The role of Cx43 Carboxyl-terminus in the regulation of endothelial barrier function

项目总结/摘要 大多数形式的心脏病都与心脏内皮屏障的破坏有关， 损害性渗漏和血管与心脏组织之间的液体积聚（也称为心脏水肿）。 治疗主要集中在作为治疗靶点的心肌组织上，但内皮细胞，其起着重要的作用。 在心脏健康中的重要作用，一直被忽视。多项研究表明， Cx43在内皮屏障功能和通透性中的作用。初步数据显示， (CT)的Cx43与紧密连接支架蛋白闭锁小带1（ZO-1）以及天然的 CX43本身。此外，有证据表明，内皮细胞可能产生自由作用的小CT， 片段作为一种内源性机制，以自我调节屏障性能。使用经典和自动化 在体外测量细胞单层电阻/阻抗的方法中，我们证明α CT 1，一个Cx43 CT模拟肽可防止VEGF介导的屏障功能破坏，并稳定细胞间紧密连接。 视网膜相关黄斑变性（AMD）模型。此外，利用电细胞-基底阻抗 传感（ECIS），一种自动化的，基于阻抗的方法，用于研究在培养中生长的细胞的活动， 真实的时间，我们确定了在人微血管内皮细胞系中， （HMEC 1）。我们实验室最近的实验表明， 出现α CT 1样Cx43 CT片段，其可能包装在小的细胞外囊泡中，称为 外来体我们的首要目标是确定内皮细胞如何利用内源性产生的 α CT 1样片段影响屏障功能。我们的具体目标是：1）检验Cx43 CT 模拟肽通过与1）ZO-1和/或2）Cx43和2）测试假设的相互作用来保护屏障功能 内皮细胞产生天然存在的α CT 1样Cx43 CT片段。Cx43的外源应用 CT模拟肽、α CT 1和变体（不能结合ZO-1和/或Cx43）将用作 作为一种工具，指导我们了解内源性Cx43的稳态功能和机制及其与 有约束力的伙伴Western印迹、免疫沉淀、共聚焦显微镜、外泌体分离、冷冻电镜 将采用显微镜和串联质谱法来确定是否存在α CT 1样 来源于表达野生型Cx43和Cx43-eGFP的Hela细胞系的外来体中的Cx43 CT片段 表达HMEC 1的细胞系，以及来自离体心脏制备物。了解Cx43 CT如何影响 屏障功能可能是推进新的内皮屏障稳定疗法治疗心脏病的关键 水肿该项目的培训将利用弗吉尼亚理工大学的核心设施和资源共享 弗吉尼亚州罗阿诺克的研究所和弗吉尼亚理工大学布莱克斯堡的主校区。一批高 弗吉尼亚理工大学的经验丰富的学者，以及弗吉尼亚理工大学以外的机构，准备帮助 通过提供培训和其他关键资源来执行这一为期3年的项目。

Project Summary/Abstract Most forms of heart disease are associated with a breakdown of the cardiac endothelial barrier, resulting in a damaging leakage and buildup of fluid between blood vessels and the heart tissue (also known as cardiac edema). Treatment has largely focused on heart muscle tissue as a therapeutic target but the endothelium, which plays a critical role in the health of the heart, has been neglected. Multiple studies have demonstrated the involvement of Cx43 in endothelial barrier function and permeability. Preliminary data shows that the Carboxyl-terminus (CT) of Cx43 interacts with both tight junction scaffolding protein Zonula Occludens 1(ZO-1) as well as native Cx43 itself. Furthermore, evidence suggests that endothelial cells might generate freely acting small CT fragments as an endogenous mechanism to self-regulate barrier properties. Using both classical and automated in vitro methods of measuring resistance/impedance across cell monolayers, we demonstrated that αCT1, a Cx43 CT mimetic peptide, prevented VEGF-mediated breakdown of barrier function and stabilized tight junctions in a model of Age-Related Macular Degeneration (AMD). Moreover, utilizing Electric Cell-substrate Impedance Sensing (ECIS), an automated, impedance-based method used to study the activities of cells grown in culture in real time, we determined that ɑCT1 recovered barrier function in a human microvascular endothelial cell line (HMEC1). Recent experiments from our lab indicated the presence of hitherto uncharacterized, naturally occurring αCT1-like Cx43 CT fragments, which might be packaged within small, extracellular vesicles called exosomes. Our overarching goal is to determine how endothelial cells might utilize an endogenously generated αCT1-like fragment to affect barrier function. Our specific aims are to: 1) Test the hypothesis that Cx43 CT mimetic peptides protect barrier function via interaction with: 1) ZO-1 and/or 2) Cx43 and 2) Test the hypothesis that endothelial cells generate naturally occurring αCT1-like Cx43 CT fragments. Exogenous application of Cx43 CT mimetic peptides, αCT1 and variants (that are either ZO-1 and/or Cx43-binding incompetent) will be used as a tool, to instruct us on the homeostatic functions and mechanisms of endogenous Cx43 and its interactions with binding partners. Western blotting, immunoprecipitation, confocal microscopy, exosome isolation, cryo-electron microscopy and tandem mass spectroscopy will be employed to determine the presence or absence of αCT1-like Cx43 CT fragments in exosomes derived from Cx43-eGFP Hela cell line expressing wildtype Cx43 and a Cx43- expressing HMEC1 cell line, as well as from ex vivo cardiac preparations. Understanding how the Cx43 CT affects barrier function could be key to advancing novel endothelial barrier stabilizing therapeutics for treating cardiac edema. Training for this project will utilize core facilities and resources shared across Virginia Tech Carilion Research Institute in Roanoke, VA and Virginia Tech's main campus in Blacksburg, VA. A number of highly experienced academics at Virginia Tech, and institutions outside of Virginia Tech, are prepared to aid in the execution of this 3-year project by providing training and other key resources.