Gap Junction Mediated Signaling and Vascular Responses

描述（由申请人提供）：该项目的长期目标是了解间隙连接介导的通讯在调节血管反应中的作用，并确定特定间隙连接蛋白的贡献。间隙连接是由连接蛋白（Cx）组成的细胞间通道簇，连接相邻细胞的细胞质，允许小分子直接在细胞间转移。Cx表达和功能的改变与高血压、缺血、炎症、动脉粥样硬化、心房颤动和心力衰竭有关。此外，Cx突变或其他基因突变引起的Cx表达改变可能是导致心脏和血管系统先天性缺陷的原因。本项目利用敲除小鼠缺乏特定血管Cxs的可用性来研究Cx在脉管系统中的功能。在第一个特定目标中，研究了Cx37和Cx40在血管发育和血管生成中的作用。多种方法用于探索缺乏Cx37和Cx40的小鼠异常血管形成的机制，包括使用内皮细胞特异性报告基因，细胞增殖试验和凋亡试验。制备原代内皮细胞培养物，直接比较在存在或不存在特异性Cxs的情况下的增殖和凋亡。在细胞培养中也检测了外源表达xs对细胞生长和凋亡的影响。使用体内实验和外植体方法比较了cx缺陷小鼠和野生型小鼠的血管生成反应。第二个目的是利用电生理和生化方法确定Cx37和Cx40是否在血管内皮细胞中形成混合成分和独特功能特性的通道。异质通道可能在决定血管发育、血管生成反应和血管舒缩反应过程中传递的信号类型方面发挥重要作用。了解Cx缺陷小鼠的表型可能有助于了解人类先天性血管缺陷，也可能阐明调节正常血管发育的过程。更好地了解血管x射线可能会带来新的见解，治疗和预防先天性血管缺陷和心血管疾病。最后，这些研究还可能导致在其他重要临床环境中控制血管生成和血管生成的新方法，例如用于对抗癌症的抗血管生成治疗。

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the role that gap junction-mediated communication plays in regulating vascular responses and to define the contributions of specific gap junction proteins. Gap junctions are clusters of intercellular channels, comprised of connexin (Cx) proteins, that connect the cytoplasm of adjacent cells, allowing the direct cell-to-cell transfer of small molecules. Alterations in Cx expression and function have been correlated with hypertension, ischemia, inflammation, atherosclerosis, atrial fibrillation, and heart failure. In addition, Cx mutations or altered Cx expression caused by other gene mutations could be responsible for congenital defects of the heart and vasculature. This project takes advantage of the availability of knockout mice lacking specific vascular Cxs to study Cx function in the vasculature. In the first specific aim, the role of Cx37 and Cx40 in vascular development and angiogenesis is investigated. A variety of methods are used to explore the mechanism of abnormal vessel formation in mice lacking both Cx37 and Cx40, including use of an endothelial cell-specific reporter gene, cell proliferation assays, and apoptosis assays. Primary endothelial cell cultures are prepared to directly compare proliferation and apoptosis in the presence or absence of specific Cxs. The effect of exogenous expression of Cxs on cell growth and apoptosis is also examined in cell cultures. Angiogenic responses in Cx-deficient and wild-type mice are compared using both an in vivo assay and an explant approach. A second aim is to determine if Cx37 and Cx40 form channels of mixed composition and unique functional properties in vascular endothelial cells, using electrophysiological and biochemical methods. Heteromeric channels could play an important role in determining the types of signals that are communicated during vascular development, angiogenic responses, and vasomotor responses. Understanding the phenotypes of Cx deficient mice may provide insight into congenital vascular defects in humans and could also shed light on the processes that regulate normal vascular development. A better understanding of vascular Cxs could lead to new insights, treatments, and prevention for congenital vascular defects and cardiovascular disease. Finally, these studies could also lead to new approaches to control vasculogenesis and angiogenesis in other clinically important settings, such as anti-angiogenesis treatments designed to fight cancer.