RU2531947C1 - Method for prediction of risk of cardiovascular mortality in patients suffering from chronic ischemic heart failure combined with type 2 diabetes mellitus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology, and can be used to predict a risk of the cardiovascular mortality in patients with post-infarction chronic heart failure combined with type 2 diabetes mellitus. That is ensured by measuring circulating progenitors of the antigenic phenotype CD34+, CD133 dim, CD45-. If the measured cell count is less than 300 per one million leukocytes, a poor prognosis manifested by progressing heart failure and the increasing risk of the cardiovascular lethality is done.

EFFECT: invention provides predicting the survival rate values in the given group of patients by using the new blood biomarker.

2 cl, 2 dwg

Description

FIELD OF THE INVENTION

The invention relates to medicine, namely cardiology, can be used to determine the risk of cardiovascular mortality in patients with chronic heart failure (CHF) of ischemic etiology, combined with type 2 diabetes mellitus, by determining the number of progenitor cells of the immunophenotype CD34 +, CD 133 dim, CD45 - in peripheral blood. This parameter can be used as a prognostic criterion for assessing the probability of an unfavorable prognosis for cardiovascular mortality.

State of the art

Chronic heart failure (CHF) is widespread in the population, which is an important medical, social and economic problem. CHF is a complex syndrome, which is the outcome of many cardiovascular diseases, such as myocardial infarction, leading to the loss of part of the myocardium, which is not restored due to endogenous reparative processes; arterial hypertension, leading to the development of diffuse fibrosis of the heart; inflammatory and autoimmune, genetic diseases of the myocardium, heart defects. The vast majority of patients with heart failure are represented by post-infarction heart failure. Although significant progress has been made in the treatment of heart failure over the past 20 years, its prognosis remains fatal, and all types of available therapy are aimed only at limiting its progression. This is largely due to the fact that the most important subgroups of patients with heart failure have not been sufficiently investigated. This is especially true for patients in whom CHF is combined with metabolic disorders such as metabolic syndrome, type 2 diabetes mellitus (T2DM) and obesity. It is known that patients with heart failure combined with insulin resistance and type 2 diabetes are characterized by a much more severe course and poor prognosis, which necessitates an earlier diagnosis of this combination and more aggressive management of such patients. The quality and life expectancy of a patient with heart failure depends largely on the presence, severity and combination of the so-called risk factors (RF) of an unfavorable prognosis. Objective risk-stratification in patients with heart failure taking into account independent RF is not always possible due to the different etiology of heart failure, individual characteristics of its course, the presence of a concomitant pathology that affects the prognostic value of RF. The RF includes a number of indicators, such as age, the presence of coronary heart disease, diabetes mellitus, the severity of heart failure, tachycardia, weight loss, low left ventricular ejection fraction, decreased test results of a 6-minute walk, increased levels of cerebral natriuretic peptide, uric acid, creatinine , troponin, indicators of “remodeling” of heart chambers, etc. [Swedberg K. The task force for the diagnosis and treatment of chronic heart failure of the European Society of Cardiology. Guidelines for the diagnosis and treatment of chronic heart failure: executive summary (update 2005). Eur. Heart J. 2005; 26 (22): 1115-40].

In recent years, special attention has been drawn to the study of cellular markers of the severity of various diseases, including cardiovascular, due to a large number of studies on the state of stem and progenitor cells in various pathologies and the discovery of the relationship between the quantitative and qualitative characteristics of various types of these cells and the clinical characteristics of patients .

According to modern concepts, the key role in the regenerative processes in the body is played by stem cells capable of self-reproduction, differentiation and reproduction of various cells of other species, i.e., transformation through the stages of progenitor cells into highly differentiated organ-specific cells, for example, into vascular endothelial cells, cardiomyocytes, blood cells, hepatocytes and others [Iwami Y., Masuda N., Asahara T. Endothelial progenitor cells: past, state of the art, and future. J Cell Mol Med 2004; 8: 488-497]. In addition, the most important mechanism for stimulating tissue regeneration processes by stem and progenitor cells is their ability to produce a large number of biologically active molecules that stimulate regenerative processes in tissues.

