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

项目摘要/摘要 大多数形式的心脏病与心脏内皮屏障的破坏有关，导致 血管和心脏组织之间液体的破坏性渗漏和积聚(也称为心源性水肿)。 治疗在很大程度上集中在心肌组织作为治疗目标，但内皮，它发挥着 在心脏健康中的关键作用，一直被忽视。多项研究已经证明了这一点 Cx43在内皮屏障功能和通透性中的作用。初步数据显示，羧基末端 Cx43的(CT)与紧密连接支架蛋白ZO-1(ZO-1)以及天然蛋白相互作用 Cx43本身。此外，有证据表明，内皮细胞可能会产生自由作用的小CT 片段作为自我调节屏障属性的内源性机制。同时使用经典和自动 在测量细胞单层电阻/阻抗的体外方法中，我们证明了αCT1，一种Cx43 CT模拟肽，阻止血管内皮生长因子介导的屏障功能破坏并稳定紧密连接 老年性黄斑变性(AMD)模型。此外，利用电池-衬底阻抗 传感(ECIS)，一种基于阻抗的自动化方法，用于研究在培养条件下生长的细胞的活动 实时，我们确定ɑCT1在人微血管内皮细胞系中恢复了屏障功能 (HMEC1)。我们实验室最近的实验表明，到目前为止还没有特征的，自然存在 出现αCT1样Cx43CT片段，这些片段可能被包装在称为 外显体。我们的首要目标是确定内皮细胞如何利用内源性产生的 αCT1样片段影响屏障功能。我们的具体目标是：1)检验Cx43 CT的假设 模拟多肽通过与以下物质相互作用保护屏障功能：1)ZO-1和/或2)Cx43和2)检验假设 内皮细胞产生自然产生的αCT1样Cx43CT片段。Cx43的外源应用 CT模拟多肽、αCT1和变异体(ZO-1和/或Cx43结合不能)将被用作 一个工具，指导我们内源性Cx43的动态平衡功能和机制及其与 有约束力的伙伴。蛋白质印迹、免疫沉淀、共聚焦显微镜、外切小体分离、冷冻电子 显微镜和串联质谱学将被用来确定是否存在αCT1样 表达野生型Cx43和A型Cx43的Hela细胞系Cx43-EGFP细胞外体中的Cx43 CT片段 表达HMEC1细胞系，以及来自体外心脏制剂。了解Cx43 CT如何影响 屏障功能可能是推动新型内皮屏障稳定疗法治疗心脏疾病的关键 浮肿。该项目的培训将利用弗吉尼亚理工大学Carilion共享的核心设施和资源 弗吉尼亚州罗阿诺克的研究学院和弗吉尼亚理工大学位于弗吉尼亚州布莱克斯堡的主校区。多个高度 弗吉尼亚理工大学经验丰富的学者和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，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.