REACTIVE SPECIES IN VASCULAR DISEASE--INJURY MECHANISMS

我们建议研究血管损伤的分子机制 由一氧化氮（NO）、超氧阴离子（O2）和过氧亚硝酸盐（ONOO）介导。 内皮素是NO的主要来源，NO已被证明介导 重要的生理功能，如血管舒张，抑制 血小板聚集和中性粒细胞粘附。 然而，出版 报告还暗示NO的过量产生作为 导致细胞毒性的因素。 因为NO有一个未配对的 电子，它以扩散限制速率与超氧化物反应形成 ONOO。 过氧亚硝酸盐是一种高活性分子， 许多生物分子和诱导细胞和组织损伤。 的 ONOO与蛋白质的反应导致在蛋白质中添加硝基， 酪氨酸残基的邻位形成硝基酪氨酸加合物。 硝基酪氨酸已在人类和动物血管中检测到， 病理性疾病如动脉粥样硬化，败血症， 炎症和缺血再灌注表明， 过氧亚硝酸盐在体内是合理的。 此外， 硝基酪氨酸可能干扰由 酪氨酸激酶。 过氧亚硝酸盐还表现出与以下物质的选择性反应性： 关键的细胞靶点，如硫醇、铁硫中心和锌指。 ONOO的这种选择性反应性也可能调节重要的细胞 呼吸、信号转导和转录等功能 因素 我们推测NO的毒性是通过 ONOO的形成，然后作为细胞信号的选择性调节剂 转导和转录事件。 评估关键方面 我们建议：1）定义过氧亚硝酸盐在 血管内皮损伤; 2）检测过氧亚硝基阴离子对血管内皮损伤的影响。 介导的酪氨酸硝化对酪氨酸激酶诱导的信号 转导事件和3）研究过氧亚硝酸盐的特异性作用 与超氧化物相反，在转录因子的激活中， 过氧化氢和一氧化氮。 拟议的实验将 建立并表征细胞模型，以检查重要的新方面 过氧亚硝酸盐介导的病理学对血管内皮的影响。 的 整合我们现有的过氧亚硝酸盐介导的 具有关键细胞功能的生化事件将迅速发展 了解致病机制，并为 治疗血管医学中的重要问题，包括败血症， 动脉粥样硬化、炎症和缺血-再灌注损伤。

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.