Many, if not all, diseases of the cardiovascular system are associated with vascular endothelial dysfunction. A special role in the process of restoring the endothelial layer and in maintaining its normal function is assigned to the endothelial cell precursors (PEC) circulating in the peripheral blood [Wassmann S., Werner N., Czech T., Nikening G. Improvement of endothelial function by systemic transfusion of vascular progenitor cells. Circ Res 2006; 99: E74]. For the first time, the PEC population was isolated from the circulating mononuclear fraction in 1997 by Asahara et al. [Asahara T., Murohara T., Sullivan A. et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: 964-966]. These cells were characterized by the authors as cells with low expression or complete absence on their surface of the common leukocyte antigen CD45 and the presence of specific cell precursors (CD34) and mature endothelium (type 2 VEGF receptors (VEGF-R2)). It was subsequently demonstrated that these cells are circulated by several subpopulations, in particular, the subpopulation carrying the CD133 + marker represents early endothelial cell precursors mobilized from the bone marrow and under certain conditions able to differentiate into cells with an endothelial phenotype [Yang C, Zhang ZH, Li ZJ et al. Enhancement of neovascularization with cord blood CD133 + cell-derived endothelial progenitor cell transplantation. Thromb Haemost. 2004: 91 (6): 1202-1212].

More recently, it has been shown that an increase in the number of circulating endothelial cell precursors (PECs) may reflect the presence of vascular pathology, endothelial dysfunction and serve as a biomarker of vascular diseases, and a decrease in the PEC pool can lead to impaired adaptive angiogenesis. It was shown that metabolic syndrome and T2DM can significantly affect the number and functional characteristics of PEC in these pathologies [Vasa M., Fichtlscherer S., Aicher A. et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001; 89: E1-E7].

Patients with heart failure often have endothelial dysfunction [Agnoletti L, Curello S, Bachetti T, Malacarne F, Gaia G, Comini L, Volterrani M, Bonetti P, Parrinello G, Cadei M, Grigolato PG, Ferrari R. Serum from patients with severe heart failure downregulates eNOS and is proapoptotic: role of tumor necrosis factor-alpha. Circulation. 1999 Nov 9; 100 (19): 1983-91]. However, there is very little work devoted to the study of populations of circulating precursor cells in heart failure and their results are contradictory [Shantsila E, Wrigley BJ, Shantsila A, Tarr LD, Gill PS, Lip GY. Monocyte-derived and CD34 + / KDR + endothelial progenitor cells in heart failure. J Thromb Haemost. 2012 Jul; 10 (7): 1252-61]. Recently it has been shown that type 2 diabetes mellitus has a damaging effect on PECs and their mobilization from the "marrow niche", which can lead to a decrease in the number of these cells in the circulation, impaired participation in the repair of vascular endothelium and angiogenesis, and therefore in tissue repair after ischemic damage [Terreg OM, Galiano RD, Capla JM et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002; 106: 2781-2786]. This can lead to the progression of ischemic diseases and the deterioration of their prognosis. However, methods for predicting cardiovascular mortality in patients with CHF associated with diabetes mellitus have not been described by the number of stem and progenitor cells circulating in the blood.

The prior art method for predicting the risk of cardiovascular mortality in patients with various forms of chronic heart failure by the level of cerebral natriuretic peptide (NT-proBNP) and tissue matrix metalloprotease inhibitor (TIMP-I) in peripheral blood. Increasing the level of NT-proBNP≥1520fmol / ml and TIMP-I≥865ng / mL allows distinguishing among high-risk patients with heart failure who require intensive treatment of both heart failure and complications (Solomakhina N.I., Belenkov Yu.N. The prognostic value of a matrix inhibitor of matrix metalloproteinases-1 (TIMP-I) in patients with heart failure // Heart failure. - 2010. - No. 5. - P.281 - 284 .; Solomakhina NI. Levels of NT-proBNP and cardiovascular mortality in patients with chronic heart failure of the elderly and senile age // Cardiovascular therapist ia and prevention. - 2009.- No. 8, S2. - P.303).

