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，大约有1.215亿美国人被诊断患有心脏病。 心脏病退伍军人在年轻时患心血管疾病的比率明显较高， 与非退伍军人相比，患心血管疾病的几率高出42%。率较高 在年轻时心血管疾病发病率较高的退伍军人中， 早期心血管疾病的死亡率。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术的数量将是2010年的两倍多。 在2008年进行，根据不同的手术，30-75%的手术发生需要再次介入的再狭窄。 治疗区。进行的血管手术数量增加相似，再狭窄率相似 发生在退伍军人中。 当用血管成形术治疗血管时，内皮细胞（EC）被去除。细胞必须 从伤口边缘迁移到伤口区域进行愈合。如果愈合延迟， 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，并抑制EC迁移在体外和体内。有限的再内皮化 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（[Ca 2 +]i）的不适当增加 通过典型瞬时受体电位（TRPC）通道，特别是TRPC 6。TRPC 6的激活 导致[Ca 2 +]i增加，导致TRPC 5活化和[Ca 2 +]i延长增加。的 增加的[Ca 2 +]i激活钙蛋白酶，钙蛋白酶分解抑制EC迁移的细胞骨架蛋白。研究 TRPC 6-/-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。再内皮化 与普通食物相比，高脂肪饮食的野生型（WT）小鼠中损伤的颈动脉显著减少， 喂食的小鼠中，但在TRPC 6-/-小鼠中，高胆固醇血症不抑制损伤的再内皮化。 相当大的努力已经指向鉴定特异性TRPC 6抑制剂，但没有成功。非 已经开发出选择性TRPC抑制剂，但它们影响TRPC 3、TRPC 6和TRPC 7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（PI 3 K），其产生磷脂酰肌醇（3，4，5）-三磷酸（PIP 3）。PIP 3锚定在细胞中 细胞膜并促进TRPC 6转运至细胞膜并导致[Ca 2 +]i增加。基于 我们正在进行的研究表明，PIP 3和TRPC 6之间的相互作用是由衔接蛋白介导的， 初步数据表明这是Grb 2相关结合蛋白1（Gab 1）。重要的是， adaptor提出了一种阻断TRPC 6激活并恢复EC迁移的方法，同时最小化脱靶 PI 3 K抑制剂或非选择性TRPC通道抑制的作用。我们假设TRPC 6的激活是由 脂质氧化产物需要衔接蛋白的相互作用以连接膜中的TRPC 6和PIP 3。 作为推论，抑制TRPC 6-PIP 3相互作用可阻断TRPC 6活化并恢复EC迁移 在脂质氧化产物的存在下。为了验证这一点，我们将1）确定介导PI 3 K的机制- 产生TRPC 6在质膜中的PIP 3锚定，特别是衔接蛋白的作用，和 2)确定lysoPC诱导的、衔接蛋白介导的TRPC 6-PIP 3相互作用的机制。 长期目标是改善血管介入治疗的结果，促进血管内皮细胞的生长。 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， 可以开发治疗方案，过渡到临床试验，并最终进行临床试验。 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。