O33 Oxidative stress induced both apoptosis and necroptosis in endothelial cells

Abstract

Oxidative stress plays an essential role in mediating endothelial cell death while the underlying mechanisms remain elusive. Previous reports suggested that ROS overproduction induced both apoptosis and necrosis. Herein, the pro-death effect of tert-butyl hydroperoxide (t-BHP), an alternative for H2O2, on human umbilical vein endothelial cells was investigated with a low concentration (50 μM) of t-BHP (t-BHPL) and a high concentration (500 μM) of t-BHP (t-BHPH). T-BHPL induced caspase-dependent apoptosis and ROS generation, which was inhibited by N-acetyl- l -cysteine (NAC). Furthermore, NADPH oxidase inhibitor diphenyleneiodonium (DPI), NOX4 siRNA, and NOX4 inhibitor GKT137831 reduced t-BHPL-induced ROS generation while mitochondrial respiratory chain inhibitors rotenone (Rot), 2-thenoyltrifluoroacetone (TTFA), and antimycin A (AA) failed to do so. NOX4 overexpression resulted in increased ROS generation and Akt expression but decreased sensitivity to t-BHPL. In contrast, RIP1 inhibitor necrostatin-1, MLKL inhibitor necrosulfonamide and silencing RIP1, RIP3, and MLKL inhibited t-BHPH-induced cell death while pan-caspase inhibitor Z-VAD-FMK showed no effect. t-BHPH-induced ROS production was inhibited by TTFA, AA and Rot while DPI showed no effect. T-BHPH induced RIP1/RIP3 interaction, which was decreased by Rot, TTFA, and AA. Silencing RIP1 and RIP3, but not MLKL, inhibited t-BHPH-induced mitochondrial membrane potential (MMP) decrease and ROS production. In summary, t-BHP induced both apoptosis and necroptosis in endothelial cells which was mediated by ROS. ROS derived from NADPH oxidase and mitochondria contributed to t-BHPL and t-BHPH-induced apoptosis and necroptosis, respectively.