Gap Junctions in Vascular Smooth Muscle: Growth Control

项目说明 这项研究的长期目标是：1)了解支配细胞生长的分子机制 血管细胞缝隙连接的通透性和生长抑制特性及其发展 了解以内皮细胞连接蛋白表达为靶点的基因治疗的基本原理 限制对缺血性损伤的敏感性并促进其恢复。三种缝隙连接蛋白是 通常在血管细胞中表达，连接蛋白(Cx)37、Cx40和Cx43；在内皮中表达Cx37和Cx40 Cx37在正常情况下占主导地位，但在血管生成过程中，以及在应激、损伤和疾病中，Cx37下调 Cx43表达上调。这一表述变化对船舶形成新的能力的影响 在血管重塑期间，血管和维持血管功能仍不确定。在我们之前的研究中， 我们证明了这些连接蛋白形成的缝隙连接通道具有截然不同的渗透率和选择性。 受生长因子激活的信号调节的生长抑制特性在连接蛋白- 具体的方式。在目前的方案中，我们假设连接蛋白特异的、磷酸化依赖的 连接膜选择性的调节为血管细胞维持协调提供了一种策略 血管的收缩/松弛功能，同时支持细胞的增殖反应 在那里。我们在目标1中通过研究磷酸化的机制基础来解决这一假设 羧基末端结构域(CT)中的事件导致相关孔的渗透性选择性改变 域。在目标2中，我们扩展了目标1的观察，并确定了生长抑制 这些连接蛋白的特性取决于它们的通透性选择特性和/或它们与蛋白质的直接相互作用 参与细胞周期控制和进展。这些生长研究利用了缺乏Cx的细胞系， 从野生型或Cx37缺陷小鼠分离的内皮细胞，以及体内后肢缺血损伤模型， 询问Cx37是否与Cx43协同或相反地调节血管生成反应 由伤害诱导，同时保持连接的渗透性选择特性。电生理学和电学的结合 荧光显微镜将被用来量化连接和分子的渗透选择特性 将使用方法来确定连接蛋白结构域和连接蛋白之间相互作用的基本区域/位置 在细胞周期机制的连接蛋白和元件之间。分离内皮细胞与体内缺血 模型将用于确定连接蛋白表达或沉默的程度和速度的益处 损伤后的血管重塑。我们的研究有望为我们提供新的机制方面的见解 依赖于磷酸化的调节通透性和生长抑制功能的基础 血管连接蛋白和可能使用基因治疗来操纵连接蛋白的表达。 在血管损伤和疾病的情况下，适当地最大限度地/最大限度地减少血管生成。

Project Description The long-term goals of this research are: 1) to understand the molecular mechanisms that govern the permeability and growth suppressive properties of vascular cell gap junctions and 2) to develop from that knowledge the rationale for gene therapies that target connexin expression in endothelial cells with the goal of limiting susceptibility to and facilitating recovery from ischemic injury. Three gap junction proteins are commonly expressed in vascular cells, connexin (Cx) 37, Cx40 and Cx43; in the endothelium Cx37 and Cx40 normally predominate, but during vasculogenesis and with stress, injury and disease, Cx37 is down-regulated and Cx43 up-regulated. The consequences of this change in expression on the vessel's ability to form new vessels and to maintain vessel functions during vascular remodeling remain uncertain. In our previous studies, we demonstrated that these connexins form gap junction channels with vastly different permselective and growth suppressive properties that are regulated by growth factor activated signaling cascades in a connexin- specific manner. In the current proposal we hypothesize that connexin-specific, phosphorylation-dependent regulation of junctional permselectivity provides vascular cells a strategy for maintaining coordinated contraction/relaxation functions of vessels while simultaneously supporting the proliferative response of cells therein. We address this hypothesis in Aim 1 by examining the mechanistic basis for how phosphorylation events in the carboxyl terminal domain (CT) lead to altered permselective properties of the associated pore domain. In aim 2 we extend the observations of Aim 1 and determine whether the growth suppressive properties of these connexins rely on their permselective properties and/or their direct interactions with proteins involved in cell cycle control and progression. These growth studies make use of Cx-deficient cell lines, endothelial cells isolated from wild type or Cx37 deficient mice, and an in vivo hindlimb ischemic injury model, asking whether Cx37 works in conjunction with or in opposition to Cx43 to regulate the angiogenic response induced by injury while preserving junctional permselective properties. A combination of electrophysiology and fluorescence microscopy will be used to quantify the permselective properties of junctions and molecular approaches will be used to identify essential regions/sites of interaction between connexin domains and between connexins and elements of the cell cycle machinery. Isolated endothelial cells and an in vivo ischemia model will be used to determine the benefit of connexin expression or silencing to the extent and speed of vascular remodeling following injury. Our studies can be expected to lend new insights on the mechanistic basis for phosphorylation-dependent regulation of the permeability and growth suppressive functions of the vascular connexins and to the possible use of gene therapy to manipulate connexin expression in the endothelium to maximize/minimize angiogenesis, as appropriate, in settings of vascular injury and disease.