Olga A. Denisenko

Background. A severe form of coronary heart disease (CHD) is ischemic cardiomyopathy (ICMP), the pathogenesis of which has not been fully studied. Disturbances in the mobilization of endothelial progenitor cells (EPC) due to an imbalance of angiogenesis mediators may exacerbate ischemia in ICMP. The aim — to establish the peculiarities of changes in the balance of early and late EPС and subpopulations of VEGFR2+ cells in the blood and bone marrow in relation to the content of angiogenesis mediators and the number of desquamated endothelial cells (DEK) in the blood of patients with CHD, suffering and not suffering from ICMP. Methods. A single-stage, clinical, controlled (case-control) study was conducted from March 2019 to June 2022. 52 patients with CHD who had a history of myocardial infarction were examined: 30 people suffering from ICMP and 22 people not suffering from ICMP, as well as 15 healthy donors. The content of VEGFR2+, VEGFR2+CD34+CD14+ (early EPC), VEGFR2+CD34+CD14– (late EPC), VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells in patients with CHD in the blood (before surgery) and bone marrow (sampling was performed at the beginning of coronary bypass) and in healthy individuals in the blood, as well as (in both groups) the content of CD45–CD146+ DEC in the blood was determined by flow cytometry. The concentration of SDF-1, VEGF-A, MCP-1, GM-CSF, G-CSF in blood plasma was measured by multiplex immunofluorescence analysis. Results. The development of CHD without cardiomyopathy was accompanied by an increase in the content of VEGFR2+CD34+CD14+ and VEGFR2+ cells (0.74 [0.46; 1.23]% and 10.00 [8.20; 11.60]%, respectively, versus 0.19 [0.13; 0.32]%, p 0.001 and 5.40 [4.30; 6.50]%, p = 0.005) and concentrations of SDF-1, MCP-1, GM-CSF (respectively 60.00 [50.00; 81.00] pg/ml; 223.0 [180.0; 297.0] pg/ml; 2.10 [1.45; 3.40] pg/ml versus 30.00 [5.00; 45.00] pg/ml, p = 0.041; 175.1 [140.0; 204.0] pg/ml, p = 0.046; 0.96 [0.46; 1.41] pg/ml, p = 0.038) in the blood relative to the norm. No such changes were observed in patients with ICMP. Regardless of the presence of ICMP, the content of VEGFR2+CD34+CD14–, VEGFR2+CD34–CD14+, VEGFR2+CD34–CD14– cells, VEGF-A, G-CSF in the blood of patients with CHD varied within physiological values, and the number of DEC exceeded the norm (7.26 [5.43; 17.94]×105/l, p = 0.039). The number of VEGFR2+ cells and their immunophenotypes in the bone marrow of patients with ICMP did not differ from the parameters in patients with CHD without cardiomyopathy. Prolonged bleeding from the venopuncture area was registered in one CHD patient without cardiomyopathy. Conclusion. The development of ICMP is associated with the absence of a compensatory response to atherogenesis in the form of increased mobilization of early EPC from the bone marrow due to the absence of a reaction associated with hyperproduction of SDF-1, MCP-1, GM-CSF, which is characteristic of CHD without cardiomyopathy. The content of EPС, VEGFR2+CD34–CD14+ and VEGFR2+CD34–CD14– cells, VEGF-A and G-CSF in the blood in СHD corresponds to physiological values, regardless of the presence of ICMP. The generation of EPC in the bone marrow in ICMP is not impaired.

