Endothelial Healing is Inhibited by PI3 Kinase-Induced Activation of TRPC6

PI3 激酶诱导的 TRPC6 激活抑制内皮愈合

• 批准号: 9240762
• 负责人: Michael Aaric Rosenbaum
• 金额: --
• 依托单位: LOUIS STOKES CLEVELAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9240762
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adaptor Signaling Protein; Aging; American; Angioplasty; Animals; Antineoplastic Agents; Area; Arterial Injury; Arteries; Award; Balloon Angioplasty; Binding; Biological Assay; Biology; Biometry; Blood Vessels; Calcium ion; Calpain; Cardiovascular Diseases; Cardiovascular system; Carotid Arteries; Catalytic Domain; Cell Proliferation; Cell membrane; Cells; Centers for Disease Control and Prevention (U.S.); Cholesterol; Cicatrix; Clinical Trials; Coagulation Process; Complement; Critical Thinking; Cytoskeletal Proteins; Diagnosis; Diet; Disease; Effectiveness; Elderly; Endothelial Cells; Foundations; Functional disorder; Funding; Generations; Goals; Grant; Heart Diseases; High Fat Diet; In Vitro; Inflammatory; Injury; Intervention; Knowledge; Laboratories; Leadership; Length; Lipids; Lysophosphatidylcholines; Membrane; Mentors; Methods; Modeling; Molecular and Cellular Biology; Mus; Operative Surgical Procedures; Outcome; Patients; Phosphatidylinositols; Play; Population; Procedures; Protein Isoforms; Proteins; Pulmonary Hypertension; Quality of life; RNA; Reactive Oxygen Species; Research; Research Personnel; Role; Site; Site-Directed Mutagenesis; Small Interfering RNA; Smooth Muscle Myocytes; Stents; Surface; Systemic hypertension; TNFRSF5 gene; Techniques; Testing; Therapeutic; Thrombosis; Transfection; Treatment Protocols; United States; United States National Institutes of Health; Vascular Diseases; Vascular Graft; Veterans; Wild Type Mouse; Writing; atherogenesis; base; career; career development; cell motility; clinical practice; experience; healing; high reward; hypercholesterolemia; improved; improved outcome; in vivo; inhibitor/antagonist; injured; kinase inhibitor; macrophage; novel; overexpression; oxidation; oxidized lipid; oxidized low density lipoprotein; prevent; receptor; restenosis; skills; success; therapy outcome; voltage gated channel

Cardiovascular disease is a devastating disorder that has a major impact on length and quality of life. According to the CDC, approximately 27 million Americans carry the diagnosis of heart disease. The number of heart and vascular procedures (balloon angioplasties and vascular grafts) that will be performed in 2030 is ex- pected to be nearly twice the number performed in 2010. Similar increases will occur in the veteran population When a blood vessel is treated with angioplasty, the endothelial cells (EC) are removed. The cells must migrate from the edge of the injury into the area of injury to heal it. If healing is delayed, the chance of restenosis is increased. Lipid oxidation products accumulate in atherosclerotic arteries and at regions of injury, cause cellular dysfunction, and inhibit EC migration in vitro and in vivo. Limited re-endothelialization contributes to thrombogenicity, smooth muscle cell proliferation, and restenosis. Oxidized lipids cause an inappropriate increase in intracellular free calcium ion concentration ([Ca2+]i) through canonical transient receptor potential (TRPC) channels, specifically TRPC6. Activation of TRPC6 by causes an increase in [Ca2+]i that results in activation of TRPC5 and a prolonged increase in [Ca2+]i. The increased [Ca2+]i activates calpains that break down cytoskeletal proteins inhibiting EC migration. Studies in TRPC6-/- mice provide compelling evidence of the importance of this cascade in vivo. Re-endothelialization of injured carotid arteries is dramatically reduced in wild-type (WT) mice on a high fat diet compared with chow- fed mice, but in TRPC6-/- mice, hypercholesterolemia does not inhibit re-endothelialization of the injury. Considerable effort has been directed at identifying a TRPC6 inhibitor without success. Lipid oxidation products induce TRPC6 externalization by activating phosphatidylinositol 3-kinase (PI3K), which generates PIP3 (phosphatidylinositol (3,4,5)-trisphosphate). PIP3 is anchored in the cell membrane and binds to TRPC6, which promotes TRPC6 translocation to the cell membrane and leads to increased [Ca2+]i. We hypothesize that inhibition of PI3K can block the activation of TRPC6 channels by lipid oxidation products and restore EC migra- tion in vitro and promote EC healing of arterial injuries in vivo. To test this, we will 1) identify the PI3K iso- form(s) essential for TRPC6 activation, 2) investigate the effectiveness of isoform-specific PI3K inhibitors to re- store EC healing of arterial injuries in hypercholesterolemic animals, and 3) investigate the mechanism through which PIP3 interacts with TRPC6 to promote TRPC6 translocation to the membrane and activation to identify a more specific method of TRPC6 inhibition. The long-term scientific goal is to improve the outcome of ther- apeutic vascular interventions promoting endothelial surfacing of angioplasty sites, stents, and vascular grafts. In terms of a research career development, my short-term research goal is to develop a foundation in a focused research area that I can expand over the course of my career and which will serve as a complement to my clinical practice. My knowledge of vascular wall biology will increase through seminars and I will expand my experience in cellular molecular biology laboratory techniques over the course of the award, including protein extraction and analysis, small-inhibitory RNA transfection, and site directed mutagenesis. Over the course of the award, I will gain additional experience in biostatistics, scientific and grant writing, laboratory management and leadership, and mentoring. I will also continue to develop the critical thinking skills necessary for successful research plans. My primary long-term career goal is to transition to an independent investigator and compete for VA Merit Review and NIH funding. My long-term research goal is to more clearly define the mechanisms by which reactive oxygen species and the activation of TRPC channels inhibit endothelial cell healing. With progress in this area, mechanism-based treatment regimens can be developed, transitioned into clinical trials, and ultimately be carried into clinical practice to improve the long-term outcomes following vascular intervention.

