Endothelial Healing is Inhibited by Activation of TRPC6 Channels

TRPC6 通道的激活会抑制内皮愈合

• 批准号: 10526285
• 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: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10526285
• 关键词: Adaptor Signaling Protein; Age; Aging; American; American Heart Association; Angioplasty; Area; Arterial Injury; Arteries; Balloon Angioplasty; Binding Proteins; Blood Vessels; Calcium; Calcium ion; Calpain; Cardiovascular Diagnostic Techniques; Cardiovascular Diseases; Cardiovascular system; Carotid Artery Injuries; Cell Proliferation; Cell membrane; Cells; Cicatrix; Clinical Trials; Coagulation Process; Cytoskeletal Proteins; Data; Diagnosis; Disease; Effectiveness; Endothelial Cells; Endothelium; Functional disorder; Goals; Heart; Heart Diseases; High Fat Diet; Hyperplasia; In Vitro; Injury; Intervention; Knock-out; Length; Life; Link; Lipids; Lysophosphatidylcholines; Mediating; Membrane; Morbidity - disease rate; Mus; Operative Surgical Procedures; Outcome; PIK3CG gene; Persons; Phosphatidylinositols; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Population; Procedures; Quality of life; Role; Site; Site-Directed Mutagenesis; Small Interfering RNA; Smooth Muscle Myocytes; Stents; Surface; TRP channel; Testing; Thrombosis; Treatment Protocols; United States; Vascular Diseases; Vascular Graft; Veterans; Wild Type Mouse; atherogenesis; cell motility; clinical practice; early onset; effectiveness testing; healing; high reward; human old age (65+); hypercholesterolemia; improved; improved outcome; in vivo; inhibitor; injured; inorganic phosphate; kinase inhibitor; migration; military veteran; mortality; oxidation; oxidized lipid; oxidized low density lipoprotein; preservation; prevent; protein activation; receptor; restenosis; src-Family Kinases; success; therapeutic target; therapy outcome; thrombogenesis; voltage gated channel

Cardiovascular disease is a devastating disorder that has a major impact on length and quality of life. According to the American Heart Association, approximately 121.5 million Americans carry the diagnosis of heart disease. Veterans have significantly higher rates of cardiovascular disease starting at younger ages and have 42% higher odds of having more cardiovascular diseases compared to non-veterans. Higher rates of cardiovascular disease in veterans with a higher likelihood of cardiovascular morbidity at a younger age leads to early onset cardiovascular mortality later in life. The number of heart and vascular procedures (balloon angioplasties and vascular grafts) that will be performed in 2040 is expected to be more than twice the number performed in 2008 and restenosis requiring reintervention occurs in 30-75% of procedures depending on the treatment area. Similar increases in number of vascular procedures performed and similar restenosis rates 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 specific TRPC6 inhibitor without success. Non- selective TRPC inhibitors have developed, but they impact TRPC3, TRPC6, and TRPC7 channels. We have discovered that lipid oxidation products induce TRPC6 externalization by activating phosphatidylinositol 3- kinase (PI3K), which generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 is anchored in the cell membrane and promotes TRPC6 translocation to the cell membrane and leads to increased [Ca2+]i. Based on our ongoing studies, the interaction between PIP3 and TRPC6 is mediated by an adaptor protein, and preliminary data suggest that this is Grb2-associated binding protein 1 (Gab1). Importantly, identification of this adaptor suggests a way to block TRPC6 activation and to restore EC migration, while minimizing off-target effects of PI3K inhibitors or non-selective TRPC channel inhibition. We hypothesize that TRPC6 activation by lipid oxidation products requires the interaction of an adaptor protein to link TRPC6 and PIP3 in the membrane. As a corollary, inhibition of the TRPC6-PIP3 interaction can block TRPC6 activation and restore EC migration in the presence of lipid oxidation products. To test this, we will 1) identify the mechanism that mediates PI3K- generated PIP3 anchorage of TRPC6 in the plasma membrane, specifically the role of an adaptor protein, and 2) determine the mechanism of lysoPC-induced, adaptor protein-mediated TRPC6-PIP3 interaction. The long-term goal is to improve the outcome of therapeutic vascular interventions promoting endothelial surfacing of angioplasty sites, stents, and vascular grafts. 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 and improve veterans’ quality of life.

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。 根据美国心脏协会的数据，大约 1.215 亿美国人患有以下疾病： 退伍军人从年轻时开始患心血管疾病的几率明显更高。 与非退伍军人相比，患有更多心血管疾病的几率高出 42%。 退伍军人的心血管疾病在年轻时发病的可能性较高 心脏和血管手术（球囊）的数量。 预计 2040 年将进行的血管成形术和血管移植手术数量将增加两倍以上 2008 年进行的手术中，30-75% 的手术出现需要重新干预的再狭窄，具体情况取决于 治疗区域的血管手术数量也有类似的增加，再狭窄率也类似。 发生在退伍军人群体中。 当用血管成形术治疗血管时，必须去除内皮细胞（EC）。 从伤口边缘迁移到受伤区域来治愈它，如果愈合延迟，则有机会治愈。 再狭窄增加，脂质氧化产物在动脉粥样硬化动脉和损伤区域积聚， 导致细胞功能障碍，并在体外和体内抑制 EC 迁移。 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 的激活。 导致 [Ca2+]i 增加，从而激活 TRPC5 并延长 [Ca2+]i 增加。 [Ca2+]i 增加会激活钙蛋白酶，分解细胞骨架蛋白，抑制 EC 迁移。 TRPC6-/- 小鼠提供了体内再内皮化级联重要性的令人信服的证据。 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。 为了鉴定特定的 TRPC6 抑制剂，人们付出了相当大的努力，但没有成功。 选择性 TRPC 抑制剂已经开发出来，但它们会影响 TRPC3、TRPC6 和 TRPC7 通道。 发现脂质氧化产物通过激活磷脂酰肌醇 3- 诱导 TRPC6 外化 激酶 (PI3K)，产生磷脂酰肌醇 (3,4,5)-三磷酸 (PIP3)。 膜并促进 TRPC6 易位至细胞膜并导致 [Ca2+]i 增加。 我们正在进行的研究表明，PIP3 和 TRPC6 之间的相互作用是由接头蛋白介导的，并且 初步数据表明，这是 Grb2 相关结合蛋白 1 (Gab1)。 适配器提出了一种阻止 TRPC6 激活并恢复 EC 迁移的方法，同时最大限度地减少脱靶 我们通过 PI3K 抑制剂或非选择性 TRPC 通道抑制来突袭 TRPC6 激活。 脂质氧化产物需要接头蛋白的相互作用来连接膜中的 TRPC6 和 PIP3。 作为推论，抑制 TRPC6-PIP3 相互作用可以阻止 TRPC6 激活并恢复 EC 迁移 为了测试这一点，我们将 1) 确定介导 PI3K- 的机制。 生成 TRPC6 在质膜中的 PIP3 锚定，特别是衔接蛋白的作用，以及 2)确定lysoPC诱导的衔接蛋白介导的TRPC6-PIP3相互作用的机制。 长期目标是改善促进内皮细胞生长的治疗性血管干预的结果 随着这一领域的进展，基于机制的血管成形术部位、支架和血管移植物的表面处理。 可以开发治疗方案，过渡到临床试验，并最终实施到临床 实践以改善血管介入治疗后的长期结果并提高退伍军人的生活质量。