Gap Junctions in Vascular Smooth Muscle: Growth Control

项目描述 本研究的长期目标是：1）了解控制 血管细胞间隙连接的渗透性和生长抑制特性，以及2）从 了解靶向内皮细胞中连接蛋白表达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从缺血性损伤中恢复。三种间隙连接蛋白是 通常表达于血管细胞，连接蛋白（Cx）37、Cx40和Cx43; 通常占主导地位，但在血管发生和压力，损伤和疾病，Cx 37下调 Cx43上调。这种表达的变化对血管形成新血管的能力的影响 在血管重塑期间维持血管功能的能力仍然不确定。在我们之前的研究中， 我们证明了这些连接蛋白形成具有极大不同的选择透过性的缝隙连接通道， 生长抑制特性由连接蛋白中的生长因子激活的信号级联调节， 具体方式。在目前的建议中，我们假设连接蛋白特异性，磷酸化依赖性， 连接选择性渗透的调节为血管细胞提供了维持协调的 血管的收缩/舒张功能，同时支持细胞的增殖反应 在其中我们在目标1中通过研究磷酸化如何在细胞中发挥作用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选择性渗透性质改变 域在目标2中，我们扩展了目标1的观察结果，并确定生长抑制剂是否 这些连接蛋白的性质依赖于它们的选择透过性和/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Cx缺陷细胞系， 从野生型或Cx 37缺陷小鼠分离的内皮细胞，和体内后肢缺血性损伤模型， 询问Cx 37是否与Cx43协同或相反地调节血管生成反应 由损伤诱导，同时保持连接选择性渗透特性。结合电生理学和 荧光显微镜将被用来量化选择性渗透性能的连接和分子 方法将用于鉴定连接蛋白结构域之间相互作用的必要区域/位点， 连接蛋白和细胞周期机制之间的联系分离的内皮细胞和体内缺血 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见解 的渗透性和生长抑制功能的磷酸化依赖性调节的基础。 血管连接蛋白和基因治疗的可能用途，以操纵连接蛋白的表达， 在血管损伤和疾病的情况下，适当地最大化/最小化血管生成。

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.