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.
