMI, kg/m2

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Without CHF (n=44)

93.5 (95% CI=88.3– GFR, mL/min/1.73 m2

85.2 (95% CI=70.3–

100.3)

112.5)

HbA1c, %

3.8 (95% CI=3.1-4.6)

6.8 (95% CI=4.1-9.5)*

6.3 (95% CI=4.4-9.0)

7.0 (95% CI=4.3-9.2)

Fasting blood glucose, mmol/L

4.11 (95% CI=3.2-5.5)

5.20 (95% CI=3.3-9.7)

4.80 (95% CI=3.6-8.5)

5.40 (95% CI=3.4-9.1)

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65.7 (95% CI=53.1– Creatinine, µmol/L

LDL-C, mmol/L

4.1 (95% CI=3.1–5.0)

5.1 (95% CI=3.9-6.1)*

2.75 (95% CI =2.44-3.6)

3.23 (95% CI =3.11–4.4)*

1.01 (95% CI=0.92–

0.91 (95% CI = 0.89–1.12)*

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HDL-C, mmol/L 1.20)

74.9 (95% CI=65.1–90.3)

5.3 (95% CI=4.6-6.0)

5.0 (95% CI=4.2-5.8)

3.60 (95% CI =3.20–4.18)

3.02 (95% CI=2.80–3.90)

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Total cholesterol, mmol/L

70.5 (95% CI=59.6–88.3)

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72.3 (95% CI=58.7–92.6) 80.5)

0.88 (95% CI = 0.82–

0.94 (95% CI = 0.92–1.06) 0.97)

21.4 (95% CI 13.8 –

1218.5 (95% CI 154.2 –

231.2 (95% CI 104.8 –

1533.6 (95% CI 644.5 –

46.2)

3480.75)*

360.7)

2560.6)**

Systolic blood pressure, mm Hg

127±6

131±8

135±5

129±4

Heart rate, beats per 1 min.

69±3

71±3

68±3

76±6

51.30±1.55*

55.40±0,80

42.80±0.76**

14.3±1.13*

12.5±1.20

16.6±0.94**

13.7±1.12*

10.6±0.84

16.6±1.00**

Е/Аm, U

6.1±0.22

Е/Em, U

7.2±0.19

EP

65.40±0.87

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LVEF, %

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NT-pro-BNP, pg /mL

Note: * - validity of differences between parameters in the groups of healthy individuals and the groups of patients with CAD (P