Hyperglycemia Environment Differentially Affects The Functions Of Bone Marrow Endothelial Progenitor Cells And Circulating Late Endothelial Progenitor Cells In Rabbits

Backgroud Diabetic vasculopathy is characterized by high prevalence, earlydevelopment, rapid progression and impaired collateral vascular development. It is themajor cause of death in diabetes mellitus. Endothelial progenitor cells (EPC) are asubtype of bone marrow-derived progenitor cells expressing surface antigens of bothhematopoietic stem cells and endothelial cells. EPC are involved in adultvasculogenesis and maintainance of vascular integrity. Recently, it had been reportedthat the number and functions of circulating EPC altered both in type 1 and type 2diabetes mellitus. EPC dysfunction has been hailed as a novel concept in thepathogenesis of diabetic vascular complications. However the mechanism of EPCdysfunction in diabetes is puzzled. There are at least two types of EPC, bone marrowendothelial progenitor cell (BM-EPC) and circulating endothelial progenitor cellwhich has two subgroups: early EPC and late EPC. It is not definitely clear whetherhyperglycemia differentially affects the functions of BM-EPC and late EPC.Objectives In present study, it is presumed that hyperglycemia andhyperinsulinemia are two major causes impairing EPC. We investigated whetherhyperglycemia and hyperinsulinemia differentially affects the functions of BM-EPCand late EPC. We observed the effects of BM-EPC and late EPC cell infusion onischemic myocardium in diabetic rabbits.Methods 1. Peripheral blood mononuclear cells and bone marrow mononuclearcells were cultivated in presence of VEGF and bFGF. Passage 1 (P1)BM-EPC andlate EPC Surface antigens CD14, CD34, CD45, CD54, CD105, CD106, CD133, KDRand vWF were analyzed by flow cytometry. 2. BM-EPC and late EPC werecultivated under different hyperglycemia conditions (5.5 mmol/1, 11mmol/1, 30mmol/1 respectively) or under different hyperinsulinemia conditions (0nmol/1, 100nmol/1,1000nmol/1 respectively). Cell proliferation capacity was analyzed by MTT andmRNA expression of VEGF and bFGF were analyzed by Reverse transcriptasepolymerase chain reaction(RT-PCR) 3. Rabbits were divided into diabetes group (n=6)and control group (n=6) randomly. Alloxan injection was used to induce diabetes indiabetes group. BM-EPC and late EPC were cultivated to evaluate number of colonyforming unit, proliferation and adhersion ability respectively. 4. Diabetic rabbits wererandomized to control group (n=8), BM-EPC transplantation group (n=8) and lateEPC transplantation group (n=8). Anterior descending branch ligation wasperformed to induce acute myocardial infarction in each group, then control groupreceived M199 200μl injection, BM-EPC and late EPC transplantation group receivedmyocardial injection of 5×10~6 BM-EPC or late EPC respectively. After 4 weeks of celltransplantation, left ventricular function was assessed by echocardiography. Massontrichrome staining was per-formed to measure the average ratio of fibrosis area toentire LV area, irnmunohistochemistry staining withⅧfactor to analyze the capillarydensity. Vasculogenesis induced by transplant- ted EPC was assessed byimmunofluorescence analysis, mRNA expression of VEGF and bFGF wereanalyzed using real-time quantitive PCR.Results 1. BM-EPC had lower expression of CD34 and CD106, but higherexpression of CD45 and CD133 than late EPC(P＜0.05).It was observed that BM-EPCand late EPC could swallow ac-LDL and bind UEA-1.There was no significantdifference in swallowing ac-LDL (P＜0.05). 2. Hyperglycemia inhibited late EPCproliferation, mRNA expression of VEGF and bFGF(P＜0.01). Hyperglycemia did notinhibit BM-EPC proliferation and mRNA expression of VEGF(P＞0.05), buthyperglycemia (30mmol/1) inhibited bFGF expression. Hyperinsulinemia influencedproliferation function of BM-EPC and late EPC depending on culture time.Hyperinsulinemia inhibited mRNA expression of bFGF both in late EPC andBM-EPC(P＜0.05). However, it inhibited mRNA expression of VEGF in late EPC butnot in BM-EPC. 3. Diabetes induced by alloxan did not reduce the number of BM-EPC (P＞0.05), but reduced the number of late EPC (P＜0.05). Diabetes impairedproliferation and adhersion ability of late EPC, but not BM-EPC. 4. Both BM-EPCand late EPC could induce vasculogenesis in ischemic myocardium, but capillarydensity was higher in BM-EPC group than in late EPC group (P＜0.01). BM-EPCtransplantation inhibited ratio of left ventricular fibrosis and improved cardiacfunction. Real-time PCR indicated that mRNA expression of VEGF and bFGF wereaugmented in BM-EPC group than those in control and late EPC group (P＜0.05).Conclusions This study demonstrated that hyperglycemia differentially affectsfunctions of BM-EPC and late EPC. Hyperglycemia and hyperinsulinemia have moresevere impairment on late EPC than BM-EPC. Transplantation of BM-EPC mayrescue the ischemic myocardium by inducing vasculogenensis and augmenting VEGFand bFGF.