Angiotensin-convertingenzyme2Gene Transfer Inhibits The AT1Receptor Expression And STAT3Signal Pathway In Smooth Muscle Cells

Objective To explore the expression of angiotensin Ⅱ type1receptor (AT1R) signal transducer and activator of transcription3(STAT3) phosphorylation and the level of cell proliferation after recombinated lentiviral angiotensin-converting enzyme2vector transfered on vascular smooth muscle cells (VSMCs).Methods1. Tissue explant technique was used to culture vascular smooth muscle cells (VSMCs);2. cell groups:control、Ientiviral-GFP、Ang Ⅱ、lentiviral-ACE2+Ang Ⅱ、Irbesartan+Ang Ⅱ、lentiviral-ACE2+Irbesartan+Ang II lentiviral-ACE2;3.Recombinated lentiviral angiotensin-converting enzyme2(ACE2) vector、Angll and Irbesartan were added into VSMCs, then ACE2and Angiotensin Ⅱ type1(AT1) receptor mRNA expression were detected with quantative real-time-PCR;4. ACE2、AT1R protein expression and STAT3protein phosphorylation were detected with western blot after ACE2、Angll and Irbesartan were added;5.The proliferation of VSMCs after ACE2gene transfection was determined with CCK-8Kit.Results1.After recombinated lentiviral-ACE2vector was transferred into VSMCs, ACE2mRNA expression was increased in MOI dependence, when the MOI of Lentiviral-ACE2was10, ACE2mRNA expression was highest, vs control and other transferred groups (p<0.05);2. After Recombinated lentiviral-ACE2vector was transferred into VSMCs, ACE2mRNA expression was increased in time dependence, When the Lentiviral-ACE2transferred for96hours, ACE2mRNA expression was highest, vs control and other transferred groups (p<0.05);3.The AT1receptor mRNA expression and STAT3protein phosphorylation induced by Angiotensin Ⅱ were increased in dose dependence, when the concentration of AngⅡ was10-7mol/L, the AT1receptor mRNA expression and STAT3protein phosphorylation were highest, vs control and other groups (p<0.05);4. The AT1receptor mRNA expression and STAT3protein phosphorylation induced by Angiotensin Ⅱ were increased in time dependence, when Angll intervened VSMCs for8hours, the AT1receptor mRNA expression and STAT3protein phosphorylation were highest, vs control and other groups (p<0.05);5. Quantative real-time-PCR detected that AT1receptor mRNA expression was reduced in the presence of Ang Ⅱ after ACE2overexpression, vs Angll group (p<0.05). When Irbesartan intervened, cooperated with ACE2, the AT1receptor mRNA expression in the presence of Ang Ⅱ was reduced remarkably, vs AngⅡ group (p<0.05);6. Western-Blot showed that AT1receptor mRNA and protein expression were reduced in the presence of Ang Ⅱ after ACE2overexpression, vs AngⅡ group (p<0.05). Similar to AT1receptor mRNA and protein expression, the signal STAT3phosphorylation was obviously inhibited while ACE2overexpression, vs Angll group p<0.05).Besides, when Irbesartan intervened, cooperated with ACE2, the AT1receptor protein and STAT3phosphorylation expression in the presence of Ang Ⅱ were reduced remarkably, vs AngⅡ group (p<0.05);7.CCK-8Kit detection foud that ACE2gene transfer inhibit significantly the growth of VSMCs in the presence of Ang Ⅱ.Conclusion1.Overexpression of ACE2gene was able to inhibit AT1receptor expression and signal pathway of STAT3phosphorylation.2.ACE2inhibited the growth of VSMCs.3.ACE2inhibited the growth of VSMCs by downregulating AT1receptor expression and signal pathway of STAT3phosphorylation.4. Irbesartan, cooperated with ACE2, inhibited the AT1receptor protein expression. It is indicating that there maybe exsit the direct action of ACE2on AT1receptor in cultured VSMCs.