Redox Regulation of PI3K/Akt and p53 in Bovine Aortic Endothelial Cells Exposed to Hydrogen Peroxide

Abstract

To clarify the apoptotic and survival signal transduction pathways in activated vascular endothelial cells exposed to oxidative stress, the effects of inhibitors of signal transduction on hydrogen peroxide (H₂O₂)-induced apoptosis in bovine aortic vascular endothelial cells (BAEC) were examined. Treatment of BAEC with 1 mM H₂O₂ caused increases of DNA fragmentation, p53 expression, Bax/Bcl-2 ratio, and the activities of caspases 3 and 9. The increases of DNA fragmentation, Bax/Bcl-2 ratio, and caspase activities were abrogated by BAPTA-AM (an intracellular Ca²⁺ chelator) and N-acetyl-L-cysteine (an antioxidant), and augmented by wortmannin [a phosphatidylinositol 3-kinase (PI3K) inhibitor]. The increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) observed in H₂O₂-stimulated cells was unaffected by wortmannin, suggesting that the potentiating effect of wortmannin on the apoptosis was not due to an alteration of [Ca²⁺]_i. H₂O₂ increased the levels of PI3K activity and Akt phosphorylation. Both were attenuated by wortmannin and, to a lesser extent, by genistein (a tyrosine kinase inhibitor) and suramin (a growth factor receptor inhibitor), but not affected by BAPTA-AM. These results suggest that H₂O₂ induces Ca²⁺-dependent apoptosis and Ca²⁺-independent survival signals such as redox-regulated activation of PI3K/Akt, which is partly mediated by the activation of growth factor receptors in BAEC.