Lipid rafts mediate H ₂ O ₂ prosurvival effects in cultured endothelial cells

Abstract

Reactive oxygen species (ROS) generated during pathological events, such as inflammation and ischemia-reperfusion, activates both proapoptotic and antiapoptotic signaling programs in endothelial cells. Because cholesterol-rich, plasma membrane rafts serve as platforms for organizing and integrating signaling transduction processes, we asked whether these membrane regions play a mechanistic role in H2O2-induced responses. Bovine aortic endothelial cell cultures exposed to a 500-microM bolus of H2O2 showed progressive activation of caspase 3 and an increase in the number of TUNEL-positive cells. Pretreatment with either wortmannin or PD 098059 heightened these apoptotic responses, demonstrating that both PI3 kinase/Akt and ERK1/2 serve as signaling mediators to alleviate H2O2 cytotoxic effects. To investigate the role of lipid rafts in these signaling processes, endothelial cells were pretreated with methyl-beta-cyclodextrin (CD) or filipin to ablate raft structures. H2O2-induced phosphorylation of Akt and ERK 1/2 was attenuated, while caspase 3 and the number of TUNEL positive cells was enhanced in CD-pretreated cells exposed to H2O2. Reconstitution of raft domains restored H2O2-induced Akt and ERK1/2 phosphorylation, which was concomitant with reduction of caspase 3 activation and DNA fragmentation. Taken together, our findings suggest that plasma membrane compartments rich in cholesterol participate in signal transduction pathways activated by oxidative stress.