Regulation of the Nox1 NADPH Oxidase in Vascular Smooth Muscle Cells

描述(由申请人提供)： 心血管疾病的特征是血管壁上活性氧物种(ROS)的产生增加，导致信号通路的激活，最终促进细胞生长和新生内膜的形成。来自血管平滑肌细胞(SMCs)的ROS是血管疾病发展的一个主要因素，尽管抗氧化治疗只取得了有限的治疗效果。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，其中Nox1是SMC表达的主要催化NADPH氧化酶。Nox1来源的ROS与动脉粥样硬化、损伤后新生内膜形成和SMC迁移有关，但Nox1激活氧化还原依赖的信号通路的确切机制仍不完全清楚。为了开发针对Nox1的靶向治疗方法，首先必须了解Nox1激活所必须满足的参数。先前的研究表明，细胞因子激活促炎因子核因子-B(NF-B)需要通过内吞作用使Nox1内化。这项建议的目的是确定Nox1中调节其激活和氧化还原信号以调节迁移和新生内膜形成的新区域。该假说认为，在细胞因子诱导的SMC中，Nox1的膜转运和磷酸化是必要的。提出以下目的来检验中心假说：1)研究细胞因子诱导的SMC中NOX1转运的功能后果。2)确定Nox1的磷酸化如何调节其运输和激活。3)确定抑制Nox1的磷酸化或转运是否在预防新生内膜增生方面提供了治疗益处。第一个目标的拟议研究将利用NOX1中规范的内化基序的定点突变来确定细胞因子刺激如何影响NOX1的运输以及NOX1依赖的ROS的产生、核因子-B的激活和SMC的迁移。对于第二个目标，将应用系统生物学方法来量化特定氨基酸残基上Nox1磷酸化的动态变化。接下来，这些磷酸化位点将被突变，以评估磷酸化在Nox1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Nox1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Nox1中激活I SMC所必需的其他信号事件和基序。这些研究的直接临床影响是有可能发现替代方法来产生针对血管病理的Nox1靶向治疗药物。

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.