Effects Of Adenovirus-mediated Gax Gene Transfection On Biological Behaviour Of Vascular Smooth Muscle Cells And It's Mechanism

Background and objective: Homeobox genes regulate growth, differention and migration at a cellular level during embryogenesis. The homeobox gene gax (growth arrest homeobox) is mainly expressed in cardiovascular tissue. Gax expression in the vessel wall is rapidly down-regulated following a balloon injury that leads to vascular smooth muscle cell (VSMC) proliferation. In cultured VSMCs gax expression is also down-regulated during the Go to G1 transition of the cell cycle following mitogen activation, and the extent of down-regulation correlates with the mitogen's ability to stimulate DNA synthesis. This pattern of gax expression in VSMCs is similar to that of gas (growth arrest-specific) genes and gadd (growth arrest and DNA damage-inducible) genes, some of which appear to function as negative regulators of the cell cycle, so gax gene might inhibit VSMCs from growth and proliferation. The abnormal proliferation, migration and apoptosis of VSMCs are of particular interest because they are important in neointima formation of restenosis lesion after angioplasty. This experiment is designed to investigate the effects and it's mechanism of recombinant adenovirus vector encoding the gax gene on biological behaviour of VSMCs such as proliferation, migration and apoptosis, and explore the role and significance of gax gene overexpression in restenosis after angioplasty.Methods: 1. Replication-defective adenovirus type 5 (Ad5) vector encoding the rat gax gene was constructed via site specific recombination and amplified in 293 cells. The resulting adenovirus was purified for a high viral titer. 2. PrimaryVSMCs were derived from the aortic explants of Wistar rats. 3. The expression of gax mRNA and protein was evaluated by a series of methods such as reverse transcription polymerase chain reaction (RT-PCR), flow cytometry and immunohistochemical technique. 4. The effects of gax overexpression on VSMCs proliferation, migration and apoptosis were observed by a series of methods such as 3-(4,5-dimethyl-thiazol-2-yl)2,5-diphenytertrazolium bromide (MTT) colorimetric analysis, 3H