Testosterone alleviates tumor necrosis factor-alpha-mediated tissue factor pathway inhibitor downregulation via suppression of nuclear factor-kappa B in endothelial cells

We have observed earlier that testosterone at physiological concentrations can stimulate tissue factor pathway inhibitor(TFPI)gene expression through the androgen receptor in endothelial cells.This study further investigated the impact of testosterone on TFPI levels in response to inflammatory cytokine tumor necrosis factor-alpha(TNF-α).Cultured human umbilical vein endothelial cells were incubated in the presence or absence of testosterone or TNF-α.TFPI protein and mRNA levels were assessed by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction.To study the cellular mechanism of testosterone’s action,nuclear factor-kappa B(NF-κB)translocation was confirmed by electrophoretic mobility shift assays.We found that after NF-κB was activated by TNF-α,TFPI protein levels declined significantly by 37.3%compared with controls(P<0.001),and the mRNA levels of TFPI also decreased greatly(P<0.001).A concentration of 30 nmol L-1 testosterone increased the secretion of TFPI compared with the TNF-α-treated group.NF-κB DNA-binding activity was significantly suppressed by testosterone(P<0.05).This suggests that physiological testosterone concentrations may exert their antithrombotic effects on TFPI expression during inflammation by downregulating NF-κB activity.

Expressions of Tissue Factor and Tissue Factor Pathway Inhibitor in Patients with Acute Graft-versus-host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

This study examined the expressions of human serum tissue factor （TF） and tissue factor pathway inhibitor （TFPI） in patients with acute graft-versus-host disease （aGVHD） after allogeneic hematopoietic stem cell transplantation （allo-HSCT） and their clinical significance. The serum TF and TFPI levels were detected by ELISA in 28 allo-HSCT recipients before and after the transplanta-tion and the changes of TF and TFPI levels were dynamically monitored at different phases of the disease. No significant differences in the serum TF and TFPI levels were found in allo-HSCT recipi-ents in the absence of aGVHD or with gradeⅠaGVHD before and after the transplantation. The lev-els of serum TF and TFPI were substantially increased in the patients with gradeⅡ aGVHD at the peak of aGVHD （P〈0.05） and they were even higher in the patients with grade Ⅲ–Ⅳ aGVHD （P〈0.01）. When the conditions became stable after treatment with immunosuppressive agents, the serum TFPI level was decreased to the baseline level （P〉0.05） and the TF level was lowered but still higher than the baseline level （P〈0.05）. It was concluded that the levels of serum TF and TFPI were increased significantly in the patients with grade Ⅱ–Ⅳ aGVHD after allo-HSCT and decreased markedly after the treatment. Monitoring the levels of serum TF and TFPI in the patients with allo-HSCT is important to predict the occurrence, outcome and prognosis of aGVHD.

Oxidized low density lipoprotein inhibited tissue factor pathway inhibitor mRNA expression in human endothelial cells

To understand the role of oxidized low density lipoprotein (OX LDL) in the pathogenesis of thrombotic complications in atherogenesis Methods Low density lipoprotein was isolated from normal heparinized blood by density gradient ultracentrifugation and oxidized by CuCl 2 Total RNA was extracted from human umbilical vein endothelial cells (HUVECs) exposed to LDL or OX LDL, using the guanidinium isothiocyanate method The quantification of tissue factor pathway inhibitor (TFPI) mRNA in HUVECs was carried out by reverse transcriptase polymerase chain reaction (RT PCR) Results HUVECs were able to express TFPI mRNA constitutively The expression was not affected by LDL but was effectively inhibited by OX LDL in a time and dose dependent manner Conclusions The results suggest that oxidized LDL may play an important role in inducing coagulation in atherosclerotic lesions by the inhibition of expression of TFPI in vascular endothelial cells

Endogenous tissue factor pathway inhibitor in vascular smooth muscle cells inhibits arterial thrombosis

Tissue factor pathway inhibitor （TFPI） is the main inhibitor of tissue factor-mediated coagulation. TFPI is expressed by endothelial and smooth muscle cells in the vasculature. Endothefium-derived TFPI has been reported to play a regulatory role in arterial thrombosis. However, the role of endogenous TFPI in vascular smooth muscle cells （VSMCs） in thrombosis and vascular disease development has yet to be elucidated. In this TFPI^Flox mice crossbred with Sma-Cre mice were utilized to establish TFPI conditional knockout mice and to examine the effects of VSMC-directed TFPI deletion on development, hemostasis, and thrombosis. The mice with deleted TFPI in VSMCs （TFP^Sma） reproduced viable offspring. Plasma TFPI concentration was reduced 7.2% in the TFPIsma mice compared with TFPI^Flox littermate controls. Plasma TFPI concentration was also detected in the TFPI^Tle2 （mice deleted TFPI in endothefial ceils and cells of hematopoietic origin） mice. Plasma TFPI concentration of the TFPI^Tle2 mice was 80.4% lower （P 〈 0.001） than that of the TFPI^Flox mice. No difference in hemostatic measures （PT, APTT, and tail bleeding） was observed between TFPIsma and TFPI^Flox mice. However, TFP^Sma mice had increased ferric chloride-indueed arterial thrombosis compared with TFPI^Flox littermate controls. Taken together, these data indicated that endogenous TFPI from VSMCs inhibited ferric chloride-induced arterial thrombosis without causing hemostatic effects.

Comparison of Anticoagulant Effects on Vein Grafts between Human TFPI Gene Transfection and Aspirin Oral Administration

To develop a more efficient antithrombotic way after coronary artery bypass grafting （CABG）, the anticoagulant effects were compared of human tissue factor pathway inhibitor （TFPI） gene transfection and aspirin oral administration （traditional method） on vein grafts. An eukaryotic expression plasmid pCMV-（Kozak） TFPI was prepared. Animal model of carotid artery bypass grafting was constructed. In operation, endothelial cells of vein grafts in TFPI group and empty plasmid control group were transfected with pCMV-（Kozak） TFPI and empty plasmid pCMV respectively, while no transfection was conducted in aspirin control group. After operation, aspirin （2 mg·kg^-1·d^-1） was administered （i.g.） in aspirin control group. Three days later, grafts （n=10） were harvested for RT-PCR, Western blotting and immunohistochemical analyses of exogenous gene expression and for pathological, scanning electron microscopic observation of thrombus. Thirty days later, the patency rates of remnant grafts （n= 10） were recorded by vessel Doppler ultrasonography. Human TFPI gene products were detected in gene transferred vein grafts. Three days later, thrombi were found in 7 animals of aspirin control group and in 8 animals of empty plasmid control group, but in only 1 of TFPI group （P〈0.01）. Thirty days later, 5 grafts were occluded in empty plasmid control group, but none of grafts was occluded in the other groups （P〈0.05）. The endothelial surfaces of grafts in both of the control groups were covered with aggregated erythrocytes and platelets, and it were not seen in TFPI group. It was suggested that the anticoagulant effects on vein grafts of human TFPI gene transfection are better than those of aspirin.