The effect of laminar fluid flow on thrombomodulin activity and gene regulation in human endothelial cells
Panaro, Nicholas Joseph, III
McIntire, Larry V.
Doctor of Philosophy
Endothelial cell biology has become a major area of research during the last decade. An increasing body of evidence suggests that the local mechanical forces arising from blood flow play a key role in the normal physiology and pathobiology of the endothelium. In this study, monolayers of human endothelial cells were subjected to hydrodynamically-induced shear stress using a parallel plate flow chamber. This allows for an in vitro simulation of hemodynamic shear stress effects on the endothelium without pressure-induced mechanical strain, as would occur in an arterial vessel. The molecules chosen for this study were thrombomodulin and c-Fos. Thrombomodulin is a plasma membrane protein which binds thrombin and then converts protein C to activated protein C which has several anticoagulant properties. Thus, thrombomodulin is able to control the extent of thrombosis by indirectly blocking the coagulation cascade. c-Fos is a transcription factor which has been implicated in the regulation of several biomolecules synthesized by endothelial cells including tPA, PAI-1 and PDGF. A four-fold increase in thrombomodulin activity was observed in endothelial cells subjected to arterial levels of shear stress for twenty-four hours with respect to stationary controls. This is the first report of a membrane bound protein whose activity is modulated by mechanical forces. Northern blot analysis of total cellular RNA isolated from endothelial cells exposed to arterial levels of shear stress for twenty-four hours for thrombomodulin mRNA yielded no conclusive results due to weak signals. Attempts to measure c-fos mRNA in shear stress-stimulated endothelial cells yielded negative results. To provide a framework for future experiments, a nuclear transduction model concerning differential gene regulation was developed to explain the published in vitro transcription and secretion data for peptides and proteins by mechanically-stimulated endothelial cells based on the AP-1 complex. A reporter gene construct consisting of bacterial CAT under the control of the human tPA promoter was successfully introduced in to endothelial cells in vitro via an adenovirus vector. Results show a four-fold increase in CAT activity in shear stress-stimulated endothelial cells with respect to controls. This result mimics the tPA secretion and mRNA results observed in vitro.