However, the method presented does not take into account that concomitant metabolic disorders such as metabolic syndrome, obesity and type 2 diabetes mellitus can affect the prognostic value of NT-proBNP level (Taylor JA, Christenson RH, Rao K, Jorge M, Gottlieb SS. B- type natriuretic peptide and N-terminal pro B-type natriuretic peptide are depressed in obesity despite higher left ventricular end diastolic pressures. Am Heart J. 2006; 152: 1071-1076 .; Martin Andreas, Harald Zeisler, Ammon Handisurya, Maximilian V Franz , Michael Gottsauner-Wolf, Michael Wolzt, Alexandra Kautzky-Wilier. N-terminal-pro-brain natriuretic peptide is decreased in insulin dependent gestational diabetes mellitus: a prospective cohort trial. Cardiovasc Diabetol. 2011; 10:28; Susana Gonzalez, Eric S. Kilpatrick, Stephen L. Atkin. The Biological Variation of N-Terminal Pro-Brain Natriuretic Peptide in Postmenopausal Women with Type 2 Diabetes: A Case Control Study. PLoS One. 2012; 7 (11): e47191; Henrik Reinhard, Niels Wiinberg, Peter R Hansen, Andreas Kjaer, Claus L Petersen, Kaj Winther, Hans-Henrik Parving, Peter Rossing, Peter K Jacobsen. NT-proBNP levels, atherosclerosis and vascular function in asymptomatic type 2 diabetic patients with microalbuminuria: peripheral reactive hyperaemia index but not NT-proBNP is an independent predictor of coronary atherosclerosis. Cardiovasc Diabetol. 2011; 10:71). This necessitates the search for new predictors of adverse outcome for patients with heart failure, combined with metabolic disorders, especially diabetes mellitus. In addition, it has been convincingly shown that the use of several predictors in addition to the levels of natriuretic peptides can significantly increase the prediction of outcomes of cardiovascular diseases (Zethelius B, Berglund L, Sundstrom J, Ingelsson E, Basu S, Larsson A, Venge P, Arnlov J. Use of multiple biomarkers to improve the prediction of death from cardiovascular causes. N Engl J Med 2008; 358: 2107-2116), which makes it urgent to develop new predictors of an unfavorable prognosis of chronic heart failure, especially when combined with metabolic disorders that can affect P ognosticheskuyu value classic biomarkers severity and prognosis of chronic heart failure.

Disclosure of invention

The objective of the invention is to provide a new method for predicting cardiovascular mortality in patients with chronic heart failure associated with type 2 diabetes.

The technical result, to which the claimed invention is directed, is the development of a new cell marker of cardiovascular mortality in patients with chronic heart failure of ischemic etiology, with its combination with diabetes mellitus.

The prognosis of cardiovascular mortality in patients with chronic heart failure associated with type 2 diabetes mellitus is based on the measurement, for example, by flow cytometry, of the number of progenitor cells circulating in the blood of a specific immunophenotype, namely the immunophenotype CD34 +, CD133 dim, CD45- (cells are characterized by the presence of a CD34 marker on their surface, a weak presence of a CD133 marker, and the absence of a CD45 marker). The decrease in the number of circulating progenitor cells of the immunophenotype CD34 +, CD 133 dim, CD45-less than 300 cells per million leukocytes in patients with chronic heart failure of ischemic etiology, combined with type 2 diabetes, negatively affects the course of chronic heart failure and, above all, on indicators survival, and can serve as an obvious predictor of cardiovascular mortality.

Brief Description of the Drawings

The invention is illustrated by drawings, where figure 1 presents an example of detection of subpopulations of precursors among mononuclear cells of peripheral blood. At the same time on figa shows the selection of living cells based on the ratio of red (Hoechst-Red) and blue (Hoechst-Blue) fluorescence Hoechst 33342; on figb - the allocation of mononuclear cells (lyphocytes, NK cells, monocytes) on the basis of light scattering, figv Identification of CD34 + / CD45-Low precursors; on figg - determination of the baseline level of fluorescence of cells in the channels of PE and PerCP using isotopic immunoglobulins; on fig.1D - detection of KDR-positive (PE +) and C0133-positive (PerCP +) cells.

Figure 2 presents a diagram showing the mortality rate, the frequency of non-fatal cardiovascular events and non-cardiovascular events in patients with chronic heart failure of ischemic origin, depending on the initial number of circulating progenitor cells.

The implementation of the invention

Considering that both diabetes mellitus and heart failure are associated with the development of endothelial dysfunction and tissue ischemia, the level of circulating progenitor cells associated with endothelial dysfunction and ischemia can be an important biomarker of disease severity and prognosis in patients with a combination of these pathologies. However, this issue has not yet been investigated in heart failure, combined with type 2 diabetes.

