Eicosanoid Regulation in Atherosclerosis: Involvement of Inducible NO Synthase

描述(由申请人提供)：在动脉粥样硬化形成过程中，二十烷类化合物的生物合成发生变化的机制还不是很清楚。血管中产生的前列腺素(花生四烯酸代谢产物)和一氧化氮(7NO)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，也称为环氧合酶)并改变二十烷类化合物的产生。值得注意的是，诱导形式的PGHS(PGHS-2)和一氧化氮合酶(INOS)增加并共存于动脉粥样硬化病变中，最近的研究表明这两种酶结合。我们最近的研究表明，iNOS的缺乏增加了细胞内PGHS-1和PGHS-2的表达，为这些途径之间的联系提供了进一步的证据。我们已经取得的重要里程碑包括在人和小鼠的动脉粥样硬化病变中发现PGHS-1是硝化的，并依赖于iNOS。我们认为，研究忽略了iNOS在动脉粥样硬化形成中的作用。我们的中心假设是，氮氧化物物种(NOx)调节PGHS-1/-2的活性(通过蛋白质修饰或通过表达)，并改变二十烷类化合物的合成，这对动脉粥样硬化和血栓形成都有影响。在特定的目标1中，我们将确定iNOS在动脉粥样硬化和血栓形成过程中依赖PGHS的二十烷类化合物产生中的作用。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；抗动脉硬化/抗血栓)和血栓烷(TXA2；促动脉粥样硬化/促血栓形成)的平衡，有利于抑制动脉粥样硬化。利用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-/-小鼠动脉粥样硬化形成中的特定作用。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发展中的作用。在特定的目标2中，研究将提供解释二十烷类化合物合成如何与iNOS联系的机制，从而解决目标1在体内和体外的观察结果。因此，我们建议表征NOx对花生四烯酸级联酶和相关的翻译后修饰的作用。鉴于我们在人和小鼠的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硝化的位置并量化这些组织中这种修饰的程度。由于我们已经揭示了一种由血红素驱动的机制，该机制导致靶向Tyr385硝化和PGHS失活，因此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-亚硝化反应，这可以为提高二十烷类化合物的产量提供一个机制。这些研究的结果将确定NOx对PGHS功能和二十烷类化合物产生的调节作用，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作为治疗抑制炎症的新靶点。

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.