Lipid rafts mediate H2O2 prosurvival effects in cultured endothelial cells

Abstract

In response to H2O2 generated during pathological events such as inflammation and ischemia-reperfusion, the endothelium activates both pro- and anti-apoptotic signaling programs. Since cholesterol-rich, plasma membrane rafts and caveolae serve as platforms for organizing and integrating signaling transduction processes, we asked whether these membrane regions play a mechanistic role in H2O2–induced responses. BAEC exposed to a 500 μM bolus of H2O2 induced progressive activation of caspase 3 and an increase in the number of TUNEL positive cells, confirming H2O2 induced apoptosis. Pretreatment of cells with either wortmanin or PD 098059 heightened theses apoptotic responses demonstrating that both PI3 kinase/Akt and ERK1/2 serve as signaling mediators to alleviate H2O2 cytotoxic effects. To begin to investigate the role of rafts/caveolae in these signaling processes, BAEC were pretreated with methyl-β-cyclodextrin (CD) 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 cholesterol-rich plasma membrane rafts and/or caveolae participate in propagation of oxidant-induced survival signals through regulation of Akt and ERK1/2 pathways. This work was supported by NIH HL66301.