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 是一种有效的 EC 迁移刺激剂，主要通过 VEGF 2 型受体 (VEGFR2) 发挥作用。在静止的 EC 中，VEGF 刺激内皮迁移的最初反应之一是稳定的细胞-细胞接触的松动，这种接触受到 VE-钙粘蛋白酪氨酸磷酸化的调节。我们发现 VEGF 刺激 Rac1 依赖的基于 gp91phox 的 NAD(P)H 氧化酶，并且活性氧 (ROS) 参与与 EC 迁移相关的 VEGFR2 介导的信号传导。人们对潜在的分子机制知之甚少。我们最近发现 IQGAP1 是一种新型 VEGFR2 结合蛋白。它作为支架蛋白发挥作用，通过与细胞骨架和细胞间粘附蛋白（包括活性 Rac1 和 E-钙粘蛋白）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，新形成的毛细血管内皮细胞中IQGAP1表达增加； 2) IQGAP1的过表达增加EC中基础Rac1活性、ROS产生和细胞迁移； 3) VEGF 促进激活的 VEGFR2 和 Rac1 募集到粘附连接处的 IQGAP1-VE-钙粘蛋白复合物，这可能促进 ROS 依赖性细胞间接触的丧失和随后的 EC 迁移； 4) 伤口分析显示，IQGAP1 与活跃的 VEGFR2 和 gp91phox 共定位于活跃迁移的 EC 的前沿。因此，我们假设 IQGAP1 作为 VEGFR2 结合支架蛋白发挥作用，将 ROS 依赖性信号传导与 VEGF 介导的内皮迁移联系起来。 Aim1 将检查 IQGAP1 是否与 VEGFR2 和 Rac1 功能性相互作用，以将 VEGFR2 与与 EC 迁移相关的 ROS 依赖性信号耦合。 Aim2 将检查 IQGAP1 是否作为支架发挥作用，通过与 VE-钙粘蛋白结合将激活的 VEGFR2 和 Rac1 招募到粘附连接处，从而促进 ROS 依赖性细胞间接触丧失，从而启动 EC 迁移。 Aim3 将检查 IQGAP1 在定向细胞迁移过程中将 VEGFR2 和 NAD(P)H 氧化酶靶向前沿的支架作用。 Aim4 将使用小鼠后肢缺血模型评估 IQGAP1 的体内功能意义。长期目标是了解 ROS 在血管生成和内皮伤口修复背景下调节 EC 迁移的分子机制，这将有助于新治疗策略的开发。