| IntroductionCollagen is the main structural protein in extracellular matrix (ECM), andmainly generated from vascular smooth muscle cells (VSMC) which distributingin endarterium. Among all subtypes of collagen, type I and III collagens are themost common subtypes in vascular wall. A balance of collagens synthesis anddegradation is the key factor for maintaining ECM, enhancing plaque stability andreducing rupture vulnerability.It is well known that prolyl4-hydroxylase(P4H) is the key intracellularenzyme of all known types of collagen, and plays a very important role in collagenmetabolism. P4H is a tetramer with two α subunits and two β subunits, of which, αsubunit mainly determines the activity of enzyme. Therefore, P4Hα1, as thecrucial rate-limiting enzyme, is closely related to the production of collagen.Overexpression of P4Hα1could increase collagen production and the inhibition ofP4Hα1would lead to collagen degradation.In vivo rennin-angiotensin system plays a central role in the development ofatherosclerosis. AngiotensinII (AngII) can cause vasoconstriction, VSMCproliferation, hypertrophy and induce inflammatory response. It has been reportedthat AngII inhibit the activity of P4Hα1and the synthesis of collagens, promotethe degradation of ECM, and finally lead to plaque vulnerability and rupture. Onthe contrary, ACE2can convert AngII to Ang(1-7). Ang(1-7) is a endogenousantagonist factor, acting on AngII AT1acceptor and has the oppose effect to AngII. Previous studies have been reported that Ang(1-7) may stimulate vasodilation,antiproliferative, antihypertrophy and anti-inflammatory, thus indicated Ang(1-7)plays the anti-atherosclerotic effect in the progression of atherosclerosis.Uniformly, our prior studies showed that overexpression of ACE2also exerted astrong antiinflammatory effects to increase collagen production and keep plaquestability.Objective1.To investigate whether ACE2enhances the P4Hα1expression and collagenproduction in vitro.2.To elucidate the mechanisms of ACE2effects on P4Hα1and collagen invitro.MethodsPreparation of ACE2Adenovirus VectorsThe ACE2cDNA was amplified by RT-PCR from RNA of mouse kidney. Then,recombinant adenoviruses carrying the ACE2(AdACE2) or a control transgeneEGFP (AdEGFP) were created with the AdMax system (Microbix Biosystems).Cell treatmentThe cells were treated with10-6mol/L AngII for24hours, then they werestimulated by different ways.Cell stimulationTo identify ACE2regulation on P4Hα1, we treated HASMCs with106pfuAdEGFP for24hours and106pfu AdACE2for24,48and72hours before cellswere harvested for experiments. To investigate Ang(1-7) effects on P4Hα1,HASMCs were treated with10-6mol/L Ang(1-7) for2,4,8,16,24hours in timecourse study, or treated with10-5mol/L,10-6mol/L and10-7mol/L Ang(1-7) for24hours in dose dependent experiment. To elucidate the role of A779, we treated HASMCs with A779as standard treatment, the stimulations for cells as follows:106pfu AdACE2，106pfu AdACE2add10-6mol/L A779,10-6mol/L Ang(1-7),10-6mol/L Ang(1-7) add10-6mol/L A779,10-6mol/L A779. After24hours, cellswere gathered for experiment.Quantitative Real-Time RT-PCRTotal RNA was extracted from HASMCs according to the manufacturer’sinstruction. After they were reverse-transcribed, The levels of P4Hα1mRNA wasmeasured with quantitative realtime RT-PCR and were calculated from the value ofthe threshold cycle (Ct) of the real time PCR collimated by that of β-actin.Western BlotThe total cell proteins were extracted from HASMCs after treatment, separatedby10%SDS-PAGE and transferred to nitrocellulose membranes. After incubationby primary antibody and secondary antibody, the bands were visualized after theywere detected.ELISASupernatants were collected after the treatment. The levels of type I and IIIcollagen were analyzed by ELISA according to manufacture’s instruction.ResultsACE2Enhances the Expression of P4Hα1In the time-course study, we treated HASMCs with106pfu AdACE2for24,48and72hours,106pfu adEGFP for24hours. Then, cells were ingathered, andRT-PCR was carried out. For RT-PCR, it showed that the level of P4Hα1mRNAincreased distinctly after24hours treatment compared with control group andAdEGFP group. However, there was no obvious difference between48and72timepoints among all the groups.Ang(1-7) Promotes P4Hα1Expression To clarify the effect of Ang(1-7) effects on P4Hα1, HASMCs was treated with10-6mol/L Ang(1-7) for2,4,8,16,24hours, then cells were harvested and P4Ha1mRNA was measured. We found P4Hα1mRNA level was significantly increasedafter16hours treatment, and reached the peak level at24hours point.In the dose dependent experiment, we treated HASMCs with Ang(1-7) for10-5mol/L,10-6mol/L and10-7mol/L for24hours, according to the result, P4Hα1mRNA expressions could be up-regulated by10-5mol/L Ang(1-7) treatment andincreased gradually in a dose dependent manner with the administration ofAng(1-7).A779reversed ACE2and Ang(1-7) effects on P4Ha1ExpressionTo study the role of A779inhibition on ACE2and Ang(1-7) regulations onP4Hα1, we treated HASMCs with adACE2and Ang(1-7) for24hours with orwithout Ang(1-7) inhibitor A779. According to RT-PCR results, we found P4Hα1mRNA levels were significant higher in ACE2and Ang(1-7) treated groupscompared with other groups, while P4Hα1levels was much higher in Ang(1-7)group than in ACE2group. Administration of A779reversed the up-regulationeffects on P4Hα1by ACE2and Ang(1-7), however, much more P4Hα1expressionswas observed in ACE2+A779group than in Ang(1-7)+A779group. In comparisonwith control group, there was no obvious change of P4Hα1productions inAng(1-7)+A779group, however, P4Hα1levels was decreased in HASMCs treatedwith A779only. Similar results have also been proved by using western blot todetect P4Hα1mRNA.ACE2increase the production of collagenThe results of ELISA indicated that the levels of type I and III collagensincreased significantly in HASMCs treated with ACE2or Ang(1-7), administrationof A779abolished about60%of ACE2and nearly100%of Ang(1-7) regulations on P4Hα1.Conclusions1.ACE2can enhance the expression of P4Hα1and the production of collagen.2.ACE2enhances P4Hα1expression both by depressing AngII and increasingAng(1-7), while the collagen synthesis will increase accordingly. |
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