Eicosanoid Regulation in Atherosclerosis: Involvement of Inducible NO Synthase

描述（由申请人提供）：在动脉粥样硬化过程中，类二十烷酸生物合成的改变发生的机制尚不清楚。前列腺素（来自花生四烯酸代谢）和一氧化氮（7NO）在血管中产生，是调节血管张力和炎症的关键介质。我们和其他人已经证明7NO调节前列腺素H2合成酶（PGHS，也称为环加氧酶）并改变类二十烷酸的产生。值得注意的是，诱导形式的PGHS （PGHS-2）和一氧化氮合酶（iNOS）在动脉粥样硬化病变中增加并共定位，最近的研究表明这两种酶结合。我们最近的研究表明，缺乏iNOS会增加细胞PGHS-1和PGHS-2的表达，这进一步证明了这些途径之间的联系。我们已经达到的重要里程碑包括发现PGHS-1在人类和小鼠动脉粥样硬化病变中被硝化，并且依赖于iNOS。我们认为研究忽略了iNOS在动脉粥样硬化发生中的作用。我们的中心假设是氮氧化物（NOx）调节PGHS-1/-2活性（通过蛋白质修饰或通过表达）并改变类二十烷合成，从而影响动脉粥样硬化和血栓形成。在Specific Aim 1中，我们将确定iNOS在动脉粥样硬化和血栓形成过程中pghs依赖性类二十烷酸生成中的作用。我们认为，在没有iNOS的情况下，PGHS产物的改变会改变前列环素（PGI2；抗动脉粥样硬化/抗血栓）和凝血烷（TxA2；促动脉粥样硬化/促血栓）的平衡，从而有利于抑制动脉粥样硬化。使用COX抑制剂，我们将确定PGHS-1和pghs -2衍生的二十烷类化合物在ApoE-/-和ApoE-/- inos -/-小鼠动脉粥样硬化中的具体作用。在缺乏iNOS的小鼠中，我们将确定PGHS和pgi2s衍生的二十烷类化合物在动脉血栓形成中的作用。在Specific Aim 2中，研究将提供解释类二十烷酸合成如何与iNOS联系的机制，从而解决Aim 1中的体内和体外观察。因此，我们提出表征NOx对花生四烯酸级联酶和相关翻译后修饰的作用。鉴于我们在人和小鼠动脉粥样硬化病变中发现了显著升高的PGHS硝化水平，我们将确定PGHS硝化的位点，并量化这些组织中这种修饰的程度。由于我们已经揭示了血红素驱动的机制，导致靶向Tyr385硝化和PGHS失活，我们将确定这一独特功能在动脉粥样硬化病变中的作用。我们还将研究PGHS酶的s -亚硝基化反应，这可能为增强类二十烷酸的产生提供一种机制。这些研究的结果将确定NOx对PGHS功能和类二十烷酸产生的调节作用，包括这些介质对动脉粥样硬化和血栓形成过程的影响，并可能突出iNOS作为治疗性炎症抑制的新靶点。

DESCRIPTION (provided by applicant): During atherogenesis, alterations in eicosanoid biosynthesis occur by mechanisms not well understood. Prostaglandins (from arachidonic acid metabolism) and nitric oxide (7NO) produced in blood vessels are critical mediators in the regulation of vascular tone and inflammation. We, and others, have shown that 7NO modulates prostaglandin H2 synthase (PGHS, also known as cyclooxygenase) and alters eicosanoid production. Notably, the inducible forms of PGHS (PGHS-2) and nitric oxide synthase (iNOS) are increased and co-localize in atherosclerotic lesions and recent developments show that both enzymes bind. Our recent studies show that the lack of iNOS increases cellular PGHS-1 and PGHS-2 expression providing further evidence that link these pathways. Important milestones that we have reached include the finding that PGHS-1 is nitrated in human and murine atherosclerotic lesions and is dependent on iNOS. We believe that studies have neglected the role of iNOS in atherogenesis. Our central hypothesis is that nitrogen oxide species (NOx) regulate PGHS-1/-2 activity (by protein modification or through expression) and alters eicosanoid synthesis, which impacts on both atherogenesis and thrombosis. In Specific Aim 1, we will determine the role of iNOS in PGHS-dependent eicosanoid production during atherosclerosis and thrombosis. We propose that in the absence of iNOS, alterations in PGHS products occur shift the balance of prostacyclin (PGI2; anti-atherosclerotic/anti-thrombotic) and thromboxanes (TxA2; pro-atherosclerotic/pro-thrombotic) to favor inhibition of atherosclerosis. Using COX inhibitors, we will identify the specific roles of PGHS-1 and PGHS-2-derived eicosanoids in atherogenesis in ApoE-/- and ApoE-/-iNOS-/- mice. In mice lacking iNOS, we will define the role of PGHS and PGI2S-derived eicosanoids in the development of arterial thrombosis. In Specific Aim 2, studies will provide mechanisms that explain how eicosanoid synthesis is linked to iNOS and thus address in vivo and ex vivo observations from Aim 1. Thus, we propose to characterize NOx actions on arachidonic acid cascade enzymes and associated posttranslational modifications. Given that we identified significantly elevated levels of PGHS nitration in human and murine atherosclerotic lesions, we will identify sites of PGHS nitration and quantify extents of this modification in these tissues. Since we have revealed a heme-driven mechanism that leads to targeted Tyr385 nitration and PGHS inactivation, we will determine the role of this unique functionality in an atherosclerotic lesion. We will also investigate S-nitrosylation reactions of PGHS enzymes, which can provide a mechanism for enhanced eicosanoid production. Results from these studies will define the modulatory effects of NOx on PGHS function and eicosanoid production including the impact of these mediators on atherogenic and thrombotic processes and may highlight iNOS as a new target for therapeutic inhibition of inflammation.