Based on the results of the studies, a criterion was found that allows a high degree of certainty to predict the risk of cardiovascular mortality in patients with chronic heart failure of ischemic etiology, combined with type 2 diabetes. As such a criterion, the quantitative content of the progenitor cells circulating in the peripheral blood of the immunophenotype CD34 +, CD133 dim, CD45-. Moreover, a decrease in the number of circulating progenitor cells below 300 cells per million leukocytes is taken as a marker for an unfavorable prognosis of the course of the disease, manifested in the progression of heart failure and an increase in cardiovascular mortality.

To measure the quantitative content of progenitor cells circulating in peripheral blood, peripheral blood is taken into a tube with EDTA as an anticoagulant, the mononuclear fraction is isolated using Hisopaque-1077 solution (Sigma-Aldrich, cat. No. 10771) in accordance with the manufacturer's instructions, the number of circulating progenitor cells of CD34 +, CD 133 dim, CD45- immunophenotype, for example, by flow cytometry and corresponding fluorescently labeled antibodies, and when a measured amount of circus is detected proliferating cells below 300 cells per million leukocytes conclude that the prognosis of the disease is poor, manifested in the progression of heart failure and an increase in cardiovascular mortality.

Brief description of the study

55 patients with chronic heart failure developed after acute myocardial infarction (MI) were included in the work. The average age was 60.1 ± 6.3 years (age range from 40 to 73 years), 47 men and 8 women. All patients were diagnosed with heart failure II-III functional class (FC) according to the classification of the New York Heart Association (NYHA) (70% of patients with FC II and 30% with FC III according to NYHA). In 25 patients, CHF was combined with type 2 diabetes. The ejection fraction (EF) of the left ventricle in all patients at the time of inclusion was less than 40%.

To assess the structural and functional state of the heart, all examinees underwent transthoracic ECHO-KG on an Philips iE ultrasound machine. To increase the accuracy of measurements in each case, the values obtained in 5 consecutive cardiac cycles were averaged. During the study, structural and functional indicators of the heart, Doppler indices of LV diastolic function were determined.

The exclusion criterion was the presence of myocardial infarction and stroke less than 12 months old, pericarditis, rheumatic heart disease, infectious endocarditis, severe concomitant pathology (malignant diseases, chronic lung diseases, renal failure, collagenoses, liver diseases).

As a control group, 20 individuals of comparable age and gender without cardiovascular diseases were examined.

The number of circulating progenitor cells (CPCs) was estimated by flow cytometry using the following procedure.

The analysis protocol of circulating precursors (markers CD45, c-Kit, CD34, KDR, CD133).

Blood sampling from the cubital vein was carried out in the morning (between 9-10 hours), on an empty stomach, after 10 minutes of rest and adaptation of the patient / donor to the environment, in a sitting position. 10 ml of blood was collected in a tube with EDTA as an anticoagulant. The resulting blood sample at room temperature was delivered to the laboratory for further analysis. The number of circulating precursors was estimated using flow cytometry method. A mononuclear fraction was isolated from blood samples using Hisopaque-1077 solution (Sigma-Aldrich, cat. No. 10771) in accordance with the manufacturer's instructions.

The resulting cells were resuspended in 150 μl of phosphate-buffered saline (PBS) with 2% bovine serum albumin (final concentration ~ 10 ⁸ / ml). The cell suspension was divided into 2 samples:

1) a sample to detect precursors (100 μl);

2) a sample with isotypic controls (50 μl of cell suspension + 50 μl of FSB with 2% albumin).

20 μl per sample of fluorescently labeled antibodies was added and incubated in a dark place with gentle stirring at room temperature for 2 hours. The following combinations of antibodies were used:

1) CD45-FITC (Beckton Dickinson, BD, cat. No. 555482), KDR-PE (R&D Systems, cat. No. FAB357P), CD133-Biot (Miltenyi Biotec, cat. No. 130-090-852), Str-PerCP (Jackson Immunoresearch, cat No. 016-120-084, was added after 1 h of incubation), CD34-APC (BD, No. 555824),

2) CD45-FITC (BD, No. 555482), IgG-PE (BD, No. 555749), IgG-PerCP (R&D, No. IC003C), Str-PerCP (Jackson Immunoresearch, cat No. 016-120-084, was added after 1 h incubation), CD34-APC (BD, No. 555824).

