Effects Of Microvesicles On Normal And Hypoxia/reoxygenation-injured H9c2 Cells

Objective:To observe the alteration of phenotype and concentration of microvesicles(MV)in rat peripheral blood after myocardial ischemia/reperfusion(I/R)and ischemic preconditioning(IPC),and investigate the effects of I/R-MV and IPC-MV on normal and hypoxia/reoxygenation(H/R)-injured H9c2 cells.Methods:1.Establishment and validation of rat I/R and IPC modelHealthy male Wistar rats were randomized into 3 groups:Sham group:a silk ligature was placed around the left anterior descending(LAD)coronary artery without tighten the slipknot;I/R group:ischemia/reperfusion was induced by tighten or loosen the slipknot to occluse or reperfuse the LAD.Rats were subjected to 30 min of ischemia followed by 120 min of reperfusion;IPC group:rats were subjected to 3 cycles of 5 min of ischemia and reperfusion before 30 min of ischemia and 120 min of reperfusion.Blood pressure and ECG were monitored throughout the operation.Myocardial infarct size was determined by 2,3,5-triphenyltetrazolium staining.2.Isolation and characterization I/R-MV and IPC-MVSham group:rats were left untreated for 45 min after a silk ligature was placed around the LAD;I/R group:rats were left untreated for 15 min and then subjected to 30 min of ischemia followed by reperfusion;IPC group:rats were left untreated for 15 min and then subjected to 3 cycles of 5 min of ischemia and reperfusion.The blood was drawn from abdominal aorta right after all the operation was done and anticoagulated with sodium citrate.Platelet-free plasma(PFP)was obtained after two centrifugation steps.Flow cytometry was used to characterize and enumber MV in PFP.The remaining PFP was further ultercentrifuged to obtain Sham-MV,I/Rst-MV and IPC-MV,respectively.3.The effects of I/Rst-MV and IPC-MV on normal H9c2 cellsH9c2 cells were cultured under nomal condition for 24 h,then divided into 4 groups and treated for 6 h:Control group:DMEM medium without FBS;Sham MV group:100 ?g/mL Sham-MV;I/Rst MV group:100 ?g/mL I/Rst-MV;? IPC MV group:100 ?g/mL IPC-MV.Cell viability and activity of LDH in culture supernatant were tested using spectrophotography.Morphology of nuclei was observed by Hoechst 33258 staining.Apoptotic rate of the target cells were observed though Annexin V/PI double staining.4.The effects of I/Rst-MV and IPC-MV on H/R injured H9c2 cellsH9c2 cells were cultured under nomal condition for 24 h,then the supernatant was discarded.H9c2 cells were divided into 5 groups and different buffer was added:Control group:control buffer;H/R group:hypoxic buffer;Sham-MV+H/R group:hypoxic buffer with 100 ?g/mL Sham MV;I/Rst-MV+H/R group:hypoxic buffer with 100 ?g/mL I/Rst-MV;IPC-MV+H/R group:hypoxic buffer with 100 ?g/mL IPC-MV.All but control group were exposed to the anoxic condition for 12 h and then to normoxia for 4 h.Cell viability was tested.Apoptosis of the target cells was observed through Hoechst 33258 staining and Annexin V/PI double staining.Further more,caspase 3 activity and p-ERK1/2 expression in H9c2 cells were also measured.Results:1.Establishment and validation of rat I/R and IPC modelIn I/R group,a dramatic elevation of ST-segment was observed.The incidences of VPC,VT and VF were 100%,100%and 62.5%,respectively.The onset of VPC was 6.08±1.01 min with the number of 203±87.The onset of VT was 8.23± 1.88 min and the duration was 0.57±0.22 min.The ratio of infarct size versus area at risk was 37.14±4.18%.Compared with I/R group,IPC group showed a significant improvement of above mentioned parameters.The elevation of ST-segment,incidences of VT and VF was decreased(37.5%vs.100%,P<0.01;0%vs.62.5%,P<0.01),onset of VPC and VT was delayed(13.95±1.63 min vs.6.08± 1.01 min,P<0.001;15.51 ±0.77 min vs.8.23± 1.88 min,P<0.001),and the duration was shortend(0.13±0.07 min vs.0.57±0.22 min,P<0.05).The ratio of infarct size versus area at risk was also decreased(18.08±3.12%vs.37.14±4.18%,P<0.001).2.Isolation and characterization I/R-MV and IPC-MVAs demonstrated by flow cytometry analysis,after two steps of centrifugation,the signal of particles smaller than 1 ?m accounted for more than 99%signal of all.Compared with sham group,I/Rst and IPC group displayed no changes in the endothelial-derived MV(EMV)but a reduction in platelet-derived MV(PMV).There was no difference in the MV concentration between I/Rst and IPC group.3.The effects of I/Rst-MV and IPC-MV on normal H9c2 cellsCompared with control group,the cell viabilily in Sham,I/Rst and IPC MV treated group was significantly decreased(96.24±2.28%,P<0.05;92.81 ±2.78%,P<0.001;91.63±2.90%,P<0.001;vs.100±0%).The cell viability in IPC MV group was lower than that of Sham MV group(91.63±2.90%vs.96.24±2.28%,P<0.001),but not different from that of I/Rst MV group.LDH activity was higher in all the MV treated groups compared with Control group,and the trend became significant in IPC MV group(114.20±7.20 U/L vs.95.54±5.54 U/L,P<0.01).Fragmented or condensed nuclei could be observed in all the MV treated groups,with more condensation in I/Rst and IPC MV groups.The apoptotic rate in all groups didn't differ significantly,but IPV MV group showed a upward trend in apoptotic rate.4.The effects of I/Rst-MV and IPC-MV on H/R injured H9c2 cellsCompared with Control group,H/R group showed a significant reduction in cell viability(68.15±5.76%vs.100±0%,P<O.OO1),an increase in nuclei fragmentation or condensation and apoptotic rate(34.37±5.16%vs.10.64± 1.51%,P<0.001).The caspase 3 activity was also increased(2387.45±136.67 IU vs.1482.93±86.45 IU,P<0.001).Compared with H/R group,I/Rst and IPC-MV treated groups showed a significant increase in cell viability(88.65±3.80%,P<0.001;86.06± 1.92%vs.68.15±5.76%,P<0·001),a reduction in nuclei fragmentation or condensation and apoptotic rate(19.76±2.16%,P<0.001;20.43±3.34%vs.34.37±.16%,P<0.001).The caspase 3 activity was decreased(1580.23±215.62 IU,P<0.01;1346.09±287.56 IU vs.2387.45±136.67 IU,P<0.001).All the above mentioned indicators didn't differ between I/Rst and IPC-MV treated groups.Western blot revealed that the p-ERK1/2 expression in IPC-MV treated group was significantly upregulated.Conclusion:1.The model of rat myocardial I/R and IPC was successifully established.Compared with Sham group,there was no alteration in I/R-EMV and IPC-EMV.An decrease in I/R-PMV and IPC-PMV was observed.2.I/Rst-MV and IPC-MV exerted slight pro-apoptotic effect on normal H9c2 cells.3.Stable hpoxia/reoxygenation injury model of H9c2 cells was established.I/Rst-MV and IPC-MV exhibited protective effects against apoptosis on H/R injured H9c2 cells,which was partly due to the inhibition of caspase 3 activity.IPC-MV could upregulated the expression of p-ERKl/2 in H/R injured H9c2 cells,indicating that ERX1/2 passway might be involved in the anti-apoptotic effect of IPC-MV on H/R injured H9c2 cells.