Effects Of Physiological Testosterone On Tissue Factor Pathway Inhibitor Expression By Cultured Endothelial Cells In Response To Inflammatory Mediators

BackgroundSignificant difference in morbidity of acute coronary syndrome (ACS), one of the critical coronary heart diseases, was found between males and females. But the role androgen played remains controversial, which becomes the interest of cardiovascular research. It was confirmed that decreased vascular endothelial anticoagulant activity and thrombosis development are the main features of acute coronary syndrome. Our previous investigation demonstrated that physiological level of testosterone has beneficial influence on hemostatic system by enhancing the anticoagulant activity through stimulating tissue factor pathway inhibitor (TFPI) secreted by cultured human umbilical vein endothelial cells (HUVEC). And testosterone also could regulate the activity of many transcription factors including nuclear factor-kappaB (NF-κB). NF-κB is one of the critical mediators in inflammation. It was confirmed that the expression of inflammatory factors mediated by the activation of NF-κB play an important role in the development of CHD. Numbers of inflammatory factors, such as tumor necrosis factor-alpha (TNF–α), could up-regulate the expression of many thrombus factors including TF. And the regulation of some thrombus factors are mediated by the activation of NF-κB. That is to say that the activity of NF-κB could be regulated by both testosterone and TNF–α. However, whether testosterone could regulate the TFPI gene expression by intervention in the activation of NF-κB mediated by TNF–αis unknown until now. The present study, therefore, is intended to investigate the effect of testosterone on TFPI gene expression and its cellular mechanism by cultured endothelial cells in response to inflammation mediated by TNF–α.ObjectiveTo investigate the impact of physiological testosterone or TNF-αon the TFPI expression and the activity of nuclear transcription factor NF-κB in HUVECs. Then, study the cellular mechanism by which testosterone at physiological concentration may influence on TFPI expression induced by inflammatory factors.Methods(1) HUVECs within 3-4 passages were divided into 7 groups: blank, controls, 30nmol/L testosterone treated group; 10ng/mL or 20ng/mL TNF-αtreated group; cells pretreated with 10ng/mL or 20ng/mL TNF-αfor 3 hours and then added 30nmol/L testosterone. After incubation for 48h, the activities of cells were detected by MTT assay. (2)HUVECs were divided into 4 groups: controls, 30nmol/L testosterone treated group; 20ng/mL TNF-αtreated group; cells pretreated with 20ng/mL TNF-αfor 3 hours and then added 30nmol/L testosterone. After incubation for 48h, the antigen levels of TFPI were detected with an enzyme-linked immunosorbent assay, and TFPI mRNA levels were assessed by real-time RT-PCR. (3) HUVECs were divided as part two and incubated for 12h. And then the nuclear proteins were isolated and the transcriptional activity of NF-kappaB was examined by electrophoretic mobility shift assay (EMSA).Results(1) 30nmol/L testosterone, 10ng/mL or 20ng/mL of TNF-αhad no influence on the activity of HUVECs; (2) TNF-α(20ng/mL) markedly down-regulated both TFPI mRNA and protein expression (P<0.001), testosterone (30nmol/L) can attenuate the down-regulation of TFPI mediated by TNF-α(P<0.05). (3) NF-κB activity was stimulated significantly by TNF-α(20ng/mL) (P<0.05), and testosterone (30nmol/L) could block TNF-αeffects on NF-κB (P<0.05).ConclusionPhysiological testosterone may cast its antithrombotic effects on TFPI expression during inflammation in endothelial cells, in which process, down-regulation of NF-kappaB activity may be closely involved.