After the staining period with antibodies, each PBS sample was diluted to a volume of 1.5 ml and precipitated by centrifugation at an acceleration of 300g × 10 min, the supernatant was removed, resuspended in 600 μl of PBS (300 μl for isotypic control) and Hoechst 33342 (Invitrogen Cat No. H21492) was added. to a final concentration of 10 μg / ml. Incubated for 20-90 min at room temperature. Cells were analyzed on a flow cytometer using excitation at wavelengths of 355, 488 and 635 nm, for example, a flow cytometer / sorter MoFlo (Beckman Colter, USA), or LSR Fortessa (BD). At the same time, at least 2 million Hoechst-positive events were accumulated for the first sample and at least 1 million for a sample with isotypic controls.

Data analysis was performed using the cytometer software (Summit for Beckman Coulter and FACSDiva for BD), or specialized software FlowJo (Treestar Inc, USA) or Kaluza (Beckman Coulter). The analysis was carried out according to the following scheme:

1. Isolation of a fraction of living blood mononuclear cells

a) On the histogram Hoechst red (650-690 nm, linear scale), Hoechst blue (420-450 nm, linear scale), the region of living cells was identified. These cells were characterized by less intense staining and the predominance of the blue Hoechst fluorescence component. The display of these cells is presented on the histogram of direct / side light scattering (Figa).

b) On the histogram (direct, lateral light scattering), the region of lymphocytes / NK cells / monocytes (mononuclear cells) was isolated and these cells were displayed on the histogram [CD45, CD34] (Fig. 1B).

2. Determination of CD34 + precursors and the content of KDR + and CD133 + cells among them

a) On the histogram [CD45, CD34], cells with high expression of CD34 and reduced expression of CD45 (CD34 + / CD45-Low, Fig. 1B) were isolated. These cells represent the majority of circulating precursors. The number of CD34 + / CD45-Low cells per 1 million mononuclear cells isolated in item 1b was calculated. These cells were displayed on a histogram of [KDR, CD133] or [IgG-PE, IgG-PerCP] for isotypic control.

b) Using a test with isotypic control, the upper limit of the basal cell fluorescence in the PE and PerCP channels was determined (Fig. 1G). On the histogram [KDR-PE, CD133-PerCP], cells were isolated whose fluorescence intensity in the PE and PerCP channels exceeded the basal fluorescence (Fig. 1E). It was calculated which% of the CD34 + cells expresses KDR and CD133.

Reproducibility and controls

CD34 + CD45-Low population is almost always clearly distinguishable and has an intense color CD34. Among 120 samples of mononuclear cells analyzed at the Faculty of Fundamental Medicine of Moscow State University M.V. Lomonosov, in 98.3% of them it was possible to clearly distinguish this population of cells. In the case when the population is indistinguishable, the alignment of the device and the correctness of registration in the APC channel were checked. If the alignment is correct and the registration parameters in the APC channel are normal, the anti-CD34 antibody is replaced. If even with new antibodies, the CD34 + CD45-Low population was not registered or was not clearly registered, all reagents used in sample preparation were checked and replaced if necessary.

Negative controls for staining with KDR, c-Kit, CD 133 are cells stained with isotypic antibodies conjugated to a fluorescent label corresponding to specific antibodies (Sterpavidin-PerCP for CD133). Positive control is not required for CD34. present in almost every sample. CD133 is expressed on almost all CD34 + CD45-Low cells, although to a lesser extent. Comparison of the level of intensity of luminescence of PerCP in the isotypic control and in the sample stained for CD133 allows us to understand whether there is a specific staining of CD133. A positive control for staining for KDR are CD45 + monocytes, as they carry all these antigens on the surface, albeit in moderation.

The reliability and reliability of population counting was evaluated using parallel sample preparation, staining and analysis of two samples from the same initial blood sample. The differences in the relative content of CD34 + CD45-Low cells were less than 10%.

Statistical processing of all research results was carried out using the statistical software packages Microsoft Excel 2003, STATISTICA 6.0, SPSS, implemented on an IBM Pentium IV PC. For comparison, the parametric method (unpaired t-test) was used for the normal distribution of variables, and the nonparametric method (Mann-Whitney test) was used for deviation from the normal one; values were expressed as mean ± standard deviation. Cumulative survival was studied using the Kaplan-Meyer method, differences between groups were evaluated taking into account the Wilcoxon criterion. The frequency of adverse events in the groups was compared using analysis of variance (one-way Fisher test).

