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.

心血管疾病是一种毁灭性的疾病，对生命的长度和质量有重大影响。 根据疾病预防控制中心 (CDC) 的数据，大约 2700 万美国人患有心脏病。数量 预计 2030 年将进行心脏和血管手术（球囊血管成形术和血管移植术） 预计将是 2010 年执行人数的近两倍。退伍军人人数也会出现类似的增长 当用血管成形术治疗血管时，内皮细胞（EC）被去除。细胞必须 从伤口边缘迁移到受伤区域以使其愈合。如果延迟愈合，则有机会 再狭窄增加。脂质氧化产物积聚在动脉粥样硬化动脉和损伤区域， 引起细胞功能障碍，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) 不适当地增加 通过经典瞬时受体电位 (TRPC) 通道，特别是 TRPC6。 TRPC6 的激活 导致 [Ca2+]i 增加，从而激活 TRPC5 并延长 [Ca2+]i 增加。这 [Ca2+]i 增加会激活钙蛋白酶，分解细胞骨架蛋白，抑制 EC 迁移。研究于 TRPC6-/- 小鼠提供了令人信服的证据，证明了体内级联的重要性。再内皮化 与饮食相比，高脂肪饮食的野生型（WT）小鼠颈动脉损伤显着减少。 喂养小鼠，但在 TRPC6-/- 小鼠中，高胆固醇血症不会抑制损伤的再内皮化。 为了鉴定 TRPC6 抑制剂，人们付出了相当多的努力，但没有成功。脂质氧化 产品通过激活磷脂酰肌醇 3-激酶 (PI3K) 诱导 TRPC6 外化，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， 它促进 TRPC6 易位到细胞膜并导致 [Ca2+]i 增加。我们假设 抑制 PI3K 可以阻断脂质氧化产物对 TRPC6 通道的激活并恢复 EC 迁移 体外并促进体内动脉损伤的 EC 愈合。为了测试这一点，我们将 1) 识别 PI3K iso- TRPC6 激活所必需的形式，2) 研究异构体特异性 PI3K 抑制剂重新激活的有效性 储存 EC 对高胆固醇血症动物动脉损伤的愈合作用，以及 3) 通过以下方式研究其机制： 其中 PIP3 与 TRPC6 相互作用，促进 TRPC6 易位至膜并激活，从而识别 TRPC6抑制的更具体方法。长期科学目标是提高治疗结果 促进血管成形术部位、支架和血管移植物的内皮表面形成的血管介入治疗。 在研究职业发展方面，我的短期研究目标是为未来的研究奠定基础。 我可以在职业生涯中扩展的重点研究领域，并将作为对 我的临床实践。我对血管壁生物学的了解将通过研讨会增加，并且我将扩展我的知识 获奖期间细胞分子生物学实验室技术的经验，包括蛋白质 提取和分析、小抑制性 RNA 转染和定点诱变。整个过程中 通过该奖项，我将获得生物统计学、科学和资助写作、实验室管理方面的额外经验 以及领导力和指导。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转型为独立调查员并争夺 VA 优异奖 审查和 NIH 资助。我的长期研究目标是更清晰地定义其机制 活性氧和 TRPC 通道的激活抑制内皮细胞愈合。随着进展 在这一领域，可以开发基于机制的治疗方案，并将其转化为临床试验，并且 最终进入临床实践，以改善血管介入治疗后的长期结果。