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Objective To investigate the effects of endothelial microvesicles(EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells(HUVECs) were treated with 10 μmol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 μm latex beads and antiPE-CD144 antibody by flow cytometry. For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Moreover, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V/PI double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles(< 1 μm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9c2 cells, and increase LDH leakage from H9c2 cells in a dose dependent manner(P<0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. Furthermore, increased apoptosis rates of H9c2 cells could be assessed through FITC-Annexin V/PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.
Objective To investigate the effects of endothelial microvesicles (EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes. Methods Human umbilical vein endothelial cells (HUVECs) were treated with 10 μmol / L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the For functional research, EMVs at different concentrations were cocultured with H9c2 cardiomyocytes for 6 h. Cell viability of H9c2 cells and the activity of LDH leaked from H9c2 cells were tested by colorimetry. Furthermore, apoptosis of H9c2 cells was observed through Hoechst 33258 staining and tested by FITC-Annexin V / PI double staining. Results EMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. membrane vesicles (<1 μm) induced by A23187 were CD144 positive. In addition, the EMVs could significantly reduce the viability of H9 c2 cells, and increased LDH leakage from H9c2 cells in a dose dependent manner (P <0.05). Condensed nuclei could be observed with the increasing concentrations of EMVs through Hoechst 33258 staining. -Annexin V / PI double staining by flow cytometry. Conclusion Microvesicles could be released from HUVECs after induced by A23187 through calcium influx, and these EMVs exerted a pro-apoptotic effect on H9c2 cells by induction of apoptosis.