Patients were observed for 3 years in the NPO FGBU RKNPK Ministry of Health of the Russian Federation from the beginning to the end of the test or until the death of each individual patient.

All patients underwent multicomponent standard therapy for heart failure and coronary heart disease, patients with diabetes received appropriate antidiabetic therapy.

In the control group for the observation period, not a single case of cardiovascular disease was noted.

In patients with CHF, there were 3 deaths due to cardiovascular causes, namely, 2 cases of sudden death on the background of CHF decompensation and one death case as a result of CHF decompensation. All deaths during the follow-up were in patients with heart failure, combined with type 2 diabetes.

In 11 cases there were non-lethal cardiovascular events - repeated hospitalizations, of which 6 patients had decompensated heart failure, 5 patients had progressive coronary heart disease (the development of acute coronary syndrome requiring intravascular revascularization - stenting). In 4 patients, the cause of hospitalization was decompensation of diabetes mellitus.

Three-year survival in the total group of CHF was 94%, with 100% survival in the control group. Moreover, in the subgroup of CHF with concomitant type 2 diabetes mellitus, the three-year survival rate was 88% with 100% survival in the CHF group without diabetes mellitus.

The number of CPC in patients of the control group averaged 649.9 ± 276.8 cells per million leukocytes, in all patients with heart failure - 793.0 ± 456.0 cells per million leukocytes.

When dividing all patients with CHF into two groups — patients with a stable course without repeated hospitalizations and fatal outcomes and patients with a progressive course and repeated hospitalizations and fatal outcomes — it was found that the CPC level in the first group was 634.9 ± 415.5 cells per million leukocytes, and in the second, 460.8 ± 411.3 cells per million leukocytes, p> 0.05, that is, there was only a tendency to a greater number of CPC in patients with chronic heart failure with a stable course.

In patients with fatal outcomes, the number of CPCs at the beginning of the study was 149.3 ± 92.7 cells per million leukocytes, and in all other patients, including cases of all hospitalizations and a stable course of heart failure, it was 610.5 ± 413.7 cells per million leukocytes, i.e. more than 4 times lower (p <0.01). At the same time, the CPC level in patients with all cases of hospitalizations for cardiovascular events and decompensation of diabetes mellitus amounted to an average of 554.3 ± 426.1 cells per million leukocytes, which was also higher than in patients with fatal outcomes, but did not significantly differ from patients with stable during CHF. In patients with a stable course, the number of CPCs was 634.9 ± 415.5 cells, which is more than 4 times higher than in patients with fatal heart failure (p <0.05). Given the significant reduction in the number of CPCs in patients who had fatal heart failure during a three-year follow-up, an analysis of all heart failure outcomes was performed depending on the initial level of CPC.

For this, all patients with CHF were divided into groups depending on the number of circulating progenitor cells. The first group included patients with heart failure, in whom the number of CPC was below the maximum dispersion of this indicator in the control group (relatively healthy people) - less than the smallest indicator in the control group. There were 12 such patients. The number of CPCs in them was less than 300 cells per million leukocytes. 43 patients constituted a group in which the initial level of CPC was within the scatter in the control group and higher. Both groups were comparable in age, gender, severity of heart failure, and other indicators.

In the first group of patients with CHF and an initially reduced amount of CPC, the survival for 3 years of observation, taking into account cardiovascular mortality, was 75%, which is significantly lower than in the group of patients with CHF with an initially normal level of CPC (100%) (p <0, 05). Among the remaining adverse events in the group with an initially reduced amount of CPC, there were three hospitalizations in connection with decompensation of heart failure and one in connection with decompensation of diabetes mellitus. Not a single hospitalization due to the progression of coronary heart disease (acute coronary syndrome or recurrent myocardial infarction was observed). In general, there were 7 adverse events in this group, including deaths, which amounted to 58.3%, cardiovascular events were 6, which amounted to 50%, 1 hospitalization was due to decompensation of diabetes mellitus.

