SHEAR STRESS EFFECTS ON ENDOTHELIAL CELL FOCAL CONTACTS

血管内皮细胞(EC)的血流动力学环境被认为起了作用 在血管疾病的发病机制中起着重要作用，包括 动脉粥样硬化和高血压。与流动相关的剪应力一直是 显示了改变EC结构和功能的各个方面，包括 细胞形态和相关的细胞骨架组织。焦点接触 是细胞形态发生变化的动态结构 通过改变与细胞外基质(ECM)的接触来调节。欧共体 涉及焦点接触蛋白的信号机制包括 纽蛋白、塔林、α-肌动蛋白和纤维连接蛋白受体 将使用设计的体外系统来检测Vitronectin受体 为EC单层提供受控水平的剪切应力。剪切 应激诱导的水平、细胞定位和 这些蛋白质的相互作用将根据不同的反应而确定 用免疫荧光法测定不同时间点的剪应力水平 本地化和Western blotting程序。剪应力相关细胞 涉及焦点接触蛋白磷酸化的信号机制 蛋白激酶C将通过免疫沉淀法检测特异性 细胞32P标记后的焦点接触蛋白。流导型 酪氨酸激酶的定位和水平的变化 酪氨酸磷酸化的蛋白质也将用特定的 抗体。ECM蛋白对剪切力的可能调制- 细胞形态变化背后的相关细胞反应将是 使用生长在不同基质上的EC进行检查。流动对生物多样性的影响 欧共体所在的ECM的组成将被调查 测量特定的ECM成分，包括IV型胶原、层粘连蛋白和 Vitronectin。欧共体可以修改其关联的另一种机制 与底物是通过选择性的，调节的蛋白质分解的一个焦点 接触蛋白或细胞外基质的一种成分。剪应力诱导 尿激酶型纤溶酶原水平和活性的变化 将检测包括胶原酶在内的激活剂和金属蛋白酶。 采用免疫学方法和十二烷基硫酸钠底物凝胶酶谱技术。

The hemodynamic environment of endothelial cells (EC) is thought to play an important role in the pathogenesis of vascular diseases including atherosclerosis and hypertension. Flow-related shear stress has been shown to alter various aspects of EC structure and function including cell morphology and associated cytoskeletal organization. Focal contacts are dynamic structures where cellular morphological changes may be modulated by modifying contacts with the extracellular matrix (ECM). EC signaling mechanisms involving the focal contact proteins including vinculin, talin, alpha-actinin and the fibronectin receptor and vitronectin receptor will be examined using an in vitro system designed to provide controlled levels of shear stress to EC monolayers. Shear stress-induced alterations in the levels, cellular localization, and interactions of these proteins will be determined in response to various levels of shear stress and at various time points using immunofluorescent localization and western blotting procedures. Shear stress-related cell signaling mechanisms involving phosphorylation of focal contact proteins by protein kinase C will be examined by immunoprecipitation of specific focal contact proteins following 32P labeling of the cells. Flow-induced alterations in the localization and levels of tyrosine kinases and tyrosine-phosphorylated proteins will also be determined using specific antibodies. The possible modulation by ECM proteins of shear stress- related cellular responses underlying changes in cell morphology will be examined using EC grown on different matrices. The effect of flow on the composition of the ECM on which the EC reside will be investigated to measure specific ECM components including collagen IV, laminin, and vitronectin. Another mechanism by which EC may modify their associations with the substratum is by selective, regulated proteolysis of a focal contact protein or a component of the ECM. Shear stress-induced alterations in the levels and activity of urokinase-type plaminogen activator and metalloproteinases including collagenases will be examined using immunological methods and SDS-substrate gel zymography.