The role of endothelial connexins in vascular wound repair

||项目摘要。 冠状动脉和隐静脉等血管的内皮损伤愈合受损是主要的 血管支架失败和致命的冠状动脉血栓形成的贡献者。识别延迟的机制 内皮修复是提高血管通畅性和患者存活率的关键。因此，这项提议 重点是确定正常内皮修复的调节因素，并了解这些因素是如何 当愈合受到损害时，就会失控。先前的研究表明，间隙连接蛋白43(Cx43)可能 调节伤口愈合。然而，连接蛋白43在内皮细胞(EC)修复中的具体作用尚不清楚。 该方案中的初步数据表明，结扎诱导的血管损伤促进了 EC Cx43在小鼠主动脉和颈动脉损伤区的表达。Cx43介导的 缝隙连接的细胞内通讯是由Cx43C末端的磷酸化控制的。我的初选 数据表明，Cx43丝氨酸(S)368的特异性磷酸化与缝隙连接关闭有关， 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。这些发现提供了Cx43表达和通道功能是 对于大动脉EC伤口的愈合至关重要。这项建议的目的是定义连接蛋白43在 内皮伤口愈合(目标1)，研究Cx43的缺失是否限制了体内的愈合(目标1)，并测试IF通道 功能，由翻译后修饰调节，改善/延迟血管修复(目标2)。这项调查 将使用一种新型的小鼠颈动脉内皮损伤存活手术在我开发的小鼠中完成，这将使 以评价Cx43在颈动脉内皮细胞愈合过程中的表达。无论是遗传还是 将使用药物策略改变小鼠损伤模型中Cx43的表达和磷酸化 在培养的人EC中，这些修饰对EC愈合的速度和质量的影响将是 量化的。此外，RNAseq方法将用于识别Cx43间隙连接依赖的信号转导 再生的内皮细胞。这些目标将在研究人员的指导下实现 在血管生物学方面拥有丰富的经验，Scott Johnstone博士、Robert Gourdie博士和Brant Isakson博士 弗吉尼亚理工大学的Fralin生物医学研究所(FBRI)。FBRI是研究设备的所在地，包括 但不限于动物设施、共焦显微镜、流式细胞仪和细胞培养设备。这种状态- 最先进的研究环境将允许进行详细和机械的调查。最终，这项研究将 描述Cx43在血管内皮细胞中的新作用。该项目旨在发现新的潜在治疗方法。 血管疾病的目标，将促进NIH改善患者健康的使命，并促进 更长的寿命，没有疾病。

|| 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.