Hypoxia potentiates nitric oxide-mediated apoptosis in endothelial cells via peroxynitrite-induced activation of mitochondria-dependent and -independent pathways.
Nitric oxide (NO*) at low concentrations is cytoprotective for endothelial cells; however, elevated concentrations of NO* (> or =1 micromol/liter), as may be achieved during inflammatory states, can induce apoptosis and cell death. Hypoxia is associated with tissue inflammation and ischemia and, therefore, may modulate the effects of NO* on endothelial function. To examine the influence of hypoxia on NO*-mediated apoptosis, we exposed bovine aortic endothelial cells (BAEC) to (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (diethylenetriamine NONOate, DETA-NO) (1 mmol/liter) under normoxic or hypoxic conditions (pO2 = 35 mm of Hg) and measured the indices of apoptotic cell death. BAEC treated with DETA-NO under normoxic conditions demonstrated increased levels of histone-associated DNA fragments, which was confirmed by terminal dUTP nick-end labeling assay, and hypoxic conditions augmented this response. To determine whether mitochondrial dysfunction was one mechanism by which NO* initiated apoptosis under hypoxic conditions, we evaluated mitochondrial membrane potential in (Psim). Exposure to DETA-NO resulted in a decrease in Psim and concomitant release of cytochrome c and caspase-9 activation, which were enhanced by hypoxia. By utilizing Rho0 BAEC (Rho0-EC), which lack functional mitochondria, we demonstrated that dissipation of Psim was associated with increased reactive oxygen species generation and peroxynitrite formation. Moreover, in Rho0-EC we identified activation of caspase-8 as part of the mitochondrial-independent pathway of apoptosis. To establish that peroxynitrite mediated mitochondrial damage and apoptosis, we treated BAEC and Rho0-EC with the peroxynitrite scavenger uric acid and found that the indices of apoptosis were decreased significantly. These findings confirm that high flux of NO* under hypoxic conditions promotes cell death via mitochondrial damage and mitochondrial-independent mechanisms by peroxynitrite.
Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Geoffrey A
GA
Walford
Rose-Laure
RL
Moussignac
Anne W
AW
Scribner
Joseph
J
Loscalzo
Jane A
JA
Leopold
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D018384:Oxidative Stress; D030421:Peroxynitrous Acid / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014226:Triazenes / Q000494:pharmacology; D051028:bcl-2-Associated X Protein
C105351:1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene; D020030:Nitric Oxide Donors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014226:Triazenes; D051028:bcl-2-Associated X Protein; D030421:Peroxynitrous Acid; D009569:Nitric Oxide