The Effects Of AT2R Gene Transfection On Neointima Hyperplasia Of Cultured Rat Carotid Artery After Injured By Balloon

Background: Percutaneous coronary intervention (PCI) is generally applied to therapy coronary atherosclerotic heart disease. However, the postprocedural restenosis(RS) is the main clincial problem which influences prostecdtive efficacy. The mechanism of postprocedural restenosis is not clear at present. It is generally regarded as a process which multiple factor and mechanism participated in. The main factors of RS includ elastic recovery of artery, thrombogenesis, remodeling of vessel wall and abnormal proliferation and migration of medial vascular smooth muscle cells(VSMCs) . Renin angiotensin system also take part in the development of RS after balloon injured. Moreover,among a number of cardiovascular mediators,the angiotoninⅡmay be important particularly. At least two subtypes of angiotonin receptor have been indentified: AT1 and AT2 which exist in the tissue of animals and human being. A lot of investigations indicate arterial intimal thickening attributed Ang Ⅱwhich mediated by AT1R.The AT2 has been shown to reverse the effects of AT1. In vitro experiments of our lab have indicated: (1) After VSMC transfeced by adenovirus vector and AT2 expressed in VSMC AT2R has inhibited the proliferation and migration of VSMC. AngII enhances such effects of the AT2R without significant concentration dependent;(2) after AT2 expressed in VSMC AT2R has hastened the apoptosis of VSMC. Such effects is not relation to AngII.The effects of inhibiting the proliferation and migration of VSMC and promoting the apoptosis of VSMC can decrease the formation of the neointinal hyperplasia of the injured artery. Previous Research is limited by cellular level. Objective: The purposes of present study are as follows: 1)to construct the adenoviral vector containing AT2R gene; 2) to investigate the effects of AT2R gene on neointima hyperplasia of rat carotid artery injured by balloon after cultured in vitro two weeks and provide data for the next experiment in vivo. Method:(1) Depend on advanced adenoviral AdEasy system the adenoviral vector containing AT2R gene was constructed by homologous recombination. Shuttle plasmid pAdTrackCMV/AT2R was linearization by enzyme and inverted into AdEasier-1 bacterium which contained supercoil adenovirus core genomic. Recombination viroplasm was selected by Kanamycin. Recombination viroplasm pAdEasy-1/AT2R transfected to 293 cell mediated by liposome . The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. (2)The artery organ culture was carried out in DMEM culture solution contain 30% fetal bovine serum and gaseous environment of 95%O2 and 5%CO2. Alive situation of the cultured artery was judged by light microscope,electron microscope and laser confocal microscopy.(3) Recombination adenoviral vector infected the rat carotid artery injured by balloon and the artery was cultured in vitro. The intimal/medial area ratio were measured. (4) The expression of AT2R in artery was evaluated by reverse polymerase chain reaction (RT-PCR),immunohistochemistry and Western blot respectively.(5)TUNEL method was used to detect the apoptosis of VSMC in the artery . Results: (1) A recombinant adenovirus carrying AT2R gene was constructed via homologous recombination in AdEasier-1 bacterium. The recombinant adenovirus proved correct by PCR and fluorescent microscope. Plaque titration on 293 cells showed titers of 1.5×1012 pfu/ml. (2) The artery was proved still alive by light microscope,electron microscope and laser confocal microscopy after cultured in DMEM culture solution contain 30% fetal bovine serum and gaseous environment of 95%O2 and 5%CO2 for tow weeks. (3) There was neointima hyperplasia of the rat carotid artery injured by balloon after cultured 14 days. (4) The green fluorescence was observed with fluorescent microscope after the injured rat carotid artery infected by recombinant adenovirus and cultured for 3 days. (5) There was no difference between the three teams after the injured rat carotid artery cultured for 7 days. At day 14 after cultured, compared with untreatment and treatment with PAd-GFP, treatment with PAdCMV/AT2R reduced I/M ratio significantly .It is indentified that PAdCMV/AT2R transfection significantly inhibited neointima hyperplasia of the ratcarotid artery injured by balloon after cultured 14 days. (6) The results of RT-PCR, immunohistochemistry and Western blot showed that the expression of AT2R mRNA and protein were detected after artery cultured 3days and increased after artery cultured 7days and 14days respectively.There were no expression of AT2R mRNA and protein in the other two team. (7) There were more apoptosis of VSMCS in the neointima and of the PAdCMV/AT2R team than the other two team. Conclusion: The results in this study suggest that (1)The model of artery organ culture in vitro is established. The artery is still alive after cultured two weeks;(2) AT2R can be efficiently expressed in cultured artery which are transfected by recombinant adenovirus PAdCMV/AT2R. (3) AT2R can significantly promotes the apoptosis of VSMCs in the injured rat carotid artery and also reduces the formation of intimal hyperplasia. These data demonstrates the clinical potential of AT2R to prevent restenosis after percutaneous coronary intervention.