Effects Of Adenoviral Vector-mediated AT2R Gene Introduction On Neointimal Hyperplasia After Rat Carotid Atrtery Balloon Injury

Background:Despite the dramatic technological advances in coronary intervention, restenosis (RS) remain a challege for clinical cardiologists. The development of antirestenotic treatments is an area of intense research activity. Balloon expandable stents have been used to relieve RS. Although lumen enlargement is achieved in almost all case, stent-mediated chronic inflammation, cellular proliferation contribute to in-stent restenosis(ISR).Intracoronary brachytherapy is demonstrated to reduce the frequency of in-stent restenosis, but late thrombosis and edge effect dampen enthusiasm for this technique. Drug-eluting stent has emerged as a promising therapy to prevent RS, The RS rate after eluting stent implantation in single de novo lesions is less than 10% at 6 month, but further evidence for long-term efficacy and safety, also in high-risk subgroups are needed. Neointimal hyperplasia is the major pathogenesis of RS, which results from phenotypic modulation, proliferation, migration of vascular smooth muscle cells(VSMCs) and extracellular matrix depoition intrigued by vascular injury. AngⅡin local tissue may participate in the development of neointimal hyperplasia, AngⅡ2type receptor(AT2R) is ubiquitous in fetal tissues and disappear after birth ,while under some pathological cardiovascular conditions such as vessel injury ,inflammation, it can be detected ,Therefore AT2R is postulated to attenuate neointimal hyperplasia . AT2R mediates growth-inhibition, migration-suppressing and apoptosis-promoting, counter-balance those of AngⅡ1type receptor(AT1R). AT2R is shown to inactivate extracellular regulated kinase(ERK) and it also downregulates AT1R, platelet-derived growth factor typeβreceptor(PDGF R -β) and transforming growth factor-βtype 1receptor(TGF-β1R)expression in cells, These AT2R researches mainly do in vitro and gene knockout animal, However, the important question remains as: how about the cross-talk between AT1R and AT2R in vivo especially in pathological cardiovascular conditions ,whether AT2R can be used in RS prevention in adult. AngⅡcan activate Basic Transcriptional Element Binding Protein-2(BTEB2) expression in VSMC and promote cellular proliferation,but the effect of AT2R on BTEB2 expression is elusive. Objective: The purposes of the present study are as follows: 1) to investigate the effects of AT2R on the expression of genes related proliferation in vitro and in vivo; 2) to investigate the cross-talk between AT1R and AT2Rin vitro and in vivo; 3)to investigate the effects of AT2R on neointimal hyperplasia after rat carotid artery balloon injury Method: ①The recombinant adenovirus of AT2R was constructed by using the method of homogenous recombination in bacteria, AT2R gene was obtained from the vector of PUHD-AT2R by PCR ,and subcloned into shuttle vector of pAdTrack-CMV, then it was linearized with PmeⅠand transformed into Adeasier-1 cell. The DNA of identified recombinant plasmid was digested with PacⅠand transfected to 293 cells to package adenovirus. PCR technique was used to detect target gene . The titre and its infection of the pAdCMV/AT2R was measured with the aid of green fluorescent protein (GFP) expression. ②After the recombinant adenovirus infected primary VSMCs, the mRNA and protein expression of AT2R were evaluated by RT-PCR , immunofluorescence staining, confocal microscope and Western blot. ③The expressions of VSMCs proliferation gene, AT1R,ERK1,ERK2,BTEB2 and proliferating cell nuclear antigen (PCNA) ,were analysed by RT-PCR, dual-influorescence and Western blot; ④AT2R gene was transduced into rat carotid arteries with pAdCMV/AT2R after the establishment of rat carotid balloon injury restenosis model. The expression of AT2R and proliferation gene were assessed by RT-PCR , immunocytochemistry and dual-influorescence strategy analysis; ⑤The intimal/medial area ratio were measured by digital analysis system. Results: ①A recombinant adenovirus with GFP report gene was constructed in 292 cell, PCR test proved the recombinant adenovirus contained the insertion of AT2R . The titre of purified recombinant adenovirus was 1.5×1012pfu/ml.②Primary cultured VSMCs identified by morphology and immunocytochemistry. Among the passage 6 to 8 of cultured cells, nearly 100% were VSMCs. ③The expression of AT2R was very obvious in VSMCs and continued for 3 day since being transfected by recombinant adenovirus pAdCMV/AT2R;④pAdCMV/AT2R transfection significantly inhibited the expression of ERK1,ERK2,BTEB2,PCNA in VSMCs (p<0.01),but no influence on AT1R expression (p>0.05); ⑤In vivo, pAdCMV/AT2R transduction efficiency in carotid artery was about40% at day14, and localized to the neointima,as well as to the media and the adventitia. ⑥pAdCMV/AT2R delivered into injured rat carotid arteries significantly up-regulated AT2RmRNA and protein expression in neointima from day 7 to 21 after injury ,the expression of AT1R was no significant difference (p>0.05);⑦pAdCMV/AT2R transfection significantly decreased the expression of ERK1,ERK2 in neointima at day 7, The most inhibitory effect of pAdCMV/AT2R on BTEB2,PCNA,PDGF-βR,TGF-β1R in neointima was at day 14;⑧At day 21, compared with no transfection group and pAd-GFP transfection group, pAdCMV/AT2R transfection reduced I/M ratio significantly (0.73±0.06 vs 1.44±0.22,1.36±0.21 respectively, p<0.01). Conclusion:①AT2R gene can be efficiently expressed in cultured VSMCs which are transfected by recombinant adenovirus pAdCMV/AT2R. ②Local endovascular delivery of pAdCMV/AT2R at the injury site is able to increase the AT2R expression, thus reduces the formation of intimal hyperplasia. ③The cross-talk between AT1R and AT2R may operate via ERK signal pathway,but not via counteraction of receptor expression.④These data demonstrate the clinical potential of AT2R to prevent restenosis after percutaneous coronary intervention...