Oxidative Alterations of Cyclooxygenase in Atherogenesis

一氧化氮(NO‘)和二十烷基类化合物是生理和病理生理过程的关键介质， 包括炎症和动脉粥样硬化。我们和其他人已经证明了NO对前列腺素的调节作用 H2合成酶(PGHS，也称为环氧合酶)，并改变二十烷类化合物的产生。我们已经决定 一种特殊的氮氧化物(NOX)物种过氧亚硝酸盐(ONOO)对PGHS有相互矛盾的影响 活性：ONOO“能增强花生四烯酸刺激的PGHS活性，但也能使该酶失活 通过酪氨酸(Tyr)硝化。S-某些氮氧化物对半胱氨酸(Cys)残基的亚硝化 以未知机制激活PGHS。提出的研究的核心假设是NOX 物种改变PGHS-1、PGHS-2和前列环素合成酶(PGI2S)活性以及二十烷类化合物的变化 合成将影响动脉粥样硬化病变的发展和血栓形成。在具体目标1中，我们 建议确定半胱氨酸残基的S亚硝化对酪氨酸自由基形成的影响。我们还提议 氮氧化物暴露下PGHS-1和PGHS-2酪氨酸硝化机理的研究 氧化应激状态。我们预测，血红素直接硝化体内酪氨酸残基，这对 将取消酶功能的催化活性，而表面暴露的Tyr残留物的硝化(通过 不依赖于血红素的机制)将不会有什么影响。通过与S.Gross博士合作(项目4)，我们将 在纯化的酶、细胞和病损中确定不同NOX物种硝化的特定Tyr位点 小鼠动脉粥样硬化模型。我们将能够就硝化反应的机理得出结论。 在动脉粥样硬化方面，这将使我们能够设计关键研究，以减少炎性氧化损害 血管酶。在具体目标2中，我们建议确定iNOS在PGHS依赖中的作用 动脉粥样硬化过程中二十烷类化合物的产生。我们认为，在没有iNOS的情况下，PGHS可能会假设 一种补偿作用，并提供有益二十烷类化合物的增加，从而导致 在载脂蛋白E、iNOS“‘”小鼠和载脂蛋白E“’”小鼠中观察到动脉粥样硬化。与A博士合作。 Marcus(项目1)，我们建议测量从ApoE“‘和ApoE”获得的主动脉组织中二十烷类化合物的水平。 APOE‘^iNOS“’”小鼠。使用COX抑制剂，我们将确定PGHS-1和PGHS-2衍生的特定角色 二十烷类化合物在载脂蛋白E“A和载脂蛋白E^‘iNOS’”小鼠动脉粥样硬化形成中的作用。 与K.Hajjar博士(项目2)一起，我们将确定PGHS和PGI2S衍生二十烷类化合物在 缺乏诱导型一氧化氮合酶的小鼠动脉血栓形成的发生。这些研究的结果将定义 氮氧化物对PGHS功能的调节作用及其对二十烷类化合物生物合成的调节 如这些介质对动脉粥样硬化和血栓形成相关过程的影响。

Nitric oxide (NO') and eicosanoids are critical mediators of physiological and pathophysiological processes, including inflammation and atherosclerosis. We, and others, have shown that NO' modulates prostaglandin H2 synthase (PGHS, also known as cyclooxygenase) and alters eicosanoid production. We have determined that a particular nitrogen oxide (NOX) species, peroxynitrite (ONOO"), has conflicting effects on PGHS activity: ONOO" can enhance arachidonic acid-stimulated PGHS activity, but can also deactivate the enzyme via tyrosine (Tyr) nitration. In addition, S-nitrosation of cysteine (Cys) residues by certain NOX species activate PGHS by unknown mechanisms. The hypothesis central to the studies proposed is that NOX species alter PGHS-1, PGHS-2 and prostacyclin synthase (PGI2S) activities and that alteration of eicosanoid synthesis will impact both atherosclerotic lesion development and thrombosis. In Specific Aim 1, we propose to determine how S-nitrosation of Cys residues impacts on Tyr radical formation. We also propose to determine mechanisms of Tyr nitration in PGHS-1 and PGHS-2 following exposure to NOX species under conditions of oxidative stress. We predict that heme directs nitration of internal Tyr residues critical to catalytic activity that will abolish enzyme function, whereas nitration of surface-exposed Tyr residues (by a heme-independent mechanism) will have little impact. In collaboration with Dr. S. Gross (Project 4) we will determine specific Tyr sites nitrated by different NOX species in purified enzyme, cells and in lesions from a mouse model of atherosclerosis. We will be able to draw conclusions concerning the mechanism of nitration in atherosclerosis, which will enable us to design key studies to reduce inflammatory oxidative damage to vascular enzymes. In Specific Aim 2, we propose to determine the role of iNOS in PGHS-dependent eicosanoid production during atherosclerosis. We propose that in the absence of iNOS, PGHS may assume a compensatory role and provide an increase in beneficial eicosanoids that lead to a reduction in atherosclerosis observed in ApoE^iNOS"'" mice compared to ApoE"'" mice. In collaboration with Dr. A. Marcus (Project 1), we propose to measure eicosanoid levels in aortic tissue obtained from ApoE"'" and ApoE'^iNOS"'" mice. Using COX inhibitors, we will identify the specific roles of PGHS-1 and PGHS-2-derived eicosanoids in the development of atherosclerosis in ApoE"A and ApoE^'iNOS"'" mice. Finally, in collaboration with Dr. K. Hajjar (Project 2), we will define the role of PGHS and PGI2S-derived eicosanoids in the development of arterial thrombosis in mice lacking iNOS. Results from these studies will define the modulatory effects of NOX species on PGHS function and the regulation of eicosanoid biosyntheses as well as the impact of these mediators on processes related to atherosclerosis and thrombosis.