心血管疾病是一种破坏性的疾病，对生活时间和质量有重大影响。 根据美国疾病控制与预防中心的数据，大约有2700万美国人被诊断患有心脏病。数量 2030年将进行的心脏和血管手术(球囊血管成形术和血管移植)不包括 预计这一数字将是2010年的近两倍。退伍军人人数也将出现类似的增长 血管成形术治疗血管时，血管内皮细胞(EC)被去除。细胞必须 从受伤的边缘迁移到受伤的区域以治愈它。如果愈合被推迟， 再狭窄增加。脂质氧化产物在动脉粥样硬化的动脉和损伤区域积聚， 导致细胞功能障碍，并抑制EC在体外和体内的迁移。有限的再内皮化贡献 血栓形成、平滑肌细胞增殖和再狭窄。 氧化脂质导致细胞内游离钙离子浓度([Ca2+]i)不适当升高 通过典型的瞬时受体电位(TRPC)通道，特别是TRPC6。TRPC6的激活方式 导致[Ca~(2+)]i升高，导致TRPC5激活和[Ca~(2+)]i持续升高。 [Ca~(2+)]i升高可激活钙蛋白酶，该酶可分解抑制EC迁移的细胞骨架蛋白。研究项目： TRPC6-/-小鼠提供了令人信服的证据，证明了这种级联反应在体内的重要性。血管再内皮化 野生型(WT)小鼠在高脂饮食的情况下，颈动脉损伤程度显著低于饲料小鼠. 但在TRPC6-/-小鼠中，高胆固醇血症并不抑制损伤的重新内皮化。 在确定TRPC6抑制剂方面已经做了相当大的努力，但没有成功。脂类氧化 产物通过激活磷脂酰肌醇3-激酶(PI3K)诱导TRPC6外化，PI3K产生 PIP3(磷脂酰肌醇(3，4，5)三磷酸)。PIP3锚定在细胞膜上并与TRPC6结合， 促进TRPC6移位到细胞膜，并导致[钙]i增加。我们假设 抑制PI3K可阻断脂质氧化产物激活TRPC6通道，恢复EC迁移。 体外抗血管内皮细胞损伤，促进血管内皮细胞体内愈合。为了测试这一点，我们将1)确定PI3K iso- 2)研究同种异构体特异性的PI3K抑制剂对TRPC6激活的影响。 保存EC对高胆固醇血症动物动脉损伤的修复作用；3)通过 哪个PIP3与TRPC6相互作用，促进TRPC6转位到膜上并激活以识别 更特异的抑制TRPC6的方法。长期的科学目标是改善治疗的结果。 治疗性血管干预促进血管成形术部位、支架和血管移植物的内皮表面。 就研究事业发展而言，我的短期研究目标是在一个 我可以在我的职业生涯中扩展的重点研究领域，这将是对 我的临床实践。我的血管壁生物学知识将通过研讨会增长，我将扩大我的 在获奖过程中有细胞分子生物学实验室技术方面的经验，包括蛋白质 提取和分析，小抑制RNA转染，定点突变。在整个过程中 获奖后，我将在生物统计学、科学和拨款撰写、实验室管理方面获得更多经验 和领导力，以及指导。我还将继续培养必要的批判性思维技能 成功的研究计划。 我的主要长期职业目标是过渡到一名独立的调查员，并竞争退伍军人管理局的荣誉 审查和国家卫生研究院的资金。我的长期研究目标是更清楚地定义 活性氧物种和TRPC通道的激活抑制内皮细胞的愈合。随着…的进展 这一领域，基于机制的治疗方案可以开发，过渡到临床试验，以及 最终应用于临床，改善血管介入治疗后的远期疗效。