| Background: Percutaneous coronary intervention(PCI) is another revolutional breakthrough to treat coronary atherosclerotic heart disease after coronary artery bypass graft(GABG). However, the postprocedural restenosis(RS) is the main clinical problem which influcences long-term efficacy. The RS rate after drug-eluting implanation in single de nove lesions is also about 10% at 6 month, and further evidences for long-term efficacy and safty, also in high-risk subgroups are needed. Therefore, it's still an important topic to explore effective methods to prevent RS. Neointimal hyperplasia is the major pathogenesis of RS, which results from proliferation, migration of vascular smooth muscle cell(VSMC) and extracellular matrix(ECM) deposition intrigued by vascular injury. Recently studies have found that Ang II 2 type receptor(AT2R) can antagonize the above-mentioned pathogenesis. It has been proved that transducing AT2R gene into rat balloon injury carotid arteries can attenuate neointimal hyperplasia by our laboratory. Normally the AT2R is expressed at low levels in adult and injury vasculature. It's very important to regulate the expression of AT2R gene according to our need when utilizing transgenic technology. Recently it has been found that mesenchymal stem cell(MSC) can differentiated into VSMC or endotheliocyte cell and then influence the vascular endothelial repair. MSC offer many advantages for developing transgenic therapeutics: ease of isolation, expansion potential, multipotential to differentiate, the low immune response, easy to obtain exogenous gene and so on.Objective: The purposes of the present study are as follows: 1) To study the effect of direct contact with VSMC on transdifferentiation of MSC; 2) To construct MSC of Dual-stable expression of AT2R gene medicated by tetracycline regulatable system.; 3) To investigate the effects of AT2R gene medicated by tetracycline regulatable system on neointimal hyperplasia in rat carotid arteries after balloon angioplasty. Method:①Rat MSC and VSMC were cultured and identified, respectively. MSC were labeled with DAPI firstly, and then co-cultivated with VSMC. The changes of morphology and ultrastructure of co-cultured cells were observed. Immunfluorescence analysis was performed by using monoclonal antibodies against specific antigen.②We established the regulatable system in two steps: a stable MSC line expressing rtTA has been constructed and characterized firstly by transfected with pUHD 17-1hyg and then selected by hygromycin B; in a second step, this line was used for trandfer the AT2R gene to MSC to get the well establishing double stable MSC lines;③The expression of AT2R regulated by doxycycline was evaluated by western blot;④The MSCs were transduced into rat carotid arteries with regulatable AT2R gene after the establishment of rat carotid balloon injury restenosis model. The intimal/medial area(I/M) ratio were measured by digital analysis system. The expression of AT2R and extracellular matrix related genes were assessed by RT-PCR, immunohistochemistry and immunfluorescence strategy analysis. TUNEL method was used to detected the apoptosis in the artery.Results:①The MSC cocultured with VSMC expressed smooth muscle a-actin, calponin and CD31, no cells positive for calponin and CD31 were detected in the control group; and a lot of filaments were observed in the co-cultured MSC by electron microscopy.②We gain dual-stable expression of AT2R gene medicated by doxycycline regulatable system of mesenchymal stem cells. The expression rate of AT2R in double stable MSC was increasing significantly by addition of doxycycline in a concentration-dependent manner by western blot, and the high expression was detected at 48 hours after induced by doxycycline and could keep up over 8 weeks.③MSCs with AT2R gene regulated by doxycycline delivered into injury carotid arteries significantly up-regulated AT2R mRNA and protein expression in neointima from day 14 to 28 after injury (P<0.01),and reduced I/M ratio significantly(P<0.01).④MSCs with AT2R gene regulated by doxycycline significantly decreased the expression of ECM including matrix metalloprotease 2(MMP-2), fibronectin(FN), laminin(LN), and osteopontin(OPN) at 14 and 28 day after injury, but increased the expression of collagenⅣ(COL-Ⅳ) at 14 day.⑤There were more cellular apoptosis in the neointima in the group of MSCs with AT2R gene regulated by doxycycline(P<0.05).Conclusion:①Cell-to-cell contact can induce rat MSC to differentiate into vascular smooth muscle-like cell and endotheliocyte-like cell.②The well established double stable MSC lines make the AT2R gene under the active control of doxycycline and can be used as a good model to study the biological effect of AT2R.③The expression of AT2R gene can be regulated efficiently in vivo by local delivery of MSC with dual-stable expression of AT2R gene, thus reduces the formation of intimal hyperplasia.④AT2R gene can influence the expression of ECM and significantly promote the cellular apoptosis in the injury rat carotid artery.⑤These data demonstrate the clinical potential of AT2R regulatable expression to prevent restenosis after PCI.
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