Reactive Oxygen Species and Endothelial Migration

活性氧和内皮迁移

• 批准号: 7097600
• 负责人: Masuko Ushio-Fukai
• 金额: $ 38.25万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7097600
• 关键词: NAD(P)H oxidoreductase; angiogenesis; binding proteins; biological signal transduction; cell cell interaction; cell migration; confocal scanning microscopy; disease /disorder model; free radical oxygen; green fluorescent proteins; growth factor receptors; immunochemistry; ischemia; laboratory mouse; protein protein interaction; small interfering RNA; tissue /cell culture; ultrasound blood flow measurement; vascular endothelial growth factors; vascular endothelium; wound healing

DESCRIPTION (provided by applicant): Endothelial cell (EC) migration is a key event for endothelial wound repair and angiogenesis. VEGF is a potent stimulator for EC migration primarily through the VEGF type2 receptor (VEGFR2). In quiescent ECs, one of the initial responses to stimulate endothelial migration by VEGF is the loosening of stable cell-cell contacts which is regulated by tyrosine phosphorylation of VE-cadherin. We showed that VEGF stimulates Rac1 -dependent gp91phox-based NAD(P)H oxidase and that reactive oxygen species (ROS) are involved in VEGFR2-mediated signaling linked to EC migration. Underlying molecular mechanisms are poorly understood. We recently identified IQGAP1 as a novel VEGFR2 binding protein. It functions as a scaffold protein that controls cell motility and morphogenesis by interacting with cytoskeletal and cell-cell adhesion proteins including active Rac1 and E-cadherin. We also found that: 1) IQGAP1 expression is increased in the newly-formed capillary ECs in a mouse hindlimb ischemia model of angiogenesis; 2) Overexpression of IQGAP1 increases basal Rac1 activity, ROS production and cell migration in ECs; 3) VEGF promotes recruitment of activated VEGFR2 and Rac1 to the IQGAP1-VE-cadherin complex at adherens junctions, which may promote ROS-dependent loss of cell-cell contacts and subsequent EC migration; 4) Wound assays reveal that IQGAP1 colocalizes with active VEGFR2 and gp91phox at the leading edge in actively migrating ECs. We thus hypothesize that IQGAP1 functions as a VEGFR2 binding scaffold protein to link ROS-dependent signaling with VEGF-mediated endothelial migration. Aim1 will examine whether IQGAP1 functionally interacts with VEGFR2 and Rac1 to couple VEGFR2 to ROS-dependent signaling linked to EC migration. Aim2 will examine whether IQGAP1 functions as a scaffold to recruit activated VEGFR2 and Rac1 to adherens junctions through binding to VE-cadherin, thereby facilitating ROS-dependent loss of cell-cell contacts, which initiates EC migration. Aim3 will examine the scaffolding role of IQGAP1 in targeting VEGFR2 and NAD(P)H oxidase to the leading edge during directed cell migration. Aim4 will assess the functional significance of IQGAP1 in vivo using mouse hindlimb ischemia model. The long-term goal is to understand the molecular mechanisms by which ROS regulate EC migration in the context of angiogenesis and endothelial wound repair, which should facilitate the development of new therapeutic strategies.

描述（由申请方提供）：内皮细胞（EC）迁移是内皮伤口修复和血管生成的关键事件。VEGF是主要通过VEGF 2型受体（VEGFR 2）进行EC迁移的有效刺激剂。在静止的内皮细胞中，VEGF刺激内皮细胞迁移的最初反应之一是稳定的细胞-细胞接触的松动，这是由VE-钙粘蛋白的酪氨酸磷酸化调节的。我们发现，VEGF刺激Rac 1依赖的gp 91 phox为基础的NAD（P）H氧化酶和活性氧（ROS）参与VEGFR 2介导的信号转导与EC迁移。基本的分子机制知之甚少。我们最近发现IQGAP 1是一种新的VEGFR 2结合蛋白。它作为一种支架蛋白，通过与细胞骨架和细胞-细胞粘附蛋白（包括活性Rac 1和E-cadherin）相互作用来控制细胞运动和形态发生。我们还发现：1）在血管生成的小鼠后肢缺血模型中，IQGAP 1在新形成的毛细血管EC中的表达增加; 2）IQGAP 1的过表达增加EC中的基础Rac 1活性、ROS产生和细胞迁移; 3）VEGF促进活化的VEGFR 2和Rac 1募集到粘附连接处的IQGAP 1-VE-钙粘蛋白复合物，这可能促进细胞-细胞接触的ROS依赖性丧失和随后的EC迁移; 4）创伤分析显示IQGAP 1与活性VEGFR 2和gp 91 phox共定位于活跃迁移的EC的前沿。因此，我们假设IQGAP 1作为VEGFR 2结合支架蛋白，将ROS依赖性信号传导与VEGF介导的内皮细胞迁移联系起来。Aim 1将检查IQGAP 1是否在功能上与VEGFR 2和Rac 1相互作用，以将VEGFR 2偶联到与EC迁移相关的ROS依赖性信号传导。Aim 2将检查IQGAP 1是否作为支架发挥作用，通过与VE-钙粘蛋白结合将活化的VEGFR 2和Rac 1募集到粘附连接处，从而促进ROS依赖性细胞-细胞接触丧失，从而启动EC迁移。Aim 3将检查IQGAP 1在定向细胞迁移过程中将VEGFR 2和NAD（P）H氧化酶靶向前沿的支架作用。目的4将使用小鼠后肢缺血模型评估IQGAP 1在体内的功能意义。长期目标是了解ROS在血管生成和内皮损伤修复背景下调节EC迁移的分子机制，这将有助于开发新的治疗策略。