Nitric oxide inhibits stress-induced endothelial cell apoptosis

Objectives: To determine a mechanism by which nitric oxide alters induction of stress-induced endothelial cell apoptosis in vitro. Apoptosis is a form of cellular suicide that has been implicated in the pathogenesis of multiple organ dysfunction syndrome. Design: Prospective, controlled trial. Setting: Research laboratory of a large, academic medical center. Subjects: Cultured primary porcine aortic endothelial cells. Interventions: Cells were treated with a range of doses of agents that either spontaneously generate nitric oxide (S nitroso-N-acetyl-D,L-penicillamine [SNAP] or (Z)-1-[2-(2- aminoethyl)-N-(Zammonioethyl)amino]diazen-1-ium-1,2-diolate [DETA-NO]) or block nitric oxide production (N(ω)-methyl-L-arginine [L-NMA]). The ability of these agents to alter the rate of cell death by apoptosis (induced by the sequence stimuli lipopolysaccharide [LPS] followed by sodium arsenite) was measured. Mechanistic studies included examining the ability of: a) nitric oxide 'donors' to alter nuclear factor kappa B (NF-κB) DNA binding activity and the level of IκBα accumulation; and b) a stable cyclic guanosine monophosphate (cGMP) analog (8-bromo-cGMP) to mimic the effect of nitric oxide donors. Measurements and Main Results: The sequence LPS/sodium arsenite increased the rate of endothelial cell apoptosis (47.4%, p< .05 vs. control), as measured by fluorescent-activated cell scanning using annexin V/propidium iodide staining. DETA-NO generated nitric oxide (as indicated by an increase in the concentration of the stable end-products of nitric oxide metabolism) and decreased the rate of endothelial cell apoptosis (20.6% at a dose of 2 mM, p = .0001 vs. control). DETA-NO also decreased NF-κB DNA binding activity and the apparent accumulation of its endogenous inhibitor, IκBα. The 8-bromo-cGMP did not mimic the effects of nitric oxide donors (DETA-NO) on apoptosis. Conclusions: These data suggest that exogenous nitric oxide can block stress-induced endothelial cell apoptosis in vitro. The mechanistic studies are consistent with our hypothesis that inhibitors of NF-κB DNA binding activity are associated with protection against apoptosis-inducing stimuli. The results do not support a role for cGMP in mediating the protective effect of DETA-NO in our model.