Effects of endothelial microvesicles induced by A23187 on H9c2 cardiomyocytes.
- Author:
Man SHANG
;
Qi ZHANG
;
Meng-Xiao ZHANG
;
Yao WANG
;
Yan CHEN
;
Yan-Na WU
;
Jun-Qiu SONG
;
Ming-Lin LIU
;
Yan-Xia LIU
- Publication Type:Journal Article
- MeSH: Annexin A5; Apoptosis; Calcimycin; pharmacology; Calcium; metabolism; Cell Line; Cell Membrane; drug effects; Coculture Techniques; Flow Cytometry; Fluorescein-5-isothiocyanate; analogs & derivatives; Human Umbilical Vein Endothelial Cells; Humans; Myocytes, Cardiac; drug effects; Staining and Labeling
- From: Chinese Journal of Applied Physiology 2013;29(6):559-564
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo investigate the effects of endothelial microvesicles (EMVs) induced by calcium ionophore A23187 on H9c2 cardiomyocytes.
METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with 10 micromol/L A23187 for 30 min. EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. EMVs were characterized using 1 and 2 microm latex beads and anti-PE-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.
RESULTSEMVs were induced by A23187 on HUVECs, and isolated by ultracentrifugation. We identified the membrane vesicles (< 1 microm) 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.
CONCLUSIONMicrovesicles 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.