|Budget Amount *help
¥4,600,000 (Direct Cost : ¥4,600,000)
Fiscal Year 1997 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Fiscal Year 1996 : ¥2,900,000 (Direct Cost : ¥2,900,000)
To investigate molecular markers in perturbed endothelium, physiological 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 (HUVEC) perturbed by cytokines, using modified cone-plate viscometer, in which well controlled and defined shear forces were geperated. Tissue factor (TF), a transmembrane glycoprotein that acts a central role in blood coagulation, are important regulators for coagulation in endothelium. Sheaur stress of 6-24 dynes/cm^2 decreased the expression of TNFalpha-induced TF in a shear intensity- and exposure time- dependent manner. Reverse transcriptase-polymerase chain reaction showed that TNFalpha-induced TF mRNA levels decreased in response to flow. TF expressed on cell surface measured by flow cytometry using an atnti-TF monoclona
l antibody, HTF-K180, was decreased to one third by shear forces of 18 dynes/cm^2 applied for 15 hours before and 6 hours after TNF stimulation. The stability of TF mRNA,however, was not changed in the absence or presence of shear stress. Tissue plasminogen activator (t-PA), which convert plasminogen to plasmin to degrade fibrin clot, and plasminogen activator inhibitor-1 (PAI-1), which inhibit t-PA function, play central roles for fibrinolysis in endothelium. Treatment of the cells with IL-1beta or TNF-alpha under static conditions had no effect on t-PA secretion, while release of PAI-1 increased. When cells were exposed to increasing shear stress up to 24 dynes/cm^2, levels of t-PA and t-PA/PAI-1 complex significantly increased relative to shear stress, while total PAI-1 and active PAI-1 secretion decreased gradually. In the presence of IL-1beta or TNF-alpha, the increase in production of t-PA and t-PA/PAI-1 complex was further augmented. Dot blot hybridization analysis of cultured cells using t-PA and PAI-1 cDNA probes revealed no t-PA mRNA in 3 mug of total RNA from static endothelial cells under resting or cytokine-stimulated conditions but abundant t-PA mRNA was detected in cells subjected to a shear force of 18 dynes/cm^2 and the increase was further augmented by addition of cytokines. In contrast, PAI-1 mRNA was detected in resting and cytokine-stimulated, non-sheared endothelial cells, but levels decreased after exposure to shear stress, even in the presence of cytokines.
These results clearly indicate that shear forces act as an important regulator of coagulation and fibrinolysis system in endothelium, to coordinately maintain aantithrombogenicity of luminal surface of blood vessels. These molecular markers might be very useful indicators for endothelial perturbation. Less