The Effect Of Angiotensin On The Tissue Factor Expression In ECs And The Intervention Of Irbesartan

BackgroundVascular Endothelial cells play a crucial role in the the development of atherosclerosis and the rupture of atherosclerotic plaque by the reduce and release some bioactivities to inhibit the activated platelet and prevent monocyte adhension to endothelial cells and reduce the inflammation factors.Tissue factor(TF),the initiator of blood coagulation ,on binding to coagulation factor VII and its active from VIIa, forms the cell surface complex responsible for the coagulation sequelae, which lead to fibrin formation plays a pivotal role in arterial thrombosis that occurs after atherosclerotic plaque fissuring. Many results strongly suggest that high levels of TF exposed on plaque rupture trigger thrombosis and myocardial infarction. Although TF is not constitutively expressed by endothelial cells and monocyte, it has been demonstrated that both cell types are capable of TF synthesis in vitro after exposure to the changed blood stress , thrombin, lip polysaccharide (LPS), tumor necrosis factor-a(TNF-a) and others. Therefore, to investigate the dangerous factors influent the expression of TF in endothelial cells is the significance to prevention of the vascular endothelial and the thrombosis. Angiotensin II (Ang II), the primary effectors of the rennin- angiotensin system (RAS), is multifunctional hormone that plays an important role in vascular function. Evidence suggests that it can reduce the TF expression in rabbit arteries.The influence of Ang II to induce the expression of TF to damage the endothelial cells and reduce normal antithrombsis is not clear. Therefore, our study to investigation of the increasing concentrations of angiotensin II-induced the effect of tissue TF coagulant activity, antigen and mRNA in HUVECs and the influent of Irbesartan, which is a angiotensin II 1 type receptor blockade by cultured HUVECs. Our object to clarify theinfluence of endothelial cells stimulated by Angll and the Irbesartan whether to prevent/inhibit the ATi binding to Angll, and reduce expression of TF, to damaged the endothelial cell .It could be effect the antithrombic of endothelial and study the new therapy of atherosclerosis.Methods and ContentsThe HUVECs were obtained the health umbilical cord veins. The cells were seeded into 25cm2 diameter dishes and maintained in M-199 containing 20% FCS, 2mmol/L L-glutamine, lOOU/mL penicillin and lOOug/mL treptomycin,40ug/mL ECGF and 5 mg/mL heparin,in humidified atmosphere of 5%CC>2 at 37 °C .Cultured medium was refreshed every other day. The HUVECs were cultured until the third passage. Purity of the endothelial cell monolayer was confirmed by the cobblestone morphological pattern and by cell staining with a monoclonal antibody specific for Vffl. Cells were used between passage numbers 3 and 5 and suspended according to the supposed of experience. The experiences were disposed of three groups. The A part: to invest the procoagulant activity and antigen of TF increasing concentration of Ang II in HUVECs. B part: to invest the procoagulant activity and antigen of TF increasing concentration of Irbesartan with or without Angll in HUVECs. C part: to invest the expression of TF mRNA stimulated by various concentration Angll with or without increasing Irbesartan. The cell viability was determined by trypanblau and MTT. The procoagulation activity was assessed by a 1-stage clotting assay. TF antigen was determined by the ELASA ( Enzyme-Linked Immunosorbnent Assay ) imubed TF kit. TF mRNA expression in HUVECs was analyzed by RT-PCR (Reverse-Transcriptase-polymerase chain reaction). All the results were repeated four times and given as mean±SD. The results were analyzed by SPSS 11.0 and performed 1-way ANOVA. Statistical significance was considered as P<0.05Results1. The different groups of cell viabilities measured by MTTThe results measured by MTT showed that the Angll at different concentrations(10"9N 10'7mol/L and 10"5) had no effects to the cell viabilities (P>0.05) in HUVECs compared with the control group (86.26±11.52,83.02±18.13 and 83.81± 16.27 vs 82.06±12.25) % .The cell viabilities also had no effect in HUVECs stimulated by only Irbesartan, respectively comparing with the control group in HUVECs. Co-cultured the increasing concentration of Irbesartan with 10-7umol/L Angll, the cell viabilities were (82.37 ±10.51) %> (79.45 + 13.54) % ,(84.67 ± 12.52) % and had no changed compared with the control group (P>0.05) .2. The effects of Angll on the tissue factor procoagulant activity expression in HUVECs and intervention of IrbesartanThe effects of TF procoagulant activity had highly increased with induced-increasing Angll. The TF procoagulant activities were (36.12 + 9.21) AU,(68.21 + 14.31) AU and (82.21 + 24.31) AU comparing with control group(P<0.05). The higher concentration of Angll, the higher procoagulant activity of TF. However, there was no change in the stimulated only Irbesartan comparing with control group. The TF procoagulant activity was highly decreased in the group of increasing concentration Irbesartan combined with Angll after coculted 6 hours (54.79 +15.32> 41.71+8.91 fP 23.47 + 10.13 vs control group, P<0.05) .3. The effects of Angll on the tissue factor antigen expression in HUVECsand intervention of IrbesartanCompared with control group, the increasing concentration of Angll-induced of TF antigen were increased(332.29±9.32pg/mL, 573.81+53.18 pg/mL and 859.12±23.98 vs 134.38±9.92)pg/mL, However, there was no changes in the stimulated only Irbesartan comparing with control group. The TF antigen was highly decreased in the group of increasing concentration combined with Angll after occulted 6 hours (588.90 + 43.11) pg/mL> (434.13 + 31.43) pg/mL and (312.45 + 37.18) pg/mL, (P< 0.05).Compared with Angll group, the tissue factor's quantity expressed on HUVEC in the Irbsartan pretreatment is lower in concentration independent way.Compared with control group,the HUVECs TF expression is higher in Irbesartan pretreatment group.4. The effect of Angll on the HUVECs' TFmRNA expression and the intervention of IrbesartanRT-PCR indicated the TF mRNA expression began to increase at the AngII( 10"9mol/L).Furthernomre, TF mRNA TF/GAPAH was highly increased at the other groug of AngII(0.194 + 0.069> 0.534 + 0.027 and 0.627+ 0.034)(P< 0.05); and there were sharply decreased stimulated by Angll cocultured with amounts of Irbesartan (P>0.05) .ConclusionOur findings indicate that Angll can stimulate the expression of TF mRNA, preduction of TF antigen and increase of TF procoagulant activity. The effect of Angll on the HUVECs TF expression maybe is mediated by ATi receptor. Moreover, Irbesartan inhibite the Angll-induced expression and production of TF may be through blockade ATi receptor.lt is considered as one of Irbersantan' antithrombosis mechanism.