ACE2 Inhibit The Expression Of AT1R By Increasing The Expression Level Of MasR In Vascular Smooth Muscle Cells

Objective To investigate the level of Mas receptor(Mas R),AT1 receptor(AT1R),signal transducer and activation of transcription 3(STAT3)and ERK1/2phosphorylation and cell migration after angiotensin-converting enzyme 2(ACE2)gene transfer on vascular smooth muscle cells(VSMCs).Analysis the relationship between Mas R and AT1 R in VSMCs.Methods 1.The murine full-length cDNA of ACE2 was amplified from RNA of mouse kidney by RT-PCR.The amplified product was cloned into a p CDH-CMV-MCS-EF1-cop GFP-Puro vector,and confirmed by enzyme digestion and sequencing.2.VSMCs were divided into 9 treatment groups such as Control group,LV-GFP group,LV-ACE2 group,LV-ACE2+Ang II(10-7 mol/L)-treated group,LV-ACE2+Ang II(10-7mol/L)+A779(10-6 mol/L)-treated group,LV-ACE2+A779(10-6mol/L)-treated group,Ang II(10-7 mol/L)-treated group,Ang-(1-7)(10-6 mol/L)-treated group and A779(10-6 mol/L)+ Ang-(1-7)(10-6 mol/L)-treated group.After incubation for 96 hours,the expression levels of Mas R,AT1 R,STAT3(signal transducer and activation of transcription 3)and ERK1/2 pathway were measured by Western blot.In addition,VSMCs' migration was assessed by Transwell chamber assay.Results Overexpression of ACE2 leads to the up-regulation of Mas R and down-regulation of AT1 R in VSMCs.Treating VSMCs with Ang II increases both the protein levels of Mas R and AT1 R,while inhibiting Mas R with A779 leads to the down-regulation of Mas R and up-regulation of AT1 R.In contrast,activating Mas R with Ang-(1-7)increased the expression level of Mas R and decreased the expression level of AT1 R although the effect was limited in a time frame of 4-6 h post the treatment.The expression level of Mas R in the LV-ACE2 group,LV-ACE2+Ang II-treated group,LV-ACE2+Ang II+A779-treated group,Ang-(1-7)-treated group and Ang II-treated group was higher than that in the LV-GFP group.AT1 R and signal STAT3,ERK1/2 phosphorylation expression level was decreased in LV-ACE2 group and Ang-(1-7)-treated group,while it was increased in Ang II-treated group.Similarly,VSMCs' migration were increased in the Ang II-treated group compared with the Control group and LV-GFP group,decreased in LV-ACE2 group,LV-ACE2+Ang II-treated group,LV-ACE2+Ang II+A779-treated group,LV-ACE2+A779-treated group but normalized in the Ang-(1-7)+A779-treated group.Conclusion ACE2 may inhibit the expression of AT1 R and the migration of VSMCs by increasing the expression level of Mas R in VSMCs.