Reactive Oxygen Species Regulation of Vascular Smooth Muscle Cell Migration

活性氧对血管平滑肌细胞迁移的调节

• 批准号: 7373675
• 负责人: DAVID S WEBER
• 金额: $ 31.38万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-07 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373675
• 关键词: Actins; Address; Adenovirus Vector; Atherosclerosis; Biochemical; Blood Vessels; Caveolae; Cell Culture System; Cells; Chemotaxis; Confocal Microscopy; Cytoskeletal Modeling; Cytoskeleton; Disease; Event; Focal Adhesions; G Actin; Generations; High Blood Pressure; In Vitro; Injury; Intervention; Life; Localized; Mediating; Mediator of activation protein; Migration Assay; Modeling; Molecular; Movement; Mus; NAD(P)H oxidase; Oxidative Stress; Pathology; Patients; Personal Satisfaction; Process; Production; Protein Dephosphorylation; Protein Overexpression; Protein phosphatase; Proteins; Reactive Oxygen Species; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Stimulus; Stress Fibers; Structure; Testing; Time; Transgenic Mice; Work; cell motility; cofilin; defined contribution; depolymerization; design; human CYBA protein; in vivo; migration; monomer; mortality; novel; p21 activated kinase; phosphoinositide-dependent kinase 1; protein activation; repaired; response; restenosis; therapeutic target; upstream kinase; vascular smooth muscle cell migration

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.

描述(由申请人提供)：血管损伤后，由于血管壁上产生的活性氧物种(ROS)增加而导致的氧化应激。在这一修复过程中，血管平滑肌细胞(VSMC)跨内弹力层的迁移增加，并对新生内膜的形成起重要作用。虽然损伤后ROS的产生增加是有文献记载的，但关于ROS促进VSMC迁移和新生内膜形成的机制却知之甚少。本课题组最近的研究已经开始剖析受ROS调控的涉及血管平滑肌细胞迁移的分子信号机制，并确定3-磷酸肌醇依赖的激酶-1(PDK1)是一个关键的调节因子。然而，我们还不清楚ROS和/或上游激酶如何调节控制细胞趋化的细胞骨架事件，也不知道这些新的机制是否在体内参与血管病理。我们的工作假设是，在VSMC迁移和损伤后的血管修复过程中，肌动蛋白细胞骨架的前沿突起是由ROS依赖的3-磷酸肌醇依赖的激酶-1(PDK1)的激活以及随后一种目前未知的蛋白磷酸酶的激活来调节的。将完成下列特定目标：1)确定PDK1的ROS依赖激活是否通过肌动蛋白解聚以及应激纤维和/或片层脂的形成来调节VSMC的迁移；2)检测PDK1的ROS依赖激活及其在Cofilin去磷酸化中的作用是否是由于其定位于细胞内的特定信号结构域；以及3)通过研究小鼠颈动脉线损伤后血管重建过程中VSMC在体内的迁移来确定ROS的贡献。为了测试这些目的，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关键的信号机制，利用体外培养的VSMCs，活的和固定的VSMCs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，以及体内电线损伤模型来评估ROS介导的VSMC迁移促进新生内膜形成的机制。预计这些研究结果将为ROS在体内外介导VSMC迁移中的作用提供重要信息，并最终确定在血管损伤和/或疾病中进行干预的潜在治疗靶点。在西方文化中，高血压、动脉粥样硬化、再狭窄等血管病变及其相关并发症对死亡率有很大影响。每种疾病的一个共同组成部分是血管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做出反应。这项建议研究了一个Well记录如何在应对血管损伤时发生的变化，增加活性氧物种的产生，增强平滑肌细胞的运动。确定这种细胞运动的机制将有助于设计更好的方法来治疗患者的并发症，并为设计药理干预措施确定目标。