Gap Junctions in Vascular Smooth Muscle: Growth Control

描述（由申请人提供）：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的分子机制，2）根据这些知识开发靶向内皮细胞连接蛋白表达的基因治疗的基本原理，目的是限制对缺血性损伤的易感性并促进其恢复。三种间隙连接蛋白通常在血管细胞中表达，连接蛋白（Cx）37、Cx40和Cx43;在内皮中，Cx 37和Cx40通常占主导地位，但在血管发生期间以及应激、损伤和疾病时，Cx 37下调，Cx43上调。这种表达变化对血管形成新血管和在血管重塑期间维持血管功能的能力的影响仍然不确定。在我们以前的研究中，我们证明了这些连接蛋白形成具有非常不同的选择透过性和生长抑制特性的间隙连接通道，其以连接蛋白特异性的方式由生长因子激活的信号级联调节。在目前的建议中，我们假设连接蛋白特异性的磷酸化依赖性调节连接选择性渗透性提供了血管细胞的策略，以维持协调的收缩/舒张功能的血管，同时支持细胞的增殖反应。我们在目的1中通过研究羧基末端结构域（CT）中的磷酸化事件如何导致相关孔结构域的选择性渗透特性改变的机制基础来解决这一假设。在目标2中，我们扩展了目标1的观察，并确定这些连接蛋白的生长抑制特性是否依赖于它们的选择透过性特性和/或它们与参与细胞周期控制和进展的蛋白质的直接相互作用。这些生长研究利用Cx缺陷型细胞系、从野生型或Cx 37缺陷型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协同或相反地工作以调节损伤诱导的血管生成反应，同时保留连接选择性渗透特性。电生理学和荧光显微镜的组合将被用来量化的选择性渗透性能的路口和分子方法将被用来确定连接蛋白结构域之间的相互作用和连接蛋白和细胞周期机制的元素之间的重要区域/网站。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对损伤后血管重塑的程度和速度的益处。我们的研究有望为磷酸化依赖性调节血管连接蛋白的渗透性和生长抑制功能的机制基础提供新的见解，并可能使用基因治疗来操纵连接蛋白在内皮细胞中的表达，以最大化/最小化血管生成，视情况而定，在血管损伤和疾病的设置。 公共卫生相关性：组织从缺血性和创伤性损伤中的成功恢复需要通过涉及血管细胞的调节性增殖的过程来恢复血流。间隙连接及其组成蛋白，连接蛋白，作为细胞增殖的调节剂。三种连接蛋白在血管细胞中表达，我们正在研究这些连接蛋白的功能和它们组成的细胞间通讯通道在正常和病理环境中是如何调节的。从我们的研究中获得的见解可能有助于制定临床策略，以促进缺血性或创伤性损伤后组织功能的更快速和完全恢复。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Successful recovery of tissues from ischemic and traumatic injury requires restoration of blood flow through a process that involves regulated proliferation of vascular cells. Gap junctions and their comprising proteins, the connexins, serve as regulators of cell proliferation. Three connexins are expressed in vascular cells; we are studying how the function of these connexins and the intercellular communication channels they comprise is regulated in normal and pathological settings. Insights gained from our studies may help in the development of clinical strategies that will facilitate more rapid and complete recovery of tissue function following ischemic or traumatic injuries.