By contrast with other capillary endothelial cells in vivo, glomerular endothelial cells are perfused under high pressure and are exposed to rapid plasma flow rates. Also, morphologic differentiation of glomerular endothelium results in an extremely attenuated layer of endothelial cytoplasm with a high density of fenestrae that lines the capillary basement membrane. Little is known about the mechanisms that control glomerular capillary endothelial cell differentiation. It is also of interest that abnormal glomerular remodeling, as observed in disease models of progressive glomerular sclerosis, may be influenced by glomerular hemodynamics. A model system was developed in this laboratory to culture glomerular capillary endothelial cells for prolonged periods of time in artificial capillaries in vitro. Preliminary experiments indicate that culture of glomerular endothelial cells under flow and superimposed alterations in the level shear stress markedly influence differentiation and lead to changes in release of soluble mediators including endothelial cell derived mitogens. The central hypothesis of this proposal is that the prevailing level of shear stress influences glomerular endothelial cell differentiation, and that alterations in the level of shear stress may participate in regulating glomerular mesangial cell growth and contractility by modifying endothelial cell mediator release. The first part of the proposal aims to explore in detail glomerular capillary endothelial cell differentiation using ultrastructural examination (transmission and scanning electron microscopy) as well as examination of the cells for expression of endothelium specific gene products and functions. Preliminary data indicate that attenuation of the cytoplasm, fenestral formation and expression of Weibel Palade body all are induced when glomerular endothelial cells are cultured in artificial capillaries under flow. The second part of the proposal aims to examine the hypothesis that endothelial cell proliferation and cytoskeletal reorganization under flow are controlled, in part, by autocrine receptor tyrosine kinase activation. The proposal therefore aims to explore the functional involvement of fibroblast growth factor activated tyrosine kinase activity in the structural reorganization and growth of glomerular endothelial cells under flow. In addition, it aims to determine whether specific mechanisms controlling secretion of soluble fibroblast growth factor are controlled by shear stress. The third part of the proposal will explore the question to what degree shear stress controls mediator synthesis and release by glomerular capillary endothelial cells. This section will specifically address endothelium-derived mediators known to influence mesangial cell growth and contractility. Preliminary data show that endothelial cells grown in artificial capillaries secrete higher levels of endothelin-1 and prostacyclin, and synthesize more platelet derived growth factor than cells grown in static conditions. Experiments in this cluster aim to examine whether increments or decrements in glomerular capillary endothelial cell shear stress actually lead to functionally significant alterations in PDGF, endothelin-1, prostacyclin and nitric oxide release.
