The role of endothelial connexins in vascular wound repair

内皮连接蛋白在血管伤口修复中的作用

• 批准号: 10751730
• 负责人: Meghan Sedovy
• 金额: $ 4.25万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2026-02-09
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10751730
• 关键词: Alanine; Antibodies; Aorta; Area; Arteries; Biological; Biology; Biomedical Research; Blood Vessels; C-terminal; Carotid Arteries; Cell Communication; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell Separation; Cells; Cessation of life; Communication; Confocal Microscopy; Connexin 43; Connexins; Coronary Thrombosis; Coronary artery; Data; Data Analyses; Defect; Deoxyuridine; Disease; Endothelial Cells; Endothelium; Ensure; Environment; Equipment; Failure; Fellowship; Female; Gap Junctions; Genetic; Goals; Health; Heart; Home; Hour; Human; Immunofluorescence Immunologic; Impaired healing; Impairment; Injections; Injury; Investigation; Knock-out; Knockout Mice; Legal patent; Life; Ligation; Link; Measures; Mediating; Mentorship; Microscope; Mission; Modeling; Modernization; Modification; Mus; Mutation; Myocardial Infarction; Operative Surgical Procedures; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Inhibition; Phosphorylation Site; Phosphotransferases; Plasmids; Post-Translational Protein Processing; Preparation; Process; Proliferating; Proliferation Marker; Proto-Oncogene Proteins c-akt; Research; Research Institute; Research Personnel; Role; Saphenous Vein; Serine; Signal Pathway; Signal Transduction; Site; Stents; Survival Rate; Technical Expertise; Techniques; Testing; Therapeutic; Thrombus; Tissues; Training; Transfection; Travel; United States National Institutes of Health; Universities; Up-Regulation; Vascular Diseases; Vascular Endothelial Cell; Vascular Graft; Virginia; Western Blotting; Work; animal facility; burden of illness; cell growth; cell injury; cell motility; design; endothelial regeneration; endothelial repair; experience; experimental study; healing; improved; in vivo; in vivo Model; injured; male; migration; mimetics; novel; pharmacologic; protein expression; repaired; response; restenosis; sex; stroke risk; therapeutic target; transcriptome sequencing; vascular injury; wound closure; wound healing

|| Project Summary . Impaired endothelial wound healing in blood vessels like the coronary artery and saphenous vein is a primary contributor to vascular stent failure and deadly coronary thrombosis. Identifying the mechanism of delayed endothelial repair is essential for improving vessel patency and patient survival rates. As such, this proposal focuses on identifying regulators of normal endothelial healing and understanding how these factors are dysregulated when healing is impaired. Previous studies have shown connexin 43 (Cx43) gap junctions may regulate wound healing. However, the specific role of connexin 43 in endothelial cell (EC) healing is unknown. Preliminary data in this proposal indicates that ligation-induced vascular injury promotes increases in the expression of EC Cx43 surrounding the damaged area in the aorta and carotid arteries in mice. Cx43-mediated gap junction intracellular communication is controlled by phosphorylation at the Cx43 C-terminus. My preliminary data demonstrate that specific Cx43 phosphorylation at its serine (s) 368, associated with gap junction closure, is present during the final stages of EC wound healing in vivo. Cx43-s368 also reduced the rate of wound closure in cultured human EC. These findings inform the hypothesis that Cx43 expression and channel functions are critical for EC wound healing in large arteries. The aims of this proposal are to define the role of connexin 43 in endothelial wound healing (Aim 1), investigate if a loss of Cx43 limits healing in vivo (Aim 1), and test if channel functions, regulated by posttranslational modifications, improve/delay vascular repair (Aim 2). This investigation will be completed using a novel mouse carotid EC injury survival surgery in mice I developed, which will allow for the assessment of Cx43 expression in carotid EC during the healing process. Both genetic and pharmacological strategies will be used to alter Cx43 expression and phosphorylation in mouse injury models and in cultured human EC, and the impact of these modifications on the rate and quality of EC healing will be quantified. Additionally, RNAseq approaches will be used to identify Cx43 gap junction-dependent signaling in the regenerating endothelium. These aims will be accomplished under the mentorship of researchers with extensive experience in vascular biology, Dr. Scott Johnstone, Dr. Robert Gourdie, and Dr. Brant Isakson, at Virginia Tech’s Fralin Biomedical Research Institute (FBRI). The FBRI is home to research equipment including but not limited to animal facilities, confocal microscopes, flow cytometers, and cell culture equipment. This state- of-the-art research environment will allow for a detailed and mechanistic investigation. Ultimately, this study will characterize a novel role for Cx43 in vascular EC. This project is designed to identify new potential therapeutic targets in vascular disease that will promote the mission of the NIH to enhance patient health and to promote longer life free of illness.

|| 项目摘要。 冠状动脉和大隐静脉等血管的内皮损伤愈合受损是主要的 导致血管支架失效和致命的冠状动脉血栓形成。识别延迟的机制 内皮修复对于改善血管开放性和患者存活率是必不可少的。因此，本提案 重点是确定正常内皮愈合的调节因子，并了解这些因子是如何 当愈合受损时，调节失调。先前的研究表明，连接蛋白43（Cx43）间隙连接可能 调节伤口愈合。然而，连接蛋白43在内皮细胞（EC）愈合中的具体作用尚不清楚。 该提案中的初步数据表明，结扎诱导的血管损伤促进了 小鼠主动脉和颈动脉损伤区周围EC Cx43的表达。Cx43介导 间隙连接细胞内通讯由Cx43 C-末端的磷酸化控制。我的初步 数据表明，与间隙连接闭合相关的， 存在于体内EC伤口愈合的最后阶段。Cx43-s368还降低了伤口闭合率 在培养的人EC中。这些发现提示了Cx43表达和通道功能是 对于大动脉中的EC伤口愈合至关重要。该提议的目的是确定连接蛋白43在以下方面的作用： 内皮伤口愈合（目标1），研究Cx43的缺失是否限制体内愈合（目标1），并测试通道 通过翻译后修饰调节的功能，改善/延迟血管修复（目的2）。这次调查 将使用我开发的一种新型小鼠颈动脉EC损伤存活手术来完成， 用于评估愈合过程中颈动脉EC中Cx43的表达。遗传和 药理学策略将用于改变小鼠损伤模型中Cx43的表达和磷酸化 以及在培养的人EC中，这些修饰对EC愈合的速率和质量的影响将是 量化。此外，RNAseq方法将用于鉴定Cx43间隙连接依赖性信号传导， 再生的内皮细胞这些目标将在研究人员的指导下实现， Scott Johnstone博士、Robert Gourdie博士和Brant Isakson博士在血管生物学方面拥有丰富的经验， 弗吉尼亚理工大学Fralin生物医学研究所（FBRI）。FBRI是研究设备的所在地， 但不限于动物设施、共聚焦显微镜、流式细胞仪和细胞培养设备。这个州- 最先进的研究环境将允许详细和机械的调查。最终，这项研究将 描述了Cx43在血管EC中的新作用。该项目旨在确定新的潜在治疗方法， 血管疾病的目标，这将促进国家卫生研究院的使命，以提高病人的健康和促进 更长的寿命免于疾病。