Reactive Oxygen Species and Endothelial Migration

描述（由申请人提供）：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是一种主要通过VEGF 2型受体（VEGFR2）促进EC迁移的有效刺激物。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Rac1依赖的gp91phox- NAD(P)H氧化酶，并且活性氧（ROS）参与了vegfr2介导的与EC迁移相关的信号传导。潜在的分子机制尚不清楚。我们最近发现IQGAP1是一种新的VEGFR2结合蛋白。它作为一种支架蛋白，通过与细胞骨架蛋白和细胞粘附蛋白（包括活性Rac1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)在小鼠后肢缺血血管生成模型中，IQGAP1在新形成的毛细血管内皮细胞中表达升高；2)过表达IQGAP1增加ECs中基础Rac1活性、ROS生成和细胞迁移；3) VEGF促进活化的VEGFR2和Rac1向粘附连接处的IQGAP1-VE-cadherin复合体募集，这可能促进ros依赖性的细胞-细胞接触丧失和随后的EC迁移；4)伤口实验显示，在主动迁移的内皮细胞中，IQGAP1与活跃的VEGFR2和gp91phox共定位在前沿。因此，我们假设IQGAP1作为VEGFR2结合支架蛋白，将ros依赖性信号与vegf介导的内皮迁移联系起来。Aim1将研究IQGAP1是否与VEGFR2和Rac1相互作用，将VEGFR2与EC迁移相关的ros依赖性信号耦联。Aim2将研究IQGAP1是否作为一个支架，通过与VE-cadherin结合，将活化的VEGFR2和Rac1招募到粘附连接，从而促进ros依赖性的细胞-细胞接触丧失，从而启动EC迁移。Aim3将研究IQGAP1在定向细胞迁移中将VEGFR2和NAD(P)H氧化酶靶向到前沿的脚手架作用。Aim4将通过小鼠后肢缺血模型来评估IQGAP1在体内的功能意义。长期目标是了解ROS在血管生成和内皮伤口修复过程中调控EC迁移的分子机制，这将有助于开发新的治疗策略。

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.