Primary Cultured Endothelial Cells Apoptosis Induced by TNF-α in the Presence of Actinomycin D

Objective To establish a model of primary cultured endothelial apoptosis induced by in- flammatory cytokine TNF-αin the presence of RNA synthesis inhibitors,actinomycin D.Methods Three or four passages of primary cultured bovine aortic endothelial cells(BAECs)were used.Cell viability was de- tected by MTT assay.After treated with TNF-α/actinomycin D for a few hours,apoptosis of endothelial cells was evaluated by DNA fragment assay,flow cytometry assay and morphological observations under fluores- cence microscopy.Bcl-2 levels and MAPK phosphorylation were detected by Western blot analysis,capase-3 activity was examined by using DEVD-p-nitroanilide as a substrate.Results TNF-αdecreased cell viability in a concentration-dependent manner in the presence of actinomycin D in MTT assay.TNF-αinduced nucle- ar condensation and fragmentation in some of endothelial cells,resulted typical DNA ladders,and increased the percent of endothelial apoptosis in flow cytometry assay.TNF-αsignificantly decreased Bcl-2 protein lev- el in a time-dependent manner and increased caspase-3 activity.TNF-αalso induced a marked dephosphory- lation of MAPK in a time-dependent manner.Conclusion The present study demonstrated an efficient mod- el of endothelial apoptosis induced by inflammatory cytokine TNF-α,which can be used to investigate the mechanism of endothelium-associated disorders(e.g.atherosclerosis,angiogenesis)in further studies.