The Relationship Between Circulating Endothelial Progenitor Cells And The Risk Factors Of CAD And Effect Of EPCs On Vascular Endothelium Repair

Vascular endothelial cells (ECs) not only provide a critical interface between the vessel itself and blood-borne elements, but also release a variety of bio-actively substance. The endothelium plays the important role in maintaining cardiovascular homeostasis. In the normal state, vascular endothelium presents a nonadhesive surface to circulating leukocytes and platelets while helping to prevent the clotting of blood, regulate vascular tone through the production and release of nitric oxide production and vascular relaxation. Endothelial cells are also crucial participants in the formation of new blood vessels. These new vessels have a beneficial role in the healing response to wounds or tissue ischemia and infarction. Vascular endothelial dysfunction is the initiation factor of coronary atherosclerosis (As). It also plays a critical role in the development and progression of coronary artery disease (CAD). Apoptosis of endothelial cells, macrophage adhesion and invasion, and smooth muscle cells migration and growth finally lead to stenosis of the targeted vessel. Accelerated reendothelialization effectively impairs smooth muscle cells proliferation and neointima formation and is therefore of special interest with regard to prevention of the early stages of atherosclerosis and restenosis after angioplasty. It was generally thought that the formation of new vessels in adults occurred exclusively through the extension of mature existing blood vessels and the associate vascular endothelium. A growing body of evidence now suggests that bone marrow-derived endothelial progenitor cells (EPCs) circulate in the blood and, at least in animal models, can play an important part in the formation of new blood vessels in ischemia tissue. Endothelial progenitor cells are primitive bone marrow (BM) cells that have the capacity to proliferate, migrate, and differentiate into various mature endothelial cell type. The first evidence indicating the presence of EPCs in the adult circulation emerged that mononuclear blood cells from healthy human volunteers were shown to acquire an endothelial cell-like phenotype in vitro and to incorporate into capillaried in vivo. These EPCs were characterized via expression of CD34, CD133 and vascular endothelial growth factor receptor-2 (VEGFR-2). EPCs may play an important role in endothelium maintenance, being implicated in both reendothelialization and neovascularization. The thoracic aortae of adult dogs that underwent BM transplantation were implanted with Dacron graft. After 3 months, the grafts were retrieved and found to be colonized with CD34+ endothelial cells of donor origin, suggesting that endothelialization arose exclusively from BM-mobilized EPCs. In humans, the surfaces of left ventricular assist devices were found to be colonized with CD133+/VEGFR-2+ cells. These studied suggest the existence of a population of EPCs in the peripheral circulation that contributes to rapid endothelialization to prevent thrombotic complications. Several studies demonstrated that BM-derived EPCs play an important role in patho-physiological neoangiogenesis and vascular regeneration after myocardial infarction. The studies demonstrated that transplanted, ex vivo expanded EPCs had a favorable impact on the preservation of ventricular function. After the induction of myocardial ischemia, labeled EPCs were injected intravenously. The EPCs were shown to accumulate in the ischemic area and to participate in myocardial neovascularization. It has been shown that the number of circulating EPCs inversely correlates with risk factors for atherosclerosis. Therefore, EPCs transplatation has become a novel way in the ischemia diseases. In this experiment, endothelial progenitor cells from circulating blood will be isolated and induced to differentiate to investigate the correlation between circulating EPCs and the risk factors of coronary artery disease as well as the severity coronary lesions and its clinical significance. EPCs will be transplanted to the rat carotid balloon-injury model and the effect on endothelium repair and neointima formation will be observed. Methods 1. The relationship between circulating endothelial progenitor cells and the risk factors of CAD: 42 patients with CAD and 36 patients excluding coronary heart disease (control) were studied. