The effect of laminar fluid flow on thrombomodulin activity and gene regulation in human endothelial cells

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