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疾病，对长度和生活质量产生重大影响。 根据疾病预防控制中心，大约有2700万美国人对心脏病进行诊断。数量 将在2030年进行的心脏和血管手术（气球血管成形剂和血管移植物） 被认为是2010年进行的数量的几乎是两倍。 当血管用血管成形术处理时，去除内皮细胞（EC）。细胞必须 从受伤的边缘迁移到伤害区域以治愈它。如果愈合延迟， 再狭窄增加。脂质氧化产物积累在动脉粥样硬化动脉和损伤区域中， 引起细胞功能障碍，并在体外和体内抑制EC迁移。有限的重新皮层化贡献 血栓形成，平滑肌细胞增殖和再狭窄。 氧化脂质会导致细胞内游离钙离子浓度不当（[CA2+] I） 通过规范瞬态受体电位（TRPC）通道，特别是TRPC6。 TRPC6激活 导致[Ca2+] i的增加导致TRPC5的激活并长时间增加[Ca2+] i。这 增加[Ca2+] I激活钙蛋白酶，破坏抑制EC迁移的细胞骨架蛋白。研究 TRPC6 - / - 小鼠提供了令人信服的证据，证明了该级联体内的重要性。重新皮层化 与Chow-Chow- 喂养小鼠，但在TRPC6 - / - 小鼠中，高胆固醇血症不会抑制损伤的重新皮层化。 已经努力识别TRPC6抑制剂而没有成功的努力。脂质氧化 产物通过激活磷脂酰肌醇3-激酶（PI3K）诱导TRPC6外部化 PIP3（磷脂酰肌醇（3,4,5） - 三磷酸盐）。 PIP3锚定在细胞膜中，与TRPC6结合， 促进TRPC6转运到细胞膜，并导致[Ca2+] i。我们假设这一点 PI3K的抑制可以通过脂质氧化产物阻止TRPC6通道的激活，并恢复EC迁移 体外体外促进动脉损伤的EC愈合。要测试这一点，我们将1）确定PI3K ISO- TRPC6激活必不可少的形式，2）研究同工型特异性PI3K抑制剂对重复的有效性 将EC的EC治愈在高胆固醇动物中的动脉损伤，3）通过 哪个PIP3与TRPC6相互作用，以促进TRPC6易位到膜并激活以识别 TRPC6抑制的更具体的方法。长期科学目标是改善治疗的结果 促进血管成形术部位，支架和血管移植的内皮表面的猿类血管干预措施。 就研究职业发展而言，我的短期研究目标是在 我可以在职业生涯中扩展的重点研究领域，并将作为补充 我的临床实践。我对血管墙生物学的了解将通过研讨会增加，我将扩大我的 在奖励过程中，在细胞分子生物学实验室技术方面具有经验，包括蛋白质 提取和分析，小型抑制性RNA转染和位置定向诱变。在整个过程中 该奖项，我将获得生物统计学，科学和赠款写作，实验室管理的额外经验 和领导和指导。我还将继续发展所需的批判性思维技能 成功的研究计划。 我的主要长期职业目标是过渡到独立调查员并竞争VA功绩 评论和NIH资金。我的长期研究目标是更清楚地定义 活性氧和TRPC通道的激活抑制内皮细胞愈合。随着进步 可以制定基于机制的治疗方案，过渡到临床试验，并 最终，进行临床实践，以改善血管干预后的长期结局。