Background. In the pathogenesis of ischemic cardiomyopathy (ICMP), angiopoiesis remains unexplored.The aim. To describe the vasculature of the heart and the imbalance of angiogenesis mediators in the coronary circulation in association with the number of endothelial progenitor cells (EPC) and desquamated endothelial cells (DEC) in the blood of patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. Fifty-two patients with CHD (30 patients with ICMP, 22 patients without ICMP), 15 healthy donors were examined. The content of EPC (CD14⁺CD34⁺VEGFR2⁺) in the blood from the cubital vein and DEC (CD45^–CD146⁺) in the blood from the coronary sinus and the cubital vein was determined by flow cytometry. The concentrations of VEGF-A (vascular endothelial growth factor A), PDGF (platelet-derived growth factor), and SDF-1 (stromal cell-derived factor 1) in blood plasma were recorded using immunofluorescence assay; the angiopoietin-2, MMP-9 (matrix metallopeptidase 9) were recorded using enzyme immunoassay. In myocardial biopsies the specific area of vessels and the expression of αSMA (smooth muscle alpha-actin) were determined by morphometric and immunohistochemical methods.Results. In the peripheral blood of patients with CHD, regardless of the presence of ICMP, the DEC content exceeded the physiological level, and the VEGF-A, PDGF, angiopoietin-2, and MMP-9 corresponded to the norm. In CHD patients without cardiomyopathy, there was an excess of SDF-1 and EPC in the blood from the cubital vein, and in ICMP, their physiological significance was noted. In the coronary blood flow in patients with CHD without cardiomyopathy, an increase in the concentration of PDGF was found, which was not determined in patients with ICMP, who had an increased content of DEC, angiopoietin-2 and MMP-9. The specific area of the vessels in the patients of the two groups was comparable; the expression of αSMA in ICMP was 6.2 times lower than in patients with CHD without cardiomyopathy.Conclusion. The development of ICMP is accompanied by impaired maturation of vessels in the myocardium, associated with the absence of a compensatory reaction of activation of cellular and humoral factors of angiogenesis.

Highlights Dysregulation of angiogenesis may be the pathogenetic factor of ischemic cardiomyopathy (ICMP). Aim. To determine the content of growth factors and desquamated endothelial cells (DEC) in the blood from the coronary sinus and ulnar vein in association with the number of progenitor endothelial cells (PEC) in the blood from the ulnar vein in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Methods. The study included 30 patients with ICMР and 22 patients with CHD, and 18 healthy donors. The content of DEC (CD45^–CD146⁺) was determined in blood from the cubital vein (peripheral) and coronary sinus, and the content of DEC (CD14⁺CD34⁺VEGFR2⁺) was determined in peripheral blood by flow cytometry (antibodies “BD Biosciences”, USA). The concentrations of VEGF-A, VEGF-B, PDGF, SDF-1, SCF, FGF-1, TGF-β1 in blood plasma from both locations were evaluated by multiplex analysis (set “Cloud-Clone Corp.”, USA).Results. The content of DEC in peripheral blood was elevated in patients with CHD of both groups, and in patients with ICMP in sinus blood was higher than in peripheral. At the same time, in patients with CHD without cardiomyopathy, an excess of PEC and SDF-1 in the blood from the ulnar vein was established in combination with an increase in the concentration of PDGF and a decrease in the content of VEGF-B in the blood from the coronary sinus relative to the parameters of systemic blood flow. In patients with ICMP, these changes were not detected, but there was an increase in the concentration of TGF-β1 in sinus blood compared with peripheral blood. Regardless of the presence of ICMP, the concentration of SCF, FGF-1, VEGF-A in the blood from the ulnar vein corresponded to the norm and that in sinus blood; the content of VEGF-A in the coronary bloodstream exceeded its systemic level.Conclusion. In patients with ICMP, desquamation of the coronary vascular endothelium is enhanced against the background of violations of its repair processes due to insufficient (relative to CHD without cardiomyopathy) mobilization of PEC from the bone marrow due to the absence of an excess of SDF-1 in the blood and their insufficient homing into the myocardium due to weak PDGF production in the heart.

