Effects Of Hypoxia/reoxygenation-induced Endothelial Microvesicles On H9c2 Cells

With deeper insights into the study of cardiovascular diseases,relationship between vascular endothelial function and cardiovascular diseases has rasied considerable concern.It is generally accepted that endothelial dysfunction is involved in the initial and core process of the pathogenesis of cardiovascular disease.Research indicated that microvesicles(MV)were released from endothelial cells during activation or apoptosis,and the elevation of endothelial MV(EMV)in peripheral blood was observed in various pathological conditions,including coronary artery diseases.Myocardial ischemia/reperfusion,as the pathophysiologic basis of coronary artery diseases,induced severe injury,apoptosis and necrosis on myocytes.However,the effects of hypoxia/reoxygenation induced EMV on H9c2 cells had not been studied.In the current research,a novel approach to the generation of EMV induced by hypoxia/reoxygenation(H/R)in vitro was explored,then the role of H/R-EMV in H9c2 cells was investigated in order to explicate the mechanism of myocardial ischemia/reperfusionObjective:It was hypothesized that hypoxia/reoxygenation(H/R)induced the generation of endothelial microvesicles and they exerted apoptotic effects on H9c2 cells.Therefore,the present study aimed to determine the effects of endothelial microvesicles and further explore the apoptotic effects on H9c2 cells.Methods:1.Hypoxia/reoxygenation was used to establish the injury model of HUVECsHUVECs were cultured with DMEM containing 10%FBS for 24 hours,then HUVECs were treated under different hypoxia/reoxygenation conditions.Cell viability was tested by MTT assay and proper hypoxia/reoxygenation time was determined.2.The generation,flow cytometric chacterization and quantitative analysis of MV induced by H/R from HUVECsSupernatants of subconfluent HUVECs were replaced with a mimic hypoxic buffer.HUVECs were exposed to the anoxic condition in the hypoxic chamber for 12 h and then to normoxia for 4 h at 37?.EMV were collected from culture supernatant and identified by flow cytometry.Absolute number of EMV was calculated with standard beads and EMV protein were quantified by BCA assay.3.Apoptotic effects of H/R-EMV on H9c2 cellsH9c2 cells were cultured with DMEM containing 10%FBS for 24 h,then subconfluent cells were treated with different concentrations of H/R-EMV for 6 h.H9c2 cells were randomly divided into 4 groups.They were Control group?10?30 and 60 ?g/mL H/R-EMV group.These groups were cultured with DMEM?10?30 and 60 ?g/mL H/R-EMV respectively.Cell viability was tested by MTT assay and activity of LDH.Moreover,apoptotic rates of the target cells were observed through Hoechst 33258 staining and Annexin V-FITC/PI double staining.Evidence for H9c2 apoptosis was detected by caspase 3 activity measurement and Bcl-2/Bax expression was assessed by western blot.Results:1.Hypoxia/reoxygenation was used to establish the injury model of HUVECsAfter 12 hours of hypoxia and 4 hours of reoxygenation(H12/R4),HUVECs showed shrinkage and distortion.Compared with control,viability of HUVECs was significantly decreased(70.53%±2.61%vs 100%±0%,P<0.05).With moderate decrease of cell viability,H12/R4 was appropriate for induction of H/R model on HUVECs.2.The generation,flow cytometric chacterization and quantitative analysis of MV induced by H/R from HUVECsMV were induced under the condition of H12/R4 from HUVECs.Flow Cytometry identified that vesicles induced by H/R were CD 144 positive with a size of<1pm.The concentration of EMV was 24014±322/?L,and the pelleted EMV protein was quantified at 0.37±0.07 ?g/?L.3.Apoptotic effects of H/R-EMV on H9c2 cellsCompared with control,cell viability in H/R-EMV 30 and H/R-EMV 60 group reduced significantly(90.03%±6.06%,78.74%±7.36%vs 100%±0%,P<0.05 or P<0.001),while LDH activity in H/R-EMV 60 group notably increased(142.1 ±9.99 vs 108.9±7.10,P<0.001),whereas LDH activity in H/R-EMV 10 decreased(92.32±18.69 vs 108.9±7.10,P<0.01).Fragmented or condensed nuclei could be observed with the increasing concentration of EMV through Hoechst33258 staining.Meanwhile,H/R-EMV 60 group displayed a significant increase with 17.71 percentage of apoptotic cells through annexin V-FITC/PI double-staining.Western blot showed a significant decrease in the Bcl-2 expression with the increasing concentration of EMV.Compared with Control group,the Bcl-2/Bax level was obviously down-regulated in H/R-EMV 30 and H/R-EMV 60 group.Conclusion:1.Stable H12/R4 injury model of HUVECs was established.2.EMV were generated from HUVECs under the condition of H12/R4,and EMV were characterized as CD144+ vesicles with diameter<1 ?m.And the pelleted EMV protein was quantified at 0.37±0.07 ?g/?L.3.H9c2 cells treated with H/R-EMV exhibited low viability but high LDH activity,among them,the effect of H/R-EMV 60 was most obvious.H/R-EMV increased the apoptosis rate and caspase 3 activity of H9c2 cells.And Bcl-2/Bax expression was down regulated,which indicated the proapoptotic effect was mediated by the increasing cleavage of caspase 3 and down-regulation of Bcl-2/Bax expression.