Severity of oxidative stress generates different mechanisms of endothelialcell death

The role of reactive oxygen species (ROS) in the pathogenesis of vascular d iseases is well established, but few data exist on the mechanisms by which ROS induce endothelial cell (EC) death. We examined the conditions and the mechanisms by which oxidative stress induces EC death, using cultured confl uent bovine aortic ECs exposed for 30 min to different concentrations of hy droxyl radicals (HO .) generated by hydrogen peroxide (H2O2) in the presenc e of 100 muM ferrous sulfate (FeSO4). Cell viability assays, Hoechst DNA st aining, TUNEL (TDT-mediated dUTP-biotin nick end-labeling) analysis, agaros e gel electrophoresis and annexin V assay were used to determine the effect of HO . on the viability of ECs, and to distinguish between apoptosis and necrosis. The results showed that at concentrations of up to 0.1 mM H2O2/Fe SO4, the large majority of cells are viable, except for similar to 12.5% de ath, which occurs by apoptosis. At a concentration of 0.2 mM H2O2, the cell viability is reduced to 66%, while EC apoptosis remained at comparable val ues (14%). At high oxidative stress (0.5 mM H2O2), the cell viability was d rastically reduced (similar to 39%), arid the prevalent form of death was n ecrosis; apoptosis accounted for only similar to 17%. Together, these data indicate that: (1) HO . induce EC death either by apoptosis or necrosis and (2) the mechanisms of EC death differ as a function of the concentration o f HO. Thus, the same insult can cause apoptosis and/or necrosis, as a funct ion of the intensity rather than the nature of the insult.