The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells.
The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.
Liu
Qi-Bing
QB
Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, Zhejiang University, Hangzhou, 310058, China.
Liu
Lu-Lu
LL
Lu
Ying-Mei
YM
Tao
Rong-Rong
RR
Huang
Ji-Yun
JY
Shioda
Norifumi
N
Moriguchi
Shigeki
S
Fukunaga
Kohji
K
Han
Feng
F
Lou
Yi-Jia
YJ
D000098:Acetophenones / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000494:pharmacology; D003603:Cytotoxins / Q000633:toxicity*; D042783:Endothelial Cells / Q000187:drug effects*; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects; D030421:Peroxynitrous Acid / Q000378:metabolism; D011744:Pyrimidinones / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000633:toxicity*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013113:Spin Labels; D013871:Thiones / Q000494:pharmacology
D000098:Acetophenones; D003497:Cyclic N-Oxides; D003603:Cytotoxins; D024101:Mitochondrial Proteins; D011744:Pyrimidinones; D017382:Reactive Oxygen Species; D013113:Spin Labels; D013871:Thiones; C501517:UCF 101; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; C056165:acetovanillone; D019255:NADPH Oxidases; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; C001803:tempol