Effects of shear stress and cyclic strain on the expression of thrombin receptor gene in human vascular endothelial and smooth muscle cells
Nguyen, Kytai Truong
McIntire, Larry V.
Doctor of Philosophy
Mechanical forces such as shear stress and cyclic strain have been shown to regulate expression of many genes that can alter vascular functions such as cell proliferation, leading to the development of vascular diseases including atherosclerosis. Thrombin receptor gene, protease-activated receptor-1 (PAR-1), mediates many important vascular functions such as thrombin-stimulated thrombosis, inflammation, and proliferation of vascular cells; however, the regulation of PAR-1 by mechanical forces has not previously been studied. This thesis investigates effects of shear stress and cyclic strain on gene regulation of PAR-1 in human vascular cells such as endothelial (ECs) and smooth muscle cells (VSMCs) and the molecular mechanisms involved in this regulation. This work finds that shear stress and cyclic strain differentially regulated PAR-1 expression in vascular cells, leading to alterations of cell functions in response to thrombin, and that these processes were mediated through various signaling pathways. Cultured cells were exposed to different levels of shear stress or cyclic strain using the parallel flow plate chamber or uni-axial cyclic strain system. After exposure, PAR-1 mRNA and protein were quantified by Northern blot and flow cytometry, respectively. In addition, inhibitors of various signal pathways such as protein kinases were used to investigate the molecular mechanisms. Arterial shear stresses decreased PAR-1 mRNA and protein both time- and dose-dependently in both macro- and microvascular ECs, leading to attenuation of thrombin-stimulated nitric oxide and endothelin-1 releases. Furthermore, protein kinase C partly mediated shear-reduced PAR-1 expression in both cell types. As in ECs, shear-downregulated PAR-1 expression in VSMCs caused decreases in thrombin-stimulated calcium mobilization and cell proliferation. The transcription mechanism, but not mRNA stability, regulated shear-reduced PAR-1 expression in VSMCs. In contrast to shear stress, high levels of cyclic strain increased PAR-1 expression in VSMCs time-dependently, leading to induction of cell proliferation in response to thrombin, and this process was mediated by reactive oxygen species, possibly through the NADPH pathway. These findings indicate important roles of mechanical forces in regulating vascular functions and thus provide a better understanding of how mechanical factors act to promote vascular diseases.
Cell biology; Biomedical engineering; Health sciences; Medicine; Surgery