Background: The angiopoietic endothelial dysfunction in ischemic cardiomyopathy (ICMP) remains unexplored. Aim: The identification of the imbalance of endothelial dysfunction mediators and the number of endothelial progenitor (EPC) and desquamated (EDC) cells in patients with coronary heart disease (CHD) with and without ICMP. Methods: A total of 87 patients (47 with ICMP and 40 without ICMP) were observed. The content of EPCs (CD14⁺CD34⁺VEGFR2⁺) in vein blood and EDCs (CD45⁻CD146⁺) in the blood from the coronary sinus and cubital vein was determined by flow cytometry. The contents of HIF-1α and HIF-2α in vein blood as well as that of ADMA and endothelin-1 in sinus plasma and angiopoietin-2, MMP-9 and galectin-3 in both samples were assessed using ELISA, and VEGF, PDGF, SDF-1 and MCP-1 contents using immunofluorescence. Results: ADMA and endothelin-1 levels in the sinus blood were comparable between the patient groups; a deficiency of HIF-1α and excess of HIF-2α were detected in the vein blood of ICMP patients. The EDC content in the vein blood increased in CHD patients regardless of ICMP, and the concentrations of VEGF-A, VEGF-B, PDGF, MCP-1, angiopoietin-2, and MMP-9 were normal. In ICMP patients, vein blood was characterized by an excess of galectin-3 and sinus blood by an excess of EDCs, angiopoietin-2, MMP-9 and galectin-3. Conclusion: ICMP is accompanied by angiopoietic endothelial dysfunction.

Introduction. The relationship of the violation of the subpopulation composition of blood monocytes in ischemic cardiomyopathy (ICMP) with changes in monocytopoiesis, as well as the effect of colony-stimulating factor of macrophages (M-CSF) and cytokines on the differentiation of monocytes of various immunophenotypes in the bone marrow is of great relevance.Aim – to study the role of cytokines in the mechanisms of local and distant regulation of differentiation of classical, intermediate, non-classical and transitional bone marrow monocytes in combination with the content of VEGFR2⁺-monocytes and hypoxia-induced factor-1a (HIF-1a) in the blood of patients with ischemic heart disease (IHD), suffering and not suffering from ischemic cardiomyopathy.Materials and methods. Seventy-four patients with IHD, suffering and not suffering from ICMP (30 and 44 people, respectively) were examined. The number of subpopulations of classical (CD14⁺⁺CD16^–), intermediate (CD14⁺⁺CD16⁺), nonclassical (CD14⁺CD16⁺⁺) and transitional (CD14⁺CD16^–) monocytes (in bone marrow samples) and CD14⁺VEGFR2⁺-monocytes (in blood and bone marrow) was determined by flow cytofluorimetry; the concentration of cytokines IL-10, IL-13, TNF-α, IFN-γ, M-CSF in bone marrow and blood, as well as HIF-1a in the blood, was determined by ELISA.Results. Content of hematopoietins IL-10, IL-13, TNF-α, M-CSF in the bone marrow, as well as the ability of M-CSF to activate and IL-13 to inhibit the differentiation of classical monocytes from transitional cell forms were comparable between groups of patients with IHD. In the blood of patients with ICMP the concentration of IL-10 was higher, and the content of HIF-1α and CD14⁺VEGFR2⁺-cells was lower than in patients with IHD without ICMP (IL-10 – 30.00 (26.25–34.50) pg/ mL vs. 0 (23.0–28.0) pg/mL, p < 0.05; HIF-1α – 0.040 (0.029–0.053) ng/mL vs. 0.063 (0.054–0.122) ng/mL, p < 0.05; CD14⁺VEGFR2⁺ – 7.00 (5.67–7.15) % vs. 7.80 (7.23–8.17) %, p < 0.05). A feature of monocytopoiesis in ICMP compared with patients with IHD without ICMP is a high concentration of IFN-γ in the BM and a low ratio of M-CSF/IL-13 (10.00 (0.65–18.23) and 0.02 [0–0.15) pg/mL, p < 0.001; 1.02 (0.41–2.00) and 9.00 (2.13–22.09), p < 0.05, respectively), in association with a decrease in the number of classical, intermediate monocytes and an increase in the number of transitional cells in the BM in patients with ICMP relative to patients without cardiomyopathy (21.0 (19.5–23.0) and 47 (41–61.5) %, p < 0.001; 0.3 (0.0–1.2) and 18.5 (6.5–28.0) %, p < 0.01; 76.2 (73.0–78.5) and 30.5 (13.0–41.5) %, p < 0.001, respectively). At the same time, regardless of the clinical form of IHD, IL-10 and IL-13 are distant hematopoietins, TNF-α is local hematopoietin.Conclusion. An increase in the concentration of IFN-γ and a low ratio of M-CSF/IL-13 in the bone marrow, as well as an excess of IL-10 and a lack of HIF-1a and CD14⁺VEGFR2⁺-cells in the blood of IHD patients, are associated with inhibition of differentiation of mature forms of monocytes and the development of ICMP.