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and were cultured. After 10 days cultured, the number of colony-forming units of EPCs were counted by phase-contrast microscope. The relationship between the number of colony-forming units of EPCs and the risk factors of CAD were assessed. 2. The correlation between high sensitive C reactive protein (hs-CRP) and EPCs: Highsensitivity C-reactive protein was quantitatively analyzed by particle enhanced immuoturbidimetric method . The relationship between hs-CRP and EPCs were assessed. 3. Measurement of cardiac vascular endothelial growth factor gradients in patients with CAD: Blood samples were collected from coronary sinus, aorta and circulation during PCI. The concentrations of serum VEGF were measured in different sites by an enzyme-linked immunsorbent assay (ELISA). The migration of endothelial progenitor cells (EPCs) were assayed with modified Boyden chamber assay. 4. Spleen-derived EPCs were isolated and induced to differentiate; EPCs were transplanted to the rat carotid balloon-injury model and the effect on endothelium repair and neointima formation were observed. Results 1. The number of risk factors of coronary artery disease was significantly correlated with a reduction of EPCs levels(r=-0.436, P=0.014). Smoking was associated with significantly lower EPCs levels, whereas a minor but nonsignificant reduction of EPCs levels was detected in the presence of gender, hypertension, and a positive family history of CAD. It was observed that low density lipoprotein (LDL) and uric acid were negative correlated with the number of colony-forming units of circulating EPCs(P<0.05), and a correlation between age, high density lipoprotein, apoprotein A and levels of circulating EPCs, however, this relation was not statistically significant. The number of colony-forming units of circulating EPCs in CAD groups was significantly lower than those in control group (12.77±6.34 versus 37±5.48,P<0.0001), and the circulating EPCs level of coronary artery lesion group (including single, double, triple vessels disease) was significantly lower than that of control group (P < 0. 01). 2. The hs-CRP level in the ACS group was significantly higher than those in control group, the number of colony-forming units of circulating EPCs in SAP and ACS groups were both significantly lower than those in control group (P < 0.05 ). The hs-CRP level of double and triple vessels disease groups were higher than that of control group, and the circulating EPCs level of CAD group (including single, double, triple vessels disease) was significantly lower than that of control group (P < 0. 01). It was also observed that a strong correlation between the level of high sensitive C reactive protein and the number of colony-forming units of circulating endothelial progenitor cells(r = -0.429 , P< 0.05). 3. The circulating serum VEGF levels of stable angina pectoris (SAP ) patients (208.46±44.74pg/ ml) and acute coronary syndrome (ACS) patients (267.29±93.99 pg/ ml) were much higher than that of controls group (120.77±26.25 pg/ ml , P < 0. 01). The concentrations of serum VEGF in coronary vessel were much higher than those in circulation or aorta in patients with CAD (P < 0.05). However, the levels of serum VEGF were not statistically significant in different sites in control group ( P > 0.05).The cardiac VEGF gradients were markedly higher in SAP and ACS patients than those in control patients ( P < 0.001),whereas cardiac VEGF gradients were not statistically significant between SAP group and ACS group. VEGF promote EPCs migratory capacity(P < 0.05). 4. The effect of EPCs transplantation on the neointima formation: the neointima formation relieved in EPCs transplated rats, neointima/media thickness ratio (I/M) reduced. The PCNA positive expression cells in transplantation group were less than that in balloon injured carotid group and M199 group. Conclusions 1. The level of circulating EPCs was inversely associated with the risk factor scores of CAD and the severity of coronary artery lesion. 2. Hs-CRP and EPCs were associated with CAD and the severity of coronary artery lesion, and the level of hs-CRP was negative correlated with the number of colony-forming units of circulating EPCs. 3. The cardiac VEGF gradients in CAD patients were elevated. VEGF can promote EPCs migratory capacity. It is possible to be related to the therapeutic vasculogenesis and regeneration of injured endothelium. 4. EPCs incorporated into the process of injured carotid reendothelialization, EPCs transplantation induced increase of the circulating EPCs, accelerated the process of endothelial repair and reduced neointima formation.