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: 10369226
• 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/10369226
• 关键词: 1-Phosphatidylinositol 3-Kinase; 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 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; 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; base; cell motility; clinical practice; early onset; effectiveness testing; healing; high reward; human old age (65+); hypercholesterolemia; improved; improved outcome; in vivo; inhibitor; injured; kinase inhibitor; 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疾病，对长度和生活质量产生重大影响。 根据美国心脏协会的数据，约有1.215亿美国人进行诊断 心脏病。从年轻人开始，退伍军人的心血管疾病发生率明显更高， 与非退伍军人相比，患有更多心血管疾病的几率高42％。较高的比率 年轻时心血管发病率较高的退伍军人的心血管疾病 到早期的早期发作心血管死亡率。心脏和血管过程的数量（气球 预计将在2040年进行的血管成形剂和血管移植）是数量的两倍以上 在2008年进行的，需要再干预的再狭窄发生在30-75％的手术中，具体取决于 治疗区。进行类似的血管手术数量和类似的再狭窄率增加 发生在退伍军人人口中。 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。 已经努力识别特定的TRPC6抑制剂而没有成功的努力。非- 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。我们有 发现脂质氧化产物通过激活磷脂酰肌醇3-- 激酶（PI3K），产生磷脂酰肌醇（3,4,5） - 三磷酸盐（PIP3）。 PIP3锚定在电池中 膜并促进TRPC6转运到细胞膜，并导致[Ca2+] i。基于 我们正在进行的研究，PIP3和TRPC6之间的相互作用是由衔接蛋白介导的，以及 初步数据表明，这是与GRB2相关的结合蛋白1（GAB1）。重要的是，对此的识别 适配器建议一种阻止TRPC6激活并恢复EC迁移的方法，同时最大程度地减少目标 PI3K抑制剂或非选择性TRPC抑制作用的影响。我们通过 脂质氧化产物需要适配蛋白与膜中的TRPC6和PIP3相互作用。 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 在脂质氧化产物的情况下。为了测试这一点，我们将1）确定介导PI3K-的机制 质膜中TRPC6的PIP3锚定，特别是适配器蛋白的作用， 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的热血管干预的结果 血管成形术部位，支架和血管移植物的表面。随着该领域的进展，基于机制的 可以开发治疗方案，转变为临床试验，并最终将其用于临床试验 练习血管干预后改善长期结局并改善退伍军人的生活质量。