Effects Of Adiponectin On Tissue Factor And Tissue Factor Pathway Inhibitor Expression And Its Mechanisms In Vascular Endothelial Cells

Research backgroundAdiponectin(Adp)is a novel adipose tissue-derived cytokine that has been shown to improve insulin sensitivity and protect endothelial cell function,and to exert antiatherosclerotic and antiinflammatory effects. Epidemiological studies demonstrated the inverse relationship between plasma adiponectin level and insulin resistant-disease states as well as thrombus diseases.Adiponectin inhibits inflamation cytokine and adhesion molecules expression in endothelial cells,adiponectin also inhibits macrophage-to-foam cell transformation and human aortic smooth musule cells(HASMC)proliferation and migration.Accordingly, adiponectin could protect endothelial cell function by increasing insulin sensitivity,antiatherosclerotic,antiinflammatory and anti-proliferation effects.On account of the closely relationship between thrombosis and coagulation activity,endothelial cells dysfunction can promote abnormality coagulation activation.But the influence of adiponectin on procoagulability of endothelial cells is still unknown.Tissue factor(TF) is a key initiator in the activation of coagulation cascade,TF has been recognized to be involved in the pathogenesis of atherosclerosis and coronary artery disease by promoting thrombus formation.Tissue factor pathway inhibitor(TFPI)is a direct physiological inhibitor of TF which can regulate the progression of thrombus formation.However,there is no data about whether adiponectin can affect the expression of the coagulation key factors of TF and TFPI.Therefore,it is important to invastigate the effects of adiponectin on coagulation activity,it can elucidate parts of mechanism of thrombus formation and can provide evidences for prevention and cure thrombosis diseases.Just for the above reasons,this study was designed to examine the effects of adiponectin on the tumor necrosis factor-α(TNF-α)induced TF and TFPI expression and on signal transduction pathways in TNF-α-induced human umbilical vein endothelial cells(HUVECs).Our research consists of three parts:Part 1.Effects of TNF-αon the expression of TF and TFPI in vascular endothelial cellsObjective To observe the effects of TNF-αon the TF and TFPI antigen expression in HUVECs.Methods HUVECs were cultured in medium RMPI 1640 containing 10%fetal calf serum(FCS);Cell viability was tested by trypan blue staining method;TF and TFPI antigen were determined by enzyme-linked immunoabsordent assay (ELISA);TF mRNA and TFPI mRNA were examined by reverse transcription polymerase chain reaction(RT-PCR).Results There were no significant difference in cell viability between treated groups and untreated groups;TNF-αincreased TF antigen expression in HUVECs in a dose or time-dependent manner,reaching a maximum level with TNF-α10 ng/ml after 6 hours(p＜0.01);TNF-αincreased TF mRNA expression in HUVECs in a dose or time-dependent manner,increasing significently after 3 huors(p＜0.01)and reaching a maximum level with TNF-α10 ng/ml after 6 hours(p＜0.01);There was no effect on the TFPI antigen and mRNA exprssion induced by TNF-αin HUVECs.Conclusions TNF-αcan induce the TF expression in HUVECs;There has no effect on TFPI expression induced by TNF-αin HUVECs.Part 2.Effects of adiponectin on the expression of TF and TFPI induced by TNF-αin HUVECsObjective To investigate the effects of adiponectin on the expression of TF and TFPI induced by TNF-αin HUVECs.Methods HUVECs were cultured in medium RMPI 1640 containing 10%FCS;Cell viability was tested by trypan blue staining method;TF and TFPI antigen were determined by ELISA;TF and TFPI activity were determined by chromogenic assay;TF mRNA and TFPI mRNA were examined by RT-PCR.Results There were no significant difference in cell viability between treated groups and untreated groups;Adiponectin can inhibit the TF protein and mRNA expression induced by TNF-αin a dose-dependent manner(p＜0.01),also inhibits the TF activity(p＜0.01),and adiponectin alone exhibited no effect on TF expression in HUVECs.Adiponectin can enhance TFPI antigen expression and activity in HUVECs(p＜0.01),but no significant difference was observed in the expression of TFPI mRNA between adiponectin treated and nontreated HUVECs.Conclusions Adiponectin can inhibit TF expession induced by TNF-αat mRNA and protein level;Adiponectin enhances TFPI expression might at post-transcriptional level.Part 3.Mechanisms of adiponectin on TF and TFPI expression induced by TNF-αin HUVECsObjective Our previous data demonstrated that adiponectin can inhibited TF expression at mRNA and protein level,and enhanced TFPI expression at post-transcriptional level induced by TNF-αin HUVECs. This study was to investigated the signal transduction pathways of adiponectin on TF and TFPI expression induced by TNF-αin HUVECs. Methods HUVECs were cultured in medium RMPI 1640 containing 10%FCS;Cell viability was tested by trypan blue staining method;TF and TFPI antigen were determined by ELISA;TF and TFPI activity were determined by chromogenic assay;The activation of nuclear factor-κB (NF-κB)was determined by ELISA;Intracellular cAMP was measured using an enzyme immunoassay;The PKA activity was measured using the PepTag Assay;Phosphrylation of p38MAPK,p44/42MAPK, SAPK/JNK,Akt,IκB-αwere performed by western blot analysis. Results Phosphorylation of IκB-αwas decreased by adiponectin,but phosphorylation of p38MAPK,p44/42MAPK,and SAPK/JNK were unaffected;Adiponectin increased intracellular cAMP levels and PKA activity in HUVECs in a dose-dependent manner;The inhibitory effect of adiponectin on TNF-α-induced TF expression was abrogated by pretreatment with Rp-cAMPs,a PKA inhibitor;Adiponectin enhances Akt phosphorylation level;Adiponectin can inhibits IκB-αphosphorylation level and NF-κB binding activity level significently;The inhibitory effect of adiponectin on TNF-α-induced TF expression was also abrogated in part by pretreatment with the PI₃K inhibitor LY294002, the inhibitory effects of adiponectin on the TF expression was abrogated about 95%by LY294002 combined with Rp-cAMPs,but the enhancement of TFPI antigen expression induced by adiponectin was unaffected.Conclusions The activation of cAMP-PKA and PI₃K-Akt could inhibit TF expression induced by TNF-αin HUVECs;The inhibition of NF-κB may mediate adiponectin inhibition of TF expression; The cAMP-PKA and PI₃K-Akt signal pathway are the upstream pathway of NF-κB.