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.

心血管疾病是一种破坏性的疾病，对生活时间和质量有重大影响。 根据美国心脏协会的数据，大约1.215亿美国人被诊断为 心脏病。退伍军人从年轻开始患心血管疾病的几率明显更高 与非退伍军人相比，患更多心血管疾病的几率高出42%。更高的 心血管疾病发病率较低的退伍军人的心血管疾病 到晚年心血管疾病的早发性死亡。心脏和血管手术的数量(气球 血管成形术和血管移植)预计将是2040年的两倍多 30-75%的手术中发生再狭窄需要重新介入取决于 治疗区域。进行血管手术的次数和相似的再狭窄率相似的增加 发生在退伍军人群体中。 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。 在确定特定的TRPC6抑制剂方面已经做出了相当大的努力，但没有成功。非- 选择性的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-来诱导TRPC6外化。 激酶(PI3K)，产生磷脂酰肌醇(3，4，5)-三磷酸(PIP3)。PIP3被锚定在细胞内 并促进TRPC6移位到细胞膜上，导致[钙]i增加。 我们正在进行的研究中，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)。重要的是，确认这一点 Adaptor建议了一种阻止TRPC6激活和恢复EC迁移的方法，同时将偏离目标的情况降至最低 PI3K抑制剂或非选择性TRPC通道抑制的作用。我们假设TRPC6的激活是通过 脂质氧化产物需要接头蛋白的相互作用来连接膜上的TRPC6和PIP3。 因此，抑制TRPC6-PIP3的相互作用可以阻断TRPC6的激活，恢复EC的迁移 在存在脂类氧化产物的情况下。为了测试这一点，我们将1)确定调节PI3K的机制- 产生TRPC6在质膜上的PIP3锚定，特别是接头蛋白的作用，以及 2)确定溶菌肽诱导、接头蛋白介导的TRPC6-PIP3相互作用的机制。 长期目标是改善促进血管内皮细胞生长的治疗性血管干预的结果 血管成形术部位、支架和血管移植物的表面处理。随着这一领域的进展，以机制为基础 治疗方案可以被开发，过渡到临床试验，并最终应用于临床 实践，以改善血管介入治疗后的长期结果，提高退伍军人的生活质量。