Molecular Characterization of NoxA1 in VSMC NADPH Oxidase Activation

描述（由申请方提供）：来自动物模型和人体研究的大量证据支持NADPH氧化酶衍生的氧化应激是心血管疾病病理生理学的主要贡献者的假设。特别地，Nox 1 NADPH氧化酶的失调已经涉及许多血管疾病，包括动脉粥样硬化、主动脉夹层、高血压和中风。虽然激活Nox 2（gp 91 phox）NADPH氧化酶的分子机制已经阐明，导致Nox 1 NADPH氧化酶活性失调的事件还没有很好地理解。我们以前的研究和初步数据支持的概念，NoxA 1，同源的p67 phox，在小鼠和人血管平滑肌细胞（VSMC）中表达，是一个关键的调节Nox 1 NADPH氧化酶活性。NoxA 1依赖的活性氧（ROS）在激动剂诱导的VSMC增殖和促生长、氧化还原敏感性蛋白激酶激活中起重要作用。NoxA 1在ApoE-/-小鼠的动脉粥样硬化和动脉粥样硬化病变中以及人早期颈动脉粥样硬化病变的内膜和中膜VSMC中上调。在本研究中，我们将探讨NoxA 1和p47 phox相互作用如何调节VSMC中Nox 1 NADPH氧化酶的活性，调节NoxA 1表达和翻译后修饰的机制及其在Nox 1 NADPH氧化酶激活中的作用。我们还将确定NoxA 1依赖的ROS产生在再狭窄和动脉粥样硬化中的作用。我们将筛选一个小分子库，以确定Nox 1 NADPH氧化酶活性的潜在抑制剂，以补充遗传方法。为了实现这些目标，我们已经组装了一套独特的试剂和方法，包括NoxA 1-/-，ApoE-/-/NoxA 1-/-和[SM 22-CreKI/+/NoxA 1f/+]小鼠，Nox 1 y/-，p47 phox-/-和NoxA 1-/-小鼠主动脉VSMC培养物，腺病毒NoxA 1过表达和shRNA构建体以及功能性AlphaScreen和100，000个成员的小分子文库，用于鉴定潜在的Nox 1 NADPH氧化酶抑制剂。我们的研究战略包括以下三个具体目标。 目的1：明确NoxA 1依赖的NADPH氧化酶活性在激动剂诱导的小鼠和人主动脉VSMC活性氧产生中的分子机制。目的2：探讨NoxA 1缺陷在再狭窄和动脉粥样硬化中的作用。目的3：鉴定新型特异性NoxA 1和p47 phox相互作用抑制剂，作为调节Nox 1活性的手段。总之，这些研究将establis VSMC Nox 1 NADPH氧化酶在血管病理生理学中的作用，并可能确定该酶的调节策略/化合物。

DESCRIPTION (provided by applicant): Extensive evidence from animal models and human studies supports the hypothesis that NADPH oxidase derived oxidative stress is a major contributor to the pathophysiology of cardiovascular diseases. In particular, dysregulation of Nox1 NADPH oxidase has been implicated in many vascular diseases including atherosclerosis, aortic dissection, hypertension and stroke. Although molecular mechanisms activating the Nox2 (gp91phox) NADPH oxidase have been elucidated, the events that lead to dysregulation of Nox1 NADPH oxidase activity are not well understood. Our prior studies and preliminary data support the notion that NoxA1, the homologue of p67phox, is expressed in mouse and human vascular smooth muscle cells (VSMC) and is a key regulator of Nox1 NADPH oxidase activity. NoxA1-dependent reactive oxygen species (ROS) play an essential role in agonist-induced VSMC proliferation and growth-promoting, redox-sensitive protein kinase activation. NoxA1 is upregulated in aortas and atherosclerotic lesions of ApoE-/- mice and in intimal and medial VSMC of human early carotid atherosclerotic lesions. In this proposal we will investigate how NoxA1 and p47phox interaction regulates Nox1 NADPH oxidase activity in VSMC, the mechanisms that regulate NoxA1 expression and post translational modifications and their role in Nox1 NADPH oxidase activation. We will also determine the role of NoxA1-dependent ROS production in restenosis and atherosclerosis. We will screen a small molecule library to identify potential inhibitors of Nox1 NADPH oxidase activity to complement the genetic approaches. To accomplish these goals, we have assembled a unique set of reagents and methodologies including NoxA1-/-, ApoE-/-/NoxA1-/- and [SM22α-CreKI/+/NoxA1f/+] mice, Nox1y/-, p47phox-/- and NoxA1-/- mouse aortic VSMC cultures, adenoviral NoxA1 overexpression and shRNA constructs and a functional AlphaScreen and a 100,000 member library of small molecules for identifying potential Nox1 NADPH oxidase inhibitors. Our research strategy includes the following three specific aims. Aim 1: Define the molecular mechanisms of NoxA1-dependent NADPH oxidase activity in agonist-induced ROS generation in mouse and human aortic VSMC. Aim 2: Determine the effect of NoxA1 deficiency in restenosis and atherosclerosis. Aim 3: Identify novel and specific NoxA1 and p47phox interaction inhibitors as means to regulating Nox1 activity. Together, these studies will establis the role of VSMC Nox1 NADPH oxidase in vascular pathophysiology and potentially identify strategies/compounds for the regulation of this enzyme.