Ordinarily, glomerular endothelial cells are highly flattened, non- proliferative cells that provide an anticoagulant surface, participate in forming a barrier to filtration, and produce vasoactive and growth regulating mediator. During glomerular development, and in response to some forms of immune-mediated injury, glomerular endothelial cells loose their flattened appearance and become activated. When endothelial cells are cultured in vitro, they also take on a less differentiated phenotype than that observed in vivo, behaving more like endothelial cells undergoing embryonic development. However, the chronic (greater than 9 days) application of laminar flow to endothelial cells in culture, with fluid shear stress in the physiologic range, results in a greater degree of differentiation. The current proposal seeks to further define mechanisms that lead to endothelial cell differentiation using the chronic shear stress model system. It is proposed that alterations in the responsiveness to transforming growth factor beta is one important mechanism regulating differentiation, and that this is brought about through a switch in expression of TGF-beta receptor- and postreceptor signaling molecule isoforms. A second mechanism that may operate is an alteration in the transcritional regulation of genes highly during shear stress exposure. To explore this possibility, regulation of one specific gene, eNOS, will be examined. Finally, it is proposed that regulation of protein synthesis by shear stress may also occur through changes in mRNA stability. A novel sequence has been identified in the 3"""""""" untranslated region of two differentiation-associated mRNAs, which may confer shear-stress responsiveness. Use of this element in regulating mRNA stability by shear stress will be examined. By understanding the signals for endothelial cell differentiation, it is anticipated that mechanisms will also be uncovered that signal loss of differentiation and proliferation, as is commonly observed during immune-mediated endothelial cell injury.
