Vascular endothelium, the single-cell-thick lining of the cardiovascular system, is constantly subjected to biomechanical forces derived from pulsatile blood flow. It is our working hypothesis that this endothelial interface is dynamically mutable, undergoing adaptive changes in its structure and function that are critical to normal physiological processes, and the pathogenesis of vascular disease. In vitro, a number of morphological and functional responses can be induced in cultured human and animal endothelial cells by exposure to defined fluid mechanical forces. Many of these involve modulation of gene expression at the transcriptional level. Using deletional mutation analyses, we have recently identified a cis-acting transcriptional regulatory element in the promoter of the PDGF-B gene which appears to be a """"""""shear-stress-responsive element (SSRE)"""""""". This SSRE also is present in the 5' flanking regions of several other genes that are induced by shear stress in endothelial cells. To test the functional relevance and mechanisms of action of this SSRE, the following cellular and molecular biological in vitro and in vivo approaches are proposed:
Specific Aim 1 : Test the hypothesis that endothelial genes (or reporter-gene constructs) that contain the SSRE will show differential responsiveness to biologically relevant mechanical forces (shear stresses, cyclic strain) in well-defined in vitro test systems;
Specific Aim 2 : Define the biomechanical transduction mechanisms involved in SSRE-dependent gene regulation, and, in particular, examine the role of endothelial cytoskeletal elements in this process;
Specific Aim 3 : Characterize the transcriptional regulatory function of this SSRE; clone its nuclear binding proteins (putative transcription factors); compare with other known transcription factor systems. These studies should help to better define the molecular mechanisms regulating endothelial gene expression by biomechanical forces, and thus provide new insights into vascular physiology, as well as the pathophysiology of diseases such as atherosclerosis, thrombosis and hypertension.
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