Regulation of the Nox1 NADPH Oxidase in Vascular Smooth Muscle Cells

描述（由申请人提供）： 心血管疾病的特征在于血管壁中活性氧（ROS）的产生增加，这导致信号通路的激活，最终促进细胞生长和新生内膜形成。来自平滑肌细胞（SMC）的ROS是血管疾病发展的主要促成因素，尽管抗氧化剂治疗仅取得有限的治疗益处。因此，有必要确定有针对性的方法来防止ROS的产生。NADPH氧化酶是血管系统中ROS的主要来源，Nox 1是SMC中表达的主要催化NADPH氧化酶。Nox 1衍生的ROS与动脉粥样硬化以及损伤后的新生内膜形成和SMC迁移有关，但Nox 1激活氧化还原依赖性信号通路的确切机制仍不完全确定。为了开发针对Nox 1的靶向疗法，首先需要了解Nox 1激活必须满足的参数。先前的研究表明，促炎因子核因子-B（NF-B）的细胞因子激活需要通过内吞作用使Nox 1内化。本研究的目的是确定Nox 1中调节其激活和氧化还原信号传导以介导迁移和新生内膜形成的新区域。这一假设是，膜运输和磷酸化的Nox 1是必需的尼古丁诱导的Nox 1在SMC中的激活。提出以下目标来检验中心假设：1）检查尼古丁诱导的SMC中Nox 1运输的功能后果。2)定义Nox 1的磷酸化如何调节其运输和激活。3)确定抑制Nox 1磷酸化或运输是否在预防新生内膜增生中提供治疗益处。第一个目标的拟议研究将利用Nox 1内的典型内化基序的定点突变来确定细胞因子刺激如何影响Nox 1运输以及Nox 1依赖性ROS产生、NF-B激活和SMC迁移。对于第二个目标，系统生物学方法将被应用于定量的Nox 1磷酸化在特定的氨基酸残基的动态变化。接下来，这些磷酸化位点将被突变，以评估磷酸化在Nox 1激活机制中的重要性。第三个目标将使用体内损伤模型来研究Nox 1内化和/或磷酸化在新生内膜形成中的作用。这些研究有可能确定Nox 1内的其他信号事件和基序，这些事件和基序是SMC激活所必需的。这些研究的直接临床影响是发现替代方法以产生针对血管病理学的Nox 1靶向治疗的潜力。

DESCRIPTION (provided by applicant): Cardiovascular disease is characterized by increased generation of reactive oxygen species (ROS) in the vessel wall, which results in activation of signaling pathways that ultimately promote cell growth and neointimal formation. ROS derived from smooth muscle cells (SMCs) is a major contributing factor in the development of vascular disease, though antioxidant therapies have achieved only limited therapeutic benefit. Therefore, it is necessary to identify targeted approaches to prevent ROS generation. NADPH oxidases are the predominant source of ROS in the vasculature, with Nox1 being the primary catalytic NADPH oxidase expressed in SMCs. Nox1-derived ROS have been linked to atherosclerosis as well as neointimal formation and SMC migration following injury, but the precise mechanisms by which Nox1 activates redox- dependent signaling pathways remain incompletely defined. In order to develop targeted therapeutics against Nox1, it is first necessary to understand the parameters that must be met for Nox1 activation. Previous studies demonstrate that cytokine activation of the pro- inflammatory factor nuclear factor-¿B (NF-¿B) requires internalization of Nox1 via endocytosis. The objective of this proposal is to identify novel regions within Nox1 that regulate its activatio and redox signaling to mediate migration and neointimal formation. The hypothesis is that membrane trafficking and phosphorylation of Nox1 are necessary for cytokine-induced Nox1 activation in SMCs. The following aims are proposed to test the central hypothesis: 1) Examine the functional consequences of cytokine-induced Nox1 trafficking in SMCs. 2) Define how phosphorylation of Nox1 regulates its trafficking and activation. 3) Determine whether the inhibition of Nox1 phosphorylation or trafficking provides therapeutic benefit in the prevention of neointimal hyperplasia. Proposed studies for the first aim will utilize site-directed mutagenesis o canonical internalization motifs within Nox1 to define how cytokine stimulation affects Nox1 trafficking as well Nox1-dependent ROS generation, NF-¿B activation, and SMC migration. For the second aim, a systems biology approach will be applied to quantitate the dynamic changes in Nox1 phosphorylation at specific amino acid residues. Next, these phosphorylation sites will be mutated to evaluate the importance of phosphorylation in the mechanisms of Nox1 activation. The third aim will use an in vivo model of injury to examine the role of Nox1 internalization and/or phosphorylation in neointimal formation. These studies have the potential to identify additional signaling events and motifis within Nox1 that are necessary for activation i SMCs. An immediate clinical impact of these studies is the potential to uncover alternative approaches to generate Nox1-targeted therapeutics for vascular pathologies.