Effects Of Glucose And Insulin On Cultured HUVEC Secreting Endothelial Cell Protein C Receptor(EPCR) And Thrombomodulin (TM)

Diabetic macrovascular complications are the major cause of mortality and disability among diabetes, the incidence of macrovascular disease is higher, the disease severity greater and the onset is earlier. So it is very necessary to investigate the impairment role of diabetic macrovacular and find out the way for prevention and treatment.Endothelial cells apoptosis is an initiatory and determinant in diabetic macrovascular disease,it can induce a series of changes on structure and function of vessel. Vascular endothelial function damage or dysfunction and platelet activation were regarded as early step in the development of atherosclerosis. Molecular markers of endothelial damage and platelet activation indicate not only contribute to the development and progression of vascular disease but also the increased risk of vascular disease (including microvascular and macrovascular)in patients with type 2 diabetes meliitus.Thrombomodulin,a cell surface-expressed glycoprotein, synthesized by vascular endothelial cells, is a critical cofactor for thrombin-mediated activation of protein C(PC),an event further amplified by the endothelial cell protein C receptor (EPCR).Activated PC(APC),in turn, is best known for its natural anticoagulation properties. Recent evidence has revealed that TM, APC, and EPCR have activities that impact not only on coagulation but also on inflammation, fibrinolysis, and cell proliferation.In this study, we tested sEPCR and sTM in HUVEC cultured with different glucose and insulin concentrations at different time points to investigate the possible molecular mechanisms of glucose and insulin on diabetic vascular complications. Materials and Methods1. Materials(1) CellsHUVEC ECV-304 strain(2) ReagentGlucose, Insulin, Fetal Bovine Serum, DMEM/F-12 medium, TM kit, EPCR kit.(3) Apparatusspectrophotometer, cell culture super-clean bench, CO₂ incubated trunk, low temperature super centrifuge, profound hypothermia box.2,Methods(1) Culture of cellsThe human umbilical vein endothelial cells(ECV304) were cultured at 37℃,5% CO₂ humidified environment and changed the culture medium every 24 hours. We harvested the cells when the cells had grown to 80% confluence.(2) Groups of experimentHUVEC were cultured in culture bench. When cells were at subconfluence , they were preincubated in fresh medium and then were devided into following groups at random on glucose and insulin different concentrations. Such as glucose at 0, 5.5, 20, 40mmol/L; insulin at 0, 0.1, 1, 10nmol/L.glucose 40mmol/L and stimulated with insulin (0.1, 1, 10nmol/L). The cells were incubated to different time points (0h, 6h, 24h, 48h, 72h). The group of 0mmol/L glucose and 0nmol/L insulin is the presence. Each group have 3 samples.(3) sTM and sEPCR were assessed by ELISA.(4) statistical analysisAll data were analyzed by software package of SAS. Results were summarized with mean±standard deviation. Statistical comparisons between groups were performed by the SNK and two-way ANOVA. Values of P < 0.05 were considered significant.Results1,After HUVEC were exposed to glucose at different concentrations for different time points(0h, 6h, 24h, 48h, 72h), the sEPCR Secreted was higher compared with control group.The secreting of sEPCR at high concentration of glucose was lower after the cells was erposed for 48h.2,After HUVEC were exposed to insulin at different concentrations for different time points(0h, 6h, 24h, 48h, 72h), the sEPCR Secreted was higher compared with control group.At the time 6h, the secreting sEPCR was lower compared with control group,after 24h,it all higher compared with control group.3,Low concentration of insulin may inhibit EC damaged, but high concentration of insulin had synergistic action with high glucose on EC damaged.4,Insulin and glucose had no effects on HUVEC secreting sEPCR and sTM.Conclusion1,After HUVEC were exposed to glucose at different concentrations for different time points, the sEPCR Secreted was higher compared with controlgroup, and it showed a dose-dependent manner and a time-dependent manner.2,Low concentration of insulin may inhibit EC damaged, but high concentration of insulin had synergistic action with high glucose on EC damaged.3,Insulin and glucose had no effects on HUVEC secreting sEPCR and sTM.