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: 7580946
• 负责人: DAVID S WEBER
• 金额: $ 31.18万
• 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/7580946
• 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; Hypertension; In Vitro; Injury; Intervention; Life; Mediating; Mediator of activation protein; Migration Assay; Modeling; Molecular; Movement; Mus; Oxidases; Oxidative Stress; Pathology; Patients; Process; Production; Protein Dephosphorylation; 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; overexpression; 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，血管壁中活性氧（ROS）生成增加导致氧化应激。在此修复过程中，血管平滑肌细胞（VSMC）穿过内弹性膜的迁移增加，并显著促进新生内膜形成。虽然损伤后ROS的产生增加是有据可查的，但关于ROS促进VSMC迁移和新生内膜形成的机制知之甚少。我们小组最近的研究已经开始剖析参与由ROS调节的血管平滑肌细胞迁移的分子信号传导机制，并确定了3-磷酸肌醇依赖性激酶1（PDK 1）作为关键调节因子。然而，我们还不知道如何ROS和/或上游激酶调节细胞骨架的事件，控制细胞趋化性，也不知道这些新的机制是否有助于在体内血管病变。我们的工作假设是，肌动蛋白细胞骨架突起在VSMC迁移和血管修复损伤后的前沿是由3-磷酸肌醇依赖性激酶1（PDK 1）的ROS依赖性激活和随后的激活目前未知的蛋白磷酸酶调节。将实现以下具体目标：1）确定PDK 1的ROS依赖性活化是否介导了丝切蛋白的去磷酸化以通过肌动蛋白解聚和应力纤维和/或板状伪足的形成来调节VSMC迁移，2）测试PDK 1的ROS依赖性活化及其在丝切蛋白去磷酸化中的作用是否是由于其定位在细胞中的特定信号传导结构域内，3）通过研究小鼠颈动脉钢丝损伤后血管重塑过程中VSMC的迁移来确定ROS的作用。为了测试这些目标，我们将利用siRNA、腺病毒载体和药理学策略来评估迁移并利用培养的VSMC在体外识别关键信号传导机制，对活的和固定的VSMC进行共聚焦显微镜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。预计这些研究的结果将提供有关ROS在体外和体内介导VSMC迁移中的作用的重要信息，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。在西方文化中，血管病变如高血压、动脉粥样硬化和再狭窄及其相关并发症是导致死亡率的重要原因。这些疾病中的每一种的一个共同组成部分是存在对血管发生的一定程度的损伤，这通常导致需要治疗和管理的进一步并发症。在血管损伤期间，构成血管结构的主要层的平滑肌细胞响应于各种刺激而移动。该提案探讨了如何一个很好的文件发生的变化，在响应血管损伤，增加活性氧的生产，加强平滑肌细胞的运动。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，以及确定药物干预设计的目标。