VASCULAR ENDOTHELIUM AND FLUID SHEAR STRESS IN VITRO

体外血管内皮和流体剪切应力

基本信息

批准号：
3338123
负责人：
C Forbes Dewey
金额：
$ 29.21万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-04-01 至 1992-03-31
项目状态：
已结题

项目摘要

The structural and functional responses of vascular endothelium to fluid shear stress will be investigated in vitro using cultured endothelial cells and a special apparatus we have developed for producing controlled fluid shear stress. The apparatus includes: a cone-and-plate system that produces both laminar and turbulent flow with shear stress from 10-2 to 100 dynes/cm2; and a parallel- plate system under construction that is specifically designed for live-time microscopy and image analysis of endothelial layers during exposure to shear. Controllable parameters include: fluid shear stress level, time history, and degree of turbulence; subcellular matrix; cell preconditioning by shear; biochemcial modulators of cell function; and cell type. A combination of live-time analysis and end-point fluorescent antibody visualization will provide data on the interrelationships of cell shape change, flow-axis alignment, and cytoskeletal reorganization in endothelial cells exposed to a range of laminar and turbulent flow conditions. Trans-membrane stimulus-response coupling will be determined using the following assays: (i) cytosolic ionized calcium levels, as monitored by live-time fluorescence microscopy using fura-2; (ii) intracellular pH; (iii) polyphosphoinositide metabolism; and (iv) prostacyclin metabolism. Cell-surface-related properties to be measured are: (i) apicalbasal polarity, as defined by cell surface-selective patterns of integral- and membrane-associated proteins; (ii) functional surface properties, as characterized by tissue factor procoagulant activity and surface adhesiveness for platelets; and (iii) receptor-mediated endocytosis, using the radiolabeled ligands alpha2-macroglobulin and insulin. This is a collaborative research effort that draws upon the resources, expertise, and experience of the Fluid Mechanics Laboratory, Massachusetts Institute of Technology, and the Vascular Research Division, Department of Pathology, Brigham and Women's Hospital. The research sytem used in this program, comprised of accurately-controlled fluid shear stress apparatus and well-defined cultured endothelial specimens, should yield valuable information on the mechanisms by which fluid shear stress influences vascular endothelium. Such information would provide new insights into vascular pathology and the pathogenesis of vascular diseases such as atherosclerosis and thrombosis.

血管内皮的结构和功能反应通过培养我们开发的内皮细胞和特殊设备产生受控的流体剪切应力。该设备包括：圆锥形板系统同时产生层流和湍流剪切应力从10-2到100 Dynes/cm2流动；和一个平行的专门设计的板系统正在建造内皮层的实时显微镜和图像分析在接触剪切期间。可控参数包括：流体剪切应力水平，时间病史和湍流程度；亚细胞基质；剪切细胞预处理；生化细胞功能的调节剂；和细胞类型。实时分析和终点荧光的组合抗体可视化将提供有关相互关系的数据细胞形状变化，流动轴对准和细胞骨架暴露于一系列层流的内皮细胞中的重组和湍流条件。跨膜刺激反应耦合将使用以下测定确定：（i）通过实时监测的胞质离子化钙水平使用Fura-2荧光显微镜；（ii）细胞内pH；（iii）多磷酸代谢；（iv）前列环素代谢。要测量的细胞表面相关特性为：（i）基尖的极性，由细胞表面选择性定义积分和膜相关蛋白的模式；（ii）功能表面特性，以组织因子为特征血小板的促凝活性和表面粘附性；和（iii）使用放射性标记的配体，受体介导的内吞作用 α2-巨球蛋白和胰岛素。这是一项协作研究工作，借鉴了流体力学的资源，专业知识和经验实验室，马萨诸塞州理工学院和杨树病理学系血管研究部和妇女医院。该程序中使用的研究系统，由精确控制的流体剪切应力设备组成和定义明确的培养的内皮标本，应产生关于流体剪切机制的宝贵信息压力会影响血管内皮。这样的信息会提供有关血管病理和发病机理的新见解血管性疾病，例如动脉粥样硬化和血栓形成。