Effects Of Resistin On Tissue Factor And Its Pathway Inhibitor Expression In Cultured Human Umbilical Vein Endothelial Cells

BackgroundThe prevalence of the metabolic syndrome ( MetS ), a concurrence of abdominal fat and insulin resistance, has been rising during recent years. Metabolic syndrome induced a pro-thrombotic state and was closely associated with an increased risk of arterial and venous thromboembolism. The mechanism, however, has not been fully elucidated. Tissue factor ( TF ) is a key initiator in the activation of extrinsic coagulation while tissue factor pathway inhibitor ( TFPI ) is found to be the strongest physiological anticoagulant substances in vivo and is a specific and only physiological inhibitor of TF that modulates the effect of TF-induced coagulation. In this study, we investigated whether resistin regulate the expression of TF and TFPI and to further explore the mechanism of metabolic syndrome in patients susceptible to thrombotic diseases.ObjectiveTo observe the direct effects of resistin on the TF and TFPI protein and their mRNA levels in cultured human umbilical vein endothelial cells ( HUVECs ). Then to study the cellular mechanism to investigate whether the extracellular signal-regulated kinases ( ERK ), the c-Jun NH2-terminal kinases ( JNK ) or p38 signaling pathway are associated in the regulating process of ressitin on TFPI.Methods( 1 ) HUVECs within 4-5 passages were divided into 5 experimental groups: controls ( medium only ), different concentrations resistin treated group ( 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL ). After incubation for 48h, 1 ) the activities of cells were detected by MTT assay; 2 ) the antigen levels of TF and TFPI were assessed with an enzyme-linked immunosorbent assay ( ELISA ), and mRNA levels were detected by real-time RT-PCR.( 2 ) HUVECs within 4-5 passages were divided into: culture medium only group ( control ), 50ng/mL resistin treated group, ERK inhibitor PD98059 treated group ( 10μmol/L ), PD98059 pretreated group ( PD + R ), JNK inhibitor SP600125 treated group ( 2.5μmol/L ), SP600125 pretreated group ( SP + R ), p38 inhibitor SB203580 treated group ( 10μmol/L ), SB203580 pretreated group ( SB + R ). After incubation for 48h, TFPI antigen levels were analyzed with ELISA kit.Results( 1 ) Incubation with resistin ( 10ng/mL, 25ng/mL, 50ng/mL, 100ng/ml ) didn't affect HUVECs proliferation and viability ( p>0.05 ); ( 2 ) 25ng/mL and 50ng/mL resistin markedly up-regulated TF protein expression at 6h ( p<0.05 ), and the most effective regulation was the 50ng/mL resistin. While no significant effect was found at 24h or at 48h ( p>0.05 for both ). TF mRNA was not changed significantly with the 50ng/mL resistin infusion ( p>0.05 ); ( 3 ) 50ng/mL resistin at 24h, 25ng/mL, 50ng/mL and 100ng/mL resistin at 48h reduced TFPI protein levels ( p<0.05 ), and the most effective regulation was 50ng/mL resistin at 48h. Resistin did not influence the mRNA levels of TFPI ( p>0.05 ); ( 4 )After HUVECs preteated with ERK inhibitor PD98059, JNK inhibitor SP600125 or p38 inhibitor SB203580, no significant increase was observed in the expression of TFPI antigen between pretreated groups and 50ng/mL resistin only trested HUVECs.ConclusionHuman recombined resistin induced increased TF, and reduction of TFPI levels in cultured HUVECs possibly due to a post-transcriptional regulational mechanism, suggesting a potential pathophysiology role of resistin to aggregate the thrombosis development. The signaling pathway, however, needs further investigation.