Background. Angiogenic endothelial dysfunction and progenitor endothelial cells (EPCs) in ischemic cardiomyopathy (ICMP) have not been studied enough.The aim. To establish the nature of changes in the cytokine profile and HIF-1α in blood and bone marrow associated with impaired differentiation of monocytic progenitor cells of endotheliocytes (CD14+VEGFR2+) in the bone marrow and their migration into the blood in patients with coronary heart disease (CHD), suffering and not suffering from ICMP.Materials and methods. A single-stage, single-centre, observational case-control study was conducted involving 74 patients with CHD, suffering and not suffering from ICMP (30 and 44 people, respectively), and 25 healthy donors. In patients with CHD, bone marrow was obtained during coronary bypass surgery, peripheral blood – before surgery. Healthy donors were taken peripheral blood. The number of CD14+VEGFR2+ in bone marrow and blood was determined by flow cytometry; the concentration of IL-6, TNF-α, M-CSF, GM-CSF, MCP-1 and HIF-1α – by the method of enzyme immunoassay.Results. A high content of CD14+VEGFR2+ cells in the blood of patients with CHD without cardiomyopathy was established relative to patients with ICMP against the background of a comparable number of these cells in myeloid tissue. Regardless of the presence of ICMP in the blood, patients with CHD showed an excess of TNF-α, a normal concentration of IL-6, GM-CSF, HIF-1α and a deficiency of M-CSF, and in the bone marrow supernatant, the concentration of IL-6 and TNF-α exceeded that in the blood plasma (the level of GM-CSF – only in patients without cardiomyopathy). With ICMP, the normal concentration of MCP-1 was determined in the blood plasma, and with CHD without cardiomyopathy, its elevated content was determined.Conclusion. The formation of ICMP is accompanied by insufficient activation of EPCs migration with the CD14+VEGFR2+ phenotype in blood without disruption of their differentiation in the bone marrow, which associated with the absence of an increase in the concentration of MCP-1 in blood plasma and not associated with the plasma content of M-CSF, GM-CSF, HIF-1α, IL-6 and TNF-α.

Aim. To identify disturbances of differentiation and subpopulation composition of VEGFR2+ cells in the blood and bone marrow associated with the features of the cytokine profile in the blood and bone marrow in patients with coronary artery disease (CAD) with and without ischemic cardiomyopathy (ICM).Materials and methods. The study included 74 patients with СAD with and without ICM (30 and 44 people, respectively) and 18 healthy donors. In all patients with СAD, peripheral blood sampling was performed immediately before coronary artery bypass grafting, and bone marrow samples were taken during the surgery via a sternal incision. In the healthy donors, only peripheral blood sampling was performed. In the bone marrow and blood samples, the number of VEGFR2⁺ cells (CD14⁺VEGFR2⁺ cells) and their immunophenotypes CD14⁺⁺CD16-VEGFR2⁺, CD14⁺⁺CD16⁺VEGFR2⁺, CD14⁺CD16⁺⁺VEGFR2⁺, and CD14⁺CD16-VEGFR2⁺ was determined by flow cytometry. Using enzyme-linked immunosorbent assay, the levels of VЕGF-А, TNFα, M-CSF, and IL-13, as well as the content of MCP-1 (only in the blood) and the M-CSF / IL-13 ratio (only in the bone marrow) were determined.Results. The content of CD14⁺VEGFR2⁺ cells in the blood of CAD patients with and without ICM was higher than normal values due to the greater number of CD14⁺⁺CD16-VEGFR2⁺, CD14⁺⁺CD16⁺VEGFR2⁺, and CD14⁺CD16⁺⁺VEGFR2⁺. In the bone marrow of the patients with ICM, the content of CD14⁺⁺CD16-VEGFR2⁺, CD14⁺CD16⁺⁺VEGFR2⁺, and CD14⁺CD16-VEGFR2⁺ was lower than in patients with CAD without ICM, and the number of CD14⁺⁺CD16+VEGFR2⁺ cells corresponded to that in the controls. Regardless of the presence of ICM in CAD, a high concentration of TNFα and normal levels of VEGF-A and IL-13 were observed in the blood. In CAD without ICM, an excess of MCP-1 and deficiency of M-CSF were revealed in the blood. In the bone marrow, the levels of VEGF-A, TNFα, M-CSF, and IL-13 were comparable between the groups of patients against the background of a decrease in the M-CSF / IL-13 ratio in the patients with ICM.Conclusion. Unlike CAD without cardiomyopathy, in ICM, no excess of VEGFR2⁺ cells and MCP-1 in the blood is observed, which hinders active migration of CD14⁺CD16⁺⁺VEGFR2⁺ cells from the myeloid tissue, and a decrease in the M-CSF / IL-13 ratio in the bone marrow disrupts differentiation of other forms of VEGFR2⁺ cells, preventing vascular repair.