In the group of patients with heart failure whose initial level of CPC was within the scatter in the control group or exceeded it, over 3 years of follow-up there was not a single fatal outcome, 5 hospitalizations for the development of acute coronary syndrome, 3 hospitalizations for decompensation of heart failure and 3 about decompensation of diabetes. A total of 11 adverse events over 3 years of observation (25.6%), which was two times less than in the group with a reduced initial amount of CPC (p <0.02). The results show that in patients with heart failure with an initially reduced amount of CPC (below 300 cells per million leukocytes), fatal outcomes associated with progression of heart failure are more often observed with higher reliability than in patients with an initially normal level of CPC. A decrease in CPC below 300 cells per million leukocytes was associated with an increase in mortality from cardiovascular causes by 25% (survival in the group with a low number of CPC is 75%, in other patients, including patients with a progressive course, it is 100% at 0% mortality per 3 years), the differences are significant, p <0.01, taking into account the Wilcoxon-Mann-Whitney criterion and the one-way Fisher test.

When evaluating data on all cardiovascular events (cardiovascular mortality, acute coronary syndrome, hospitalization for decompensation of heart failure), excluding hospitalizations for decompensation of diabetes mellitus, in patients with initially low values of CPC they were 50% at 18, 6% in patients with normal and elevated CPK values (p <0.01). Moreover, it should be emphasized that in the group with low CPC values there was not a single case of acute coronary syndrome, and all non-lethal cardiovascular events were represented by decompensation of heart failure. At the same time, in patients with normal and elevated levels of CPC among non-lethal cardiovascular events, more than half was the development of acute coronary syndrome, which required intravascular revascularization (stenting).

CHF that occurs after a myocardial infarction is the result of postinfarction remodeling of the heart. The development of necrosis of part of the myocardium and the subsequent formation of scar tissue leads to a change in the shape, size and thickness of the walls of the left ventricle of the heart - their thinning in the scar zone and hypertrophy of the undamaged portion of the myocardial wall in response to increased hemodynamic load. Changing the shape of the left ventricle leads to a decrease in its pumping function, which leads to the development of heart failure. A decrease in the pumping function indicator - ejection fraction of the left ventricle - is always associated with a worsening prognosis. On the other hand, the process of heart remodeling and its recovery after a heart attack can be affected by the level of adaptive angiogenesis and endothelial function, which, in particular, can be affected by circulating progenitor cells that can be mobilized from the bone marrow in response to growth factors and cytokines secreted cells of the damaged myocardium, enter the bloodstream and by the mechanism of homing come into the damaged myocardium, where they can have effects that contribute to the regeneration / repair of the myocardium: to adapt to damaged endothelium and improve its function, participate in angio-vasculogenesis and improve myocardial blood supply, and also, importantly, secrete a wide range of growth factors and cytokines that improve the functional state of cardiomyocytes and suppress their apoptosis, which helps to improve heart function and inhibits progression heart failure. Therefore, the decrease in the number of CPCs, which most likely occurs as a result of violations of their mobilization from the bone marrow under the influence of concomitant diabetes mellitus, can make a significant contribution to the prognosis of patients with heart failure, especially when combined with type 2 diabetes.

Thus, a decrease in the number of circulating progenitor cells (immunophenotype CD34 +, CD133 dim, CD45-) of less than 300 cells per million leukocytes in patients with chronic heart failure of ischemic etiology, combined with type 2 diabetes, negatively affects the course of heart failure and primarily on survival rates and may serve as an obvious predictor of cardiovascular mortality.

A method for predicting cardiovascular mortality by CPC level has been tested in the scientific advisory department of the Cardiology Institute named after A.L. Myasnikov Federal State Budgetary Institution "RKNPK" Ministry of Health of the Russian Federation. He gave significantly positive results and will find application in medical practice.

Claims (2)

1. A method for predicting the risk of cardiovascular mortality in patients with chronic heart failure of ischemic etiology combined with type 2 diabetes mellitus, comprising measuring the number of progenitor cells circulating in the peripheral blood of the immunophenotype CD34 +, CD133 dim, CD45-, while detecting a measured number of circulating progenitor cells below 300 cells per million leukocytes conclude an unfavorable prognosis of the course of the disease, manifested in the progression of heart failure chnosti and an increase in cardiovascular mortality. 2. The method according to claim 1, characterized in that the measurement of the number of circulating progenitor cells of the immunophenotype CD34 +, CD 133 dim, CD45- is carried out using flow cytofluorimetry and corresponding fluorescently labeled antibodies.

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