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）：血管损伤后发生的活性氧（ROS）产生的增加，氧化应激。在此修复过程中，血管平滑肌细胞（VSMC）在整个弹性层层中的迁移增加，并显着促进新内膜形成。虽然损伤后的ROS产量增加的增加，但对于ROS促进VSMC迁移和新内膜形成的机制知之甚少。我们小组的最新研究已开始剖析由ROS调节的血管平滑肌细胞迁移所涉及的分子信号传导机制，并已将3-磷酸肌醇依赖性激酶-1（PDK1）确定为关键调节剂。但是，我们尚不了解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，也不了解这些新型机制是否有助于体内血管病理学。我们的工作假设是，在VSMC迁移期间，肌动蛋白细胞骨架突出受伤后的前缘和血管修复受伤后的血管修复受到ROS依赖性激活3-磷酸肌醇依赖性激酶-1（PDK1）的激活，并随后对当前未知的蛋白质磷酸化酶的激活调节。将实现以下特定目标：1）确定PDK1的ROS依赖性激活介导Cofilin的去磷酸化是否是否通过肌动蛋白解聚化来调节VSMC迁移，以及应力纤维的形成和/或lamellipodia的形成和/或lamellipodia的形成，2）测试是否在pdk1及其在Cofilin中的作用在COFILIN中的作用，以及其在COFILIN中的作用。细胞中的结构域和3）通过研究小鼠颈动脉线损伤后血管重塑期间的VSMC迁移来定义ROS的贡献。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评估迁移，并确定体外利用培养的VSMC，生物和固定VSMC的共斑显微镜检查的关键信号传导机制，通过在迁移和蛋白质共核过程中进行迁移和蛋白质的迁移，并在研究中进行固定的VSMC，并在迁移过程中发生迁移，并在VIV中进行了模型，VIV在VIV中发生了模型。 VSMC迁移有助于新的形成。可以预料，这些研究的发现将提供有关ROS在体外和体内介导VSMC迁移中的作用的重要信息，并最终可能确定潜在的治疗靶标在血管损伤和/或疾病期间进行干预。高血压，动脉粥样硬化和再狭窄等血管病理及其相关并发症对西方培养物的死亡率显着贡献。这些疾病中每种疾病的一个常见组成部分是，血管发生一定程度的损伤，这通常会导致需要治疗和治疗的进一步并发症。在血管损伤期间，平滑肌细胞构成了血管结构的主要层，以响应各种刺激而移动。该提案研究了井文件如何响应血管损伤，活性氧的产生增加而发生的变化，增强了平滑肌细胞的运动。鉴定该细胞运动基础的机制将允许设计更好的方法治疗患者并发症的方法，并确定设计药理干预措施的靶标。