Highlights. The features of subsets of monocytes in combination with the levels of desquamated endotheliocytes, endothelial damage and regeneration mediators and progenitor cell migration-enhancing factors in patients with coronary heart disease and with/without ischemic cardiomyopathy were analyzed. For the first time it was shown that in patients with ischemic cardiomyopathy, compared with CHD patients without cardiomyopathy, higher desquamation of the endothelium is associated with a deficiency of non-classical monocytes and reduced migration of progenitor endothelial cells (VEGFR2+-monocytes) with regenerative potential across the bone marrow due to a deficiency of the HIF-1α mediator in the blood.Background. The development of ischemic cardiomyopathy (ICM) is an understudied process, and one of its elements may be insufficient regeneration of blood vessels due to an imbalance of subsets of monocytes in the blood.Aim. To assess subsets of monocytes and desquamated endothelial cells in combination with endothelial damage and regeneration mediators in the blood of patients with coronary heart disease (CHD) and with/without ICM.Methods. The study included 30 patients with ICM, 22 patients with coronary heart disease without cardiomyopathy aged 55–69 years, and 18 healthy donors. In whole blood, the populations of CD45^–CD146⁺ desquamated endothelial cells and progenitor endothelial cells related to CD14+VEGFR2+ monocytes, intermediate CD14⁺⁺CD16⁺ and non-classical CD14⁺CD16⁺⁺ monocytes were assessed by flow cytometry using the appropriate monoclonal antibodies (BD Biosciens, USA). In blood plasma, the levels of hypoxia-inducible factor HIF-1α, monocyte chemoattractant protein MCP-1 and matrix metalloproteinase MMP-9 were assessed by enzyme immunoassay. The results of the analysis were considered significant at pResults. The number of progenitor and desquamated endothelial cells was increased in both groups of patients with coronary artery disease. At the same time, in patients with ICM, the number of progenitor endothelial cells did not reach the number noted in patients with CHD without cardiomyopathy, while the number of desquamated endothelial cells reached the number noted in CHD patients without cardiomyopathy. There was a deficiency of non-classical monocytes and HIF-1α in the blood of patients with ICM, and an excess of intermediate monocytes and MCP-1 was observed in CHD patients without cardiomyopathy. The concentration of MMP-9 in patients with CHD corresponded to the norm, regardless of the presence of ICM.Conclusion. In ICM, in contrast to CHD without cardiomyopathy, vascular damage is associated with a deficiency of nonclassical monocytes and reduced endothelial repair due to insufficient migration of progenitor endothelial cells across the bone marrow due to HIF-1α deficiency in the blood.

Endothelial progenitor cells (EPCs) are the cells that play a key role in maintaining vascular integrity and repairing endothelial damage. It has been shown that EPCs can differentiate into mature endothelial cells and are also capable of producing various regulatory growth factors and cytokines. There are many studies focusing on the associations between coronary artery disease and circulating EPCs. However, it is still challenging to use EPCs for the development of diagnostic and treatment algorithms in cardiovascular diseases. This review presents data on EPCs subtypes, methods for obtaining them, biological characteristics of cell populations, and the factors of EPCs mobilization to the site of injury in cardiovascular diseases.