| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
To investigate the regulatory mechanism of thrombosis and hemostasis in endothelium, in vitro experiments have been undertaken. Hemodynamic forces modulate various endothelial cell functions even in the presence of cytokines under gene regulation. We have investigated the effect of shear stress on coagulation and fibrinolysis system in cultured human umbilical vein endothelial cells (HUVECs) perturbed by cytokines or lipopolysaccharide (LPS), using modified cone-plate type viscometer, in which well controlled and defined shear forces were generated. We have revealed that cytokine-induced tissue factor expression was decreased in response to flow, whereas secretion of tissue plasminogen activator was augmented under shear stress in the presence of cytokines, and the secretion of plasminogen activator inhibitor-I was increased in the presence of cytokines, but, decreased under shear stress. Furthermore, we investigated the expression of various other factors in HUVECs under shear stress
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in the presence of cytokines, using immunocytochemistry, EIA method, and laser flow-cytometry. Tissue factor pathway inhibitor (TFPI), serine protease that acts as a regulator for tissue factor, was not affected by stimulation of inflammatory cytokines, TNF, nor shear stress, but decreased by tumor promoting activator, PMA. Next, several monoclonal antibodies have been developed against human nasal-mucous microvessels. Monoclonal antibodies have been produced against two adhesive glycoproteins for E-selectin and Mel-CAM that act as important molecules for leucocyte adhesion to endothelium under inflammatory site. E-selectin was not detected in resting unstimulated HUVECs, whereas, surface membrane expression of E-selectin was increased 3-6 hours after the stimulation of TNF. In contrast, Mel-CAM expression was clearly observed in resting unstimulated HUVECs, whereas the release to culture supernatant was increased after TNF stimulation and decreased under shear stress. These results indicate that the mechanism of thrombosis and hemostasis might be regulated by complicated net-work. The cross-talk of regulatory mechanism for shear forces and cytokines, at least as a part, is important modulator for thrombogenecity and anti-thrombogenecity. Less
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