The Relationship Between Serum Insulin, Circulating Endothelial Progenitor Cells And Severity Of Coronary Stenosis In Patients With CAD

Background and Objective :Insulin resistant (IR), a common and independent risk factor for cardiovascular disorders.The mechanisms underlying the patho-physiology of IR are varied and uncertain, endothelial dysfunction/injury has been considered one of the leading mechanisms contributing to atherogenesis. EPCs are cells population with the capacity to circulate,proliferate and differentiate into mature endothelial cells but have neither acquired characteristic mature endothelial markers nor formed a lumen.Endothelial dysfunction ultimately represents an imbalance between the magnitude of injury and the capacity for repair. A variety of evidence suggests that circulating endothelial progenitor cells (EPCs) constituted one aspect of this repair process. Laboratory evidence suggests that these precursors participate in postnatal neovascularization and reendothelialization. Recently, studies have demonstrated that risk factors for coronary artery disease (CAD) correlate with reduced number and decreased functional activities of circulating EPCs. In addition, previous in vivo experiments have shown that patients with metabolic syndrome have reduced EPCs numbers.However,Is there close relationship between insulin itself and the number and activity of circulating EPCs?It is still unknown recently.To aim, we investigate the correlation between the number and activity of circulating EPCs and serum insulin levels as well as severity of coronary lesions.Methods:1. Circulating EPCs culture ,characterization and enumerationBriefly, total mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation with Ficoll separating solution. Circulating EPCs were enumerated as KDR/CD133+ cells via fluorescence- activated cell sorter analysis(FACS). Cells were plated on culture dishes coated with human fibronectin and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal-calf serum (VEGF 50ng/ml, b-FGF 5ng/mL, EGF 10ng/mL).Cell growth and morphology were evaluated. To observe the function of EPCs, FITC-ulex europaeus agglutinin(UEA-I) binding and DiI-LDL intake were performed; To identify the cell phaenotype, FACS of CD133 and KDR were carried out again.2. Selective coronary arteriography was carried out ,and assessment of the severity of coronary stenosis in the patients was estimated by the coronary score of Gensini.3. Concentration of fasting serum insulin was detected,and insulin resistance was estimated by the homeostasis model assessment-insulin resistance index (HOMA-IR).Results1 Culture and Characterization of circulating EPCs1.1 Morphology and growth of EPCsAt first day, cultured MNCs were round and lucency, with enough photonasty. Four days later, cells number increased and cell body stretched. Oval-shaped or spindle like cells appeared which accompanied with some cell process. At 7th day, number of spindle cells augmented. Cell cluster and clones appeared. At 14th day, cells connected with each other and lead to some chord or reticulate, blood capillary-shaped structure. After 21 days culture, cells mixed together, resulting in cobble-stone morphology.1.2 EPCs phaenotype analysisAfter 7 days of culture, fluorescence activated cell sorting showed expression of CD133(11.22±8.78)%, KDR (52.6±12.24)%。1.3 EPCs functional identificationAfter cellular staining of DiI-acLDL and FITC-UEA-I, red (acLDL-DiI intake) and green (FITC-UEA-I binding) fluorescence cells were found. Confocal microscopy indicated 85.34±6.07% EPCs were double positive (yellow) which were considered as differentiating2 Effects of insulin level on the number of circulating EPCs and severity of coronary stenosis in patients with CAD.2.1 The number of circulating EPCs decreased in CAD+IR group[CAD+IR vs. CAD+IS, (0.34±0.08)‰vs. (0.47±0.09)‰, P<0.01]2.2 The angiographic Gensini score decreased in CAD+IR group(CAD+IR vs. CAD+IS, 45.13±18.68 vs. 20.66±16.11, P<0.01).2.3 Relationship between HOMA-IR,circulating EPCs and Severity of CAD. HOMA-IR was inversely correlated with the number of circulating EPCs(r=-0.3785,P=0.009),and the number of circulating EPCs was inversely correlated with angiographic Gensini score(r=-0.3984,P=0.006).3 Effects of insulin level on circulating EPCs biological function and Severity of Coronary Stenosis in patients with CAD.3.1 Effects on EPCs proliferationMTT analysis showed optical density decreased in CAD+IR group (CAD+IR vs. CAD+IS, 0.27±0.09 vs. 0.39±0.11, P<0.05)3.2 Effects on EPCs migrationThe number of migrated EPCs decreased in CAD+IR group (CAD+IR vs. CAD+IS, 16.9±4.5 vs. 25.6±5.2, P<0.05)4 Effects of insulin level on the number of circulating EPCs in patients without CAD. The number of circulating EPCs weren`t significantly reduced in nCAD+IR group[CAD+IR vs. CAD+IS, (0.51±0.09)‰vs. (0.58±0.07)‰), P>0.05].Conclusion1.The number and functional capacity of circulating EPCs were impaired in patients with CAD, and were inversely correlated with the severity of coronary stenosis.2. Insulin resistence/ hyperinsulinemia reduce the number of circulating EPCs and impair their functions of proliferation and migration in CAD patients,and was inversely correlated with the number of circulating EPCs.3. Insulin resistence/ hyperinsulinemia may lead to reduce the number and activity of circulating EPCs, accelerate or aggravate atherosclerosis.