Molecular Characterization of NoxA1 in VSMC NADPH Oxidase Activation

描述（由申请人提供）：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的主要因素这一假设。特别是，Nox1 NADPH氧化酶的失调与许多血管疾病有关，包括动脉粥样硬化、主动脉夹层、高血压和中风。虽然已经阐明了激活Nox2 (gp91phox) NADPH氧化酶的分子机制，但导致Nox1 NADPH氧化酶活性失调的事件尚不清楚。我们之前的研究和初步数据支持这样的观点，即p67phox的同源物NoxA1在小鼠和人类血管平滑肌细胞（VSMC）中表达，并且是Nox1 NADPH氧化酶活性的关键调节因子。noxa1依赖性活性氧（ROS）在激动剂诱导的VSMC增殖和促生长、氧化还原敏感蛋白激酶激活中发挥重要作用。NoxA1在ApoE-/-小鼠的主动脉和动脉粥样硬化病变以及人类早期颈动脉粥样硬化病变的内膜和内侧VSMC中表达上调。在本文中，我们将研究NoxA1和p47phox相互作用如何调节VSMC中Nox1 NADPH氧化酶的活性，以及NoxA1表达和翻译后修饰的调节机制及其在Nox1 NADPH氧化酶激活中的作用。我们还将确定noxa1依赖性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文库，以确定Nox1 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NoxA1-/-， ApoE-/-/NoxA1-/-和[SM22&#945；- creki /+/NoxA1 -/-和[SM22&#945；]小鼠，NoxA1 /-， p47phox-/-和NoxA1-/-小鼠主动脉VSMC培养，腺病毒NoxA1过表达和shRNA构建，功能性AlphaScreen和100,000成员的小分子文库，用于鉴定潜在的Nox1 NADPH氧化酶抑制剂。我们的研究策略包括以下三个具体目标。目的1：明确noxa1依赖性NADPH氧化酶活性在激动剂诱导小鼠和人主动脉VSMC中ROS生成的分子机制。目的2：确定NoxA1缺乏对再狭窄和动脉粥样硬化的影响。目的3：鉴定新的特异性NoxA1和p47phox相互作用抑制剂，作为调节Nox1活性的手段。总之，这些研究将确定VSMC Nox1 NADPH氧化酶在血管病理生理中的作用，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. PUBLIC HEALTH RELEVANCE: Oxidative stress generated from dysregulated Nox1 NADPH oxidase activity has been implicated in a growing number of vascular diseases including atherosclerosis, aortic dissection, hypertension and stroke. Elucidation of molecular composition and activation mechanisms of this VSMC NADPH oxidase will yield valuable insight to develop novel approaches for the treatment of various oxidative stress-dependent vascular diseases.