REACTIVE SPECIES IN VASCULAR DISEASE--INJURY MECHANISMS

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We propose to examine the molecular mechanisms of vascular injury mediated by nitric oxide (NO), superoxide (O2) and peroxynitrite (ONOO). Endothelium is a major source of NO which has been shown to mediate important physiological functions such as vasodilatation, inhibition of platelet aggregation and neutrophil adherence. However, published reports have also implicated excessive production of NO as a contributing factor in cellular toxicity. Because NO has an unpaired electron, it reacts at a diffusion limited rate with superoxide to form ONOO. Peroxynitrite is a highly reactive molecule capable of oxidizing many biological molecules and inducing cellular and tissue injury. The reaction of ONOO with protein results in the addition of a nitro group in the ortho position of tyrosine residues to form nirotyrosine adducts. Nitrotyrosine has been detected in human and animal vascular endothelium in pathological disorders such as atherosclerosis, sepsis, inflammation and ischemia-reperfusion indicating that the formation of peroxynitrite is plausible in vivo. Moreover, the formation of nitrotyrosine may interfere with signal transduction events mediated by tyrosine-kinases. Peroxynitrite also exhibits selective reactivity with key cellular targets such as thiols, iron sulfur centers and zinc fingers. This selective reactivity of ONOO may also regulate important cellular functions such as respiration, signal transduction and transcriptional factors. We hypothesize that the toxicity of NO is mediated via the formation of ONOO which then acts as a selective modulator of cell signal transduction and transcriptional events. To evaluate the critical aspects of this hypothesis we propose to: 1) define the role of peroxynitrite in vascular endothelium injury, 2) examine the influence of peroxynitrite- mediated tyrosine nitration on tyrosine kinase-induced signal transduction events and 3) investigate the specific action of peoxynitrite in the activation of transcription factors as opposed to superoxide, hydrogen peroxide and nitric oxide. The proposed experiments will establish and characterize a cell model to examine important, new aspects of peroxynitrite-mediated pathology to vascular endothelium. The integration of our existing knowledge of peroxynitrite-mediated biochemical events with key cellular functions will rapidly advance understanding of pathogenic mechanisms and provide a basis for treatment of important problems in vascular medicine, including sepsis, atherosclerosis, inflammation and ischemia-reperfusion injury.