SHEAR STRESS EFFECTS ON ENDOTHELIAL CELL FOCAL CONTACTS

剪切应力对内皮细胞局灶性接触的影响

• 批准号: 3473520
• 负责人: PEGGY R GIRARD
• 金额: $ 9.48万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-24 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3473520
• 关键词: alpha actinin; atherosclerosis; biological signal transduction; cell adhesion molecules; collagen; collagenase; cytoskeleton; extracellular matrix proteins; fibronectins; human tissue; hypertension; immunofluorescence technique; immunoprecipitation; laminin; mechanical stress; phosphorylation; plasminogen activator; protein kinase C; protein tyrosine kinase; radiotracer; southern blotting; tissue /cell culture; vascular endothelium; vinculin; vitronectin; western blottings

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.

内皮细胞（EC）的血流动力学环境被认为是发挥 在血管疾病的发病机制中起重要作用，包括 动脉粥样硬化和高血压。 与流动相关的剪切应力已经被 显示改变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 是动态结构，其中细胞形态学变化可能是 通过修饰与细胞外基质（ECM）的接触来调节。EC 涉及局部接触蛋白的信号传导机制，包括 黏着斑蛋白、talin、α-辅肌动蛋白和纤连蛋白受体， 玻连蛋白受体将使用设计的体外系统进行检查 以向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响应于各种 水平的剪切应力和在不同的时间点，使用免疫荧光 定位和蛋白质印迹法。 剪应力相关细胞 涉及局部接触蛋白磷酸化的信号传导机制 蛋白激酶C将通过特异性免疫沉淀法进行检查。 32 P标记细胞后的局部接触蛋白。 流激 酪氨酸激酶的定位和水平的改变， 酪氨酸磷酸化的蛋白质也将使用特异性 抗体的 ECM蛋白对剪切应力的可能调节- 细胞形态学变化的相关细胞反应将是 使用在不同基质上生长的EC进行检查。 流动对 将研究EC所在ECM的组成， 测量特定的ECM成分，包括胶原蛋白IV，层粘连蛋白， 玻连蛋白。 欧共体可以修改其联系的另一种机制 是通过选择性的，调节性的局部蛋白水解， 接触蛋白或ECM的组分。 剪切力上调 尿激酶型纤溶酶原水平和活性的改变 将检查激活剂和金属蛋白酶，包括胶原酶 使用免疫学方法和SDS-底物凝胶酶谱法。