Reactive Oxygen Species Regulation of Vascular Smooth Muscle Cell Migration

描述（由申请人提供）：血管损伤后发生的氧化应激是由血管壁活性氧（ROS）生成增加引起的。在这个修复过程中，血管平滑肌细胞（VSMC）在内部弹性板上的迁移增加，并显著促进新内膜的形成。虽然损伤后ROS的产生增加已被充分记录，但ROS促进VSMC迁移和新生内膜形成的机制却知之甚少。我们小组最近的研究已经开始分析血管平滑肌细胞迁移受ROS调节的分子信号机制，并确定了3-磷酸肌醇依赖性激酶-1 （PDK1）是一个关键的调节因子。然而，我们还不了解ROS和/或上游激酶如何调节控制细胞趋化性的细胞骨架事件，也不知道这些新机制是否有助于体内血管病理。我们的工作假设是，VSMC迁移和损伤后血管修复过程中前沿的肌动蛋白细胞骨架突出是由ros依赖性的3-磷酸肌醇依赖性激酶-1 （PDK1）激活和随后未知的蛋白磷酸酶激活调节的。将实现以下具体目标：1)确定ros依赖的PDK1激活是否介导cofilin的去磷酸化，从而通过肌动蛋白解聚和应力纤维和/或板足的形成来调节VSMC迁移；2)测试ros依赖的PDK1激活及其在cofilin去磷酸化中的作用是否由于其定位于细胞中的特定信号域。3)通过研究小鼠颈动脉丝损伤后血管重构过程中VSMC在体内的迁移来确定ROS的作用。为了验证这些目标，我们将利用siRNA、腺病毒载体和药理学策略来评估迁移并确定体外培养VSMC的关键信号机制，利用活VSMC和固定VSMC的共聚焦显微镜来检查迁移过程中发生的细胞骨架重组和蛋白质共定位，并利用体内丝损伤模型来评估ros介导的VSMC迁移促进新内膜形成的机制。预计这些研究结果将为ROS在体外和体内介导VSMC迁移中的作用提供重要信息，并可能最终确定血管损伤和/或疾病期间干预的潜在治疗靶点。在西方，高血压、动脉粥样硬化、再狭窄等血管病变及其相关并发症是造成死亡率的重要原因。这些疾病的一个共同组成部分是，血管会受到一定程度的损伤，这通常会导致进一步的并发症，需要治疗和管理。在血管损伤期间，构成血管结构主要层的平滑肌细胞会对各种刺激作出反应。本研究探讨了血管损伤、活性氧产生增加、平滑肌细胞运动增强等过程中发生的变化。识别这种细胞运动的机制将允许设计更好的方法来治疗患者的并发症，以及确定设计药物干预的目标。

DESCRIPTION (provided by applicant): Oxidative stress, resulting from increased reactive oxygen species (ROS) generation in the vascular wall, occurs following vascular injury. During this repair process, vascular smooth muscle cell (VSMC) migration across the internal elastic lamina is increased and contributes significantly to neointimal formation. While the increased ROS production following injury is well documented, very little is known regarding the mechanisms by which ROS contribute to VSMC migration and neointimal formation. Recent studies by our group have begun to dissect the molecular signaling mechanisms involved in vascular smooth muscle cell migration that are regulated by ROS and have identified 3-phosphoinositide-dependent kinase-1 (PDK1) as a key regulator. However, we do not yet understand how ROS and/or upstream kinases regulate the cytoskeletal events that control cell chemotaxis, nor whether these novel mechanisms contribute to vascular pathology in vivo. Our working hypothesis is that actin cytoskeletal protrusion at the leading edge during VSMC migration and vascular repair following injury is regulated by the ROS-dependent activation of 3-phosphoinositide-dependent kinase-1 (PDK1) and subsequent activation of a currently unknown protein phosphatase. The following specific aims will be accomplished: 1) determine whether the ROS-dependent activation of PDK1 mediates the dephosphorylation of cofilin to regulate VSMC migration via actin depolymerization and the formation of stress fibers and/or lamellipodia, 2) test if the ROS-dependent activation of PDK1 and its role in cofilin dephosphorylation is due to its localization within specific signaling domains in the cell, and 3) define the contribution of ROS by studying VSMC migration in vivo during vessel remodeling following carotid wire injury in mice. To test these aims we will utilize siRNA, adenoviral vectors, and pharmacological strategies to assess migration and identify key signaling mechanisms in vitro utilizing cultured VSMCs, confocal microscopy of both live and fixed VSMCs to examine cytoskeletal reorganization and protein co-localizations occurring during migration, and an in vivo wire injury model to assess mechanisms by which ROS-mediated VSMC migration contribute to neointimal formation. It is anticipated that the findings from these studies will provide important information about the role of ROS in mediating VSMC migration in vitro and in vivo, and may ultimately identify potential therapeutic targets for intervention during vascular injury and/or disease. Vascular pathologies such as high blood pressure, atherosclerosis, and restenosis and their related complications contribute significantly to mortality in Western cultures. One common component of each of these diseases is that there is some degree of injury which occurs to the blood vessels which typically makes results in further complications requiring treatment and management. During vascular injury the smooth muscle cells, which make up a main layer of the blood vessel structure, move in response to a variety of stimuli. This proposal examines how a well documents change that occurs in response to vascular injury, increased reactive oxygen species production, potentiates the movement of smooth muscle cells. The identification of mechanisms underlying this cell movement will allow for the design of better approaches to treatment of the complications in patients as well as identify targets for the design of pharmacological interventions.