Role Of H/R-EMVs On Apoptosis And Oxidative Injury In H9c2 Cells And Its Correlation With PI3K/Akt And MAPK Signal Pathways

Objective:Endothelial microvesicles(EMVs)are sub-cellular membrane vesicles released from endothelial cell during activation or apoptosis.The EMVs carry various bioactive factors,such as cytokine,protein,RNA,DNA etc.They lead to intercellular connection by transmitting these bioactive substances to target cells or triggering a series of biological signals.EMVs play an important role in coagulation,inflammation and regulation of intracellular signal transduction.The aim of this study is to investigate the effects and mechanism of hypoxia/reoxygenation(H/R)induced endothelial microvesicles(H/R-EMVs)on apoptosis and oxidative injury of H9c2 cells.Meanwhile,we explored the effects of H/R-EMVs on PI3K/Akt,ERK1/2,p38MAPK,JNK signal pathways and examined the MMP-2 activity and ROS production of H/R-EMVs.These could provide new insights into the pathogenesis of myocardial ischemia/reperfusion injury.Methods:1.The isolation and characterization of H/R-EMVsHUVECs were cultured for 24 h.The culture medium was changed with a hypoxic buffer.Cells were cultured under the hypoxic condition of 95%N2-5%CO2 for 12 h and then reoxygention for 4 h.H/R-EMVs were collected from culture supernatant and extracted by gradient centrifugation.H/R-EMVs were characterized using 1 μm calibration beads and anti-PE-CD144 antibody by flow cytometry;Protein quantification of H/R-EMVs was carried out using a BCA protein assay.2.Effects of H/R-EMVs on apoptosis,oxidative injury and PI3K/Akt,ERKI/2,p38MAPK,JNK signal pathways in H9c2 cellsH9c2 cells were divided into 4 groups randomly after 24 h incubating.i.Control group was cultured with serum-free high glucose DMEM medium for 6 h.ii-iv were H/R-EMVs 10,30 and 60 groups.Cells were co-cultured with 10,30,60 μmol/L final concentrations of H/R-EMVs respectively for 6 h,Cell viability was tested by MTT assay;Hoechst 33258 staining,Annexin V-FITC/PI double staining and caspase-3 activity assay were applied for the detection of apoptosis;Bcl-2/Bax proteins were performed by Western blotting.Activity of lactate dehydrogenase(LDH)and total superoxide dismutase(T-SOD),content of malondialdehyde(MDA)were detected using colorimetric method.ROS production of H9c2 cells were measured by flow cytometry.p-Akt/t-Akt,p-ERK1/2/t-ERK1/2,p-p38 and p-JNK1/2 proteins were performed by Western blotting.3.Examining the MMP-2 activity and ROS carried by H/R-EMVsHUVECs were divided into 2 groups randomly after 24 h incubating.Control group was cultured with hypoxic control buffer for 16 h.H/R group was treated with hypoxic buffer and exposed to the hypoxic condition of 95%N2-5%CO2 for 12 h,and then reoxygention for 4 h.Control-EMVs(C-EMVs)and H/R-EMVs were collected from culture supernatant and extracted by gradient centrifugation.MMP-2 activity on EMVs were detected by gelatin zymography.ROS content of EMVs were measured by flow cytometry.Results:1.The isolation and characterization of H/R-EMVsFlow cytometry identified that vesicles induced by H/R were CD 144 positive with a size<1 μm.The protein of H/R-EMVs was quantified at 0.346±0.085 μg/μL.2.Effects of H/R-EMVs on apoptosis,oxidative injury and PI3K/Akt,ERK1/2,p38MAPK,JNK signal pathways in H9c2 cellsCompared with Control,cell viability in H/R-EMVs 30 and H/R-EMVs 60 groups reduced significantly(89.70±5.26%,78.74±7.87%vs.100±0%,P<0.05 or P<0.001).Meanwhile,H/R-EMVs 60 group displayed a significant increase with 18.71 percentage of apoptotic cells through Annexin V-FITC/PI double-staining(18.71±2.81%vs.6.90±0.77%,P<0.01).The activity of caspase-3(294.14±28.03 vs.140.33±29.43,P<0.01)was increased significantly.Fragmented or condensed nuclei could be observed with the increasing concentration of H/R-EMVs through Hoechst staining.The Bcl-2/Bax ratio was obviously down-regulated in H/R-EMVs 30 and H/R-EMV 60 group(P<0.001).Compared with Control,LDH activity(142.56±9.67 vs.109.23±6.54,P<0.001)and the content of MDA(2.80±0.40 vs.1.81±0.44,P<0.01)in H/R-EMVs 60 group were increased significantly.The activity of T-SOD was decreased significantly in H/R-EMV 30 and 60 groups(46.50±2.78,32.97±2.63 vs.62.66±3.40,P<0.001).With the raising concentrations of H/R-EMVs,increased the mean fluorescence intensity of ROS production could be observed.The H/R-EMVs 60 group showed the strongest fluorescence intensity(8.23±0.1 84 vs.7.51±0.16,P<0.01).Western blot showed that H/R-EMVs could decrease the expression of p-Akt in a dose dependent manner(P<0.01 or P<0.001).Meanwhile,H/R-EMVs 30 and 60 groups could also down-regulate the expression of p-ERK1/2(P<0.05).However,H/R-EMVs 30 and 60 group could upregulate the expression of p-p38MAPK(P<0.01)and p-JNK1/2(P<0.01).3.Examining the MMP-2 activity of H/R-EMVs.Gelatin zymography showed that MMP-2 activity was increased significantly in H/R group compared with Control(110.94±4.12 vs.100±0,P<0.05);H/R-EMVs exhibited higher MMP-2 activity compared with the supernatant after ultracentrifuging in H/R group(175.85±20.06 vs.100±0,P<0.01).4.Examining the ROS content of H/R-EMVsFlow cytometry showed the mean fluorescence intensity of ROS production in H/R-EMVs was increased significantly compared with C-EMVs(639.83±41.03 vs.453.67±20.42,P<0.001).Conclusion:H/R-EMVs could release from H/R-exposed HUVECs,the H/R-EMVs protein was quantified at 0.346±0.085 μg/μL.60 μg/mL H/R-EMVs induced apoptosis in H9c2 cells by down regulating the ratio of Bcl-2/Bax.60 μg/mL H/R-EMVs induced oxidative injury in H9c2 cells.10,30,60 μg/mL H/R-EMVs could down-regulate the expression of p-Akt and p-ERK1/2,up-regulate the expression of p-p38MAPK and p-JNK1/2 in a dose dependent manner.Moreover,H/R-EMVs carried MMP-2,H/R-EMVs also had higher content of ROS.