Adenovirus Mediated At2r Turn The Gene On Biological Behavior Of Vascular Smooth Muscle Cells

Background: The well known diversit of angiotensin II (AngII) action is due to the diversity of its receptors. AngII acts on at least two receptor subtypes: type I and 2 (AT1 & AT2). Although the physiological role of the AT2 receptor is still poorly defined, it may be implicated in inhibition of the proliferation and migration vascular smooth muscle cell (VSMC). which play a major role in the pathological changes of restenosis after angiopiasty. But AT2 receptor is only expressed abundantly in the fetal vasculature with rapid decline after birth. and the level of re-expression in the adult vasculature after injury is very low. So there are little understanding about the actions of AT2 receptor in vascular diseases, especially in the restenosis after injury. We hypothesize that the low- or non-expression of AT2 receptor in adult and injury vasculature is one of the important reasons of the initiation and development of restenosis after angioplasty. It may result in inhibited proliferation and migration of VSMC, and induced the occurrence of VSMC apoptosis to increase the level of AT2 receptor expression.. This result may be good for the improvement of the restenosis. In this study, AT2 receptor gene was transferred into the cultured VSMC mediated by recombinant adenoviral vector and then its action on the bio-behaviours such as proliferation, migration and apoptosis of VSC detected. Objective: The purposes of present study are as follows: I) to construct the adenoviral vector to transfer AT2 receptor gene; 2) to found the cell model of rat VSMCs to express AT2 receptor; 3) to investigate the effect of AT2 receptor on the proliferation, migration and apoptosis of VSMC in vitro; 4) to study the II effect of some interfering agents such as angiotensin II, AT1 receptor antagonist (CV1 1974) and AT2 receptor antagonist (PD 123319) on the expression and action of AT2 receptor. Method: The VSMCs. gotten from the aorta of rat. were cultured by routine method according to previous studies. Recombinant adenoviral vector. AdCMV-AT2R, containing rat AT2R gene was constructed by homologous recombination, and then it was used to transfer AT2R gene into VSMC. The VSMC expressed AT2 receptor gene was used as a cell model to study the function on the cells in vitro. The rate of AT2R expression in VSMC was analyzed by flow cytometry. The expression of mRNA and protein of AT2R was detected by RT-PCR, Western blot and itnmunohistochemistry respectively. At the same time, AT1 receptor was also determined with the same methods. To detect the change of VSMC proliferation features. flow cytometry was used to analyse the cell cycle of VSMC, and the assays including cell devision index, incorporation of 5-bromodeoxyuridine (5-BrdU) and 3-(4.5-dimethyl-thiazol -2-vl) 2,5- diphenytetrazolium bromide (MTT) were used to determine the proliferation of VSMC, respectively. The modified Bovdens chamber method was used to test the number of VSMC migration. The re-organization and expression of F-actin in VSMC was analyzed by confocal microscopy. Apoptosis in cultured VSMC was quantified by flow cvtometry and terminal deoxynucleotide transferase-mediated dUTP nick end-labeling(TUNEL) in situ. The expression of bcl-2 and bax mRNA was also analyzed by RT-PCR assay. All above indexes were also determined by the same methods after CV11 974, PD 123319 and the different concentration of AngII. given...