Signal Transduction Pathway In VSMC Proliferation Induced By Autoantibodies Against AT1-receptor From Hypertensives

Background and purpose: Agonistic AT1 receptor autoantiboies(AT1-AA) have been discovered in malignant hypertensive and preeclamptic. Some studies had showed the AT1-AA been involved in the mmunopathogenesis of hypertension. Our studies were to certify further the agonist-like activity of these autoantibodies similar to AngII (such as cell proliferation) in the pathogenesis of hypertension and investigate the signal transduction pathway caused these effects.Methods: After collecting the serums with primary hypertension whose serum AT1-AA titer was above 1:80(tested by ELISA) and purifying the AT1-AA by ammonium sulfate precipitation and affinity chromatograph, we detected the autoantibody titer by ELISA with the 96-well microtiter plates overlayed the synthetic peptide according to the second extracelluar peptide of AT1 as the antigen. Then the AT1-AA purified(1:40)were prepared to judge their effect on VSMC proliferation by the method of bromodeoxyuridine incorporation, protein expression of NF-κB and phosphorylation JAK-STAT by western blotting, binding activation of NF-κB by electrophoretic mobility shift assay. Meanwhile we erected different treatment groups.Result: AT1-AA caused a significant increase in BrdU incorporation similar to AngII during 0-24h and the most obvious was in 12h. The OD value of in 450nm was higher in AT1-AA groups(0.236±0.0118) than AG490+AT1-AA groups(0.176±0.0088), Losartan+ AT1-AA groups(0.119±0.0059),PDTC+AT1-AA groups(0.172±0.0096) and Serum Free groups(0.127±0.0064). Western blotting showed that expression of NF-κB, phosphorylation JAK2, STAT1 and STAT3 molecules in VSMCs had an more obvious increase in the AT1-AA group than in control group; the level of NF-κB in nucleic protein was high at 15min and 120min while the phosphorylation STAT1 and STAT3 in total protein is at 30 and 15min respectively. The activation of STAT3 in response to AT1-AA was more intensive than STAT1 and the gray relative ratio of pSTAT1/STAT1 and pSTAT3/STAT3 at peak time were accordingly 0.387±0.0114 and 0.734±0.0018. Meanwhile EMSA showed that the AT1-AA increased NF-κB activation similar to AngII group, compared with the control group, losartan group and PDTC group.Conclusion: Our results demonstrated that the purified AT1-AA could cause proliferation of VSMC via AT1 receptor and activation of NF-κB and JAK-STAT signal molecules was in this effect. In addition, STAT3 was the main activatory molecule in JAK-STAT signaling pathway. Our studies therefore suggested that AT1-AA have an agonist-like activity effect similar to Ang II but not all in signal transduction mechanism.