| Considerable evidence has accumulated to indicate that there is a coagulation-inflammation network in vivo. The serine proteases in blood clotting process not only participate in the activation of coagulation factors, but also result in a series of cell responses particularly involved in inflammation process through appropriate receptors. Among those, the expression of TF in activated vascular endothelial cells may present a major link between inflammation and thrombosis.Vascular endothelial cells participate in the regulation of hemeostasis through controlling the expression of procoagulant factors as well as anticoagulant factors. Tissue factor (TF) is of major biological significance for procoagulant properties in endothelial cells and monocytes, and functions as a membrane glycoprotein receptor that specially binds coagulation factor VII or Vila. It is well known that Production of TF in vivo has been associated with coagulopathy and thrombosis in damaged vessels of a number of diseases such as septic shock, atheroma and disseminated intravascular coagulation. Many factors, however, have been shown to be important in causing cell TF expression. Among these, the proinflammatory tumor necrosis factor-o, (TNF-a) is most strongly implicated in vascular disease.Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb Ligusticuum, and is generally used as an antithrombotic in Chinese clinical practice. TMP is also known to exert cytoprotective and antioxidant effects on endothelial cell and antithrombotic effects in experimental animal models. However, whether it affects the TF gene expression in endothelial cells and its exact mechanism remains unclear yet.The aim of present study was to 1) investigate the effect of TMP on the expression of tissue factor (TF) induced by tumor necrosis factor(TNF)a in human umbilical vein endothelium derived cell lineIVis-ECV304; 2) observe the role of PKC, one of intracellular signal transduction pathways, and transcription factor NF- K B in the regulation of IMP on TF expression in ECV304.Methods ECV304 were cultured in RPMI-1640. TF activity was determined with one-stage clotting assay measuring total cellular pro-coagulant activity. TF antigen was assayed by ELISA. TF mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). LDH was measured with clinical biochemistry kit. Liquid scintillation count was used to check PKC activity by radioimmunoassay. Immunohistochemical analysis was performed to evaluate the activation of NF- K. B.Results A gradual increase in TF activity and TF antigen were observed in ECV304 stimulated for 6h with increasing concentrations of TNFa (0, 250, 500, 1000 U/mL), reached the maximal levels at concentrations of 1000 U/mL of TNFa. TNFa elicited an increase in TF mRNA expression in a dose-dependent manner in ECV304. TNFa (1000 U/mL) increased TF activity, TF antigen and TF mRNA in ECV304 in a time dependent fashion, reaching a maximum level after 6h, 6h and 2h respectively. All quantitative results of TF mRNA levels were normalized to the GADPH as an internal standard. Increasing concentrations of TMP (1-1000 u g/mL) decreased the TF activity and TF antigen induced in ECV304 by TNFa (1000 U/mL) (PO.01), in a dose-dependent fashion. TF activity induced by stimulant decreased to approximately 30% after treating with 1000 u g/mL of TMP. TMP also decreased the expression of TF mRNA induced by TNFa. There were no differences in the content of LDH in endothelial cells supernatant among TMP, Staurosporine, PMA and control. TNFa could significantly increase the level of LDH (PO.01) and pretreatment of TMP could suppress this increase (PO.05), while pretreatment of Staurosporine and PMA had no effects on the levels of LDH in ECV304 stimulated with TNFa. PMA, as well as TNFa, elicited increases in TF mRNA, TF antigen and TF activity expression (PO.01), TMP alone had no marked effect on TF expression in endothelial cell, but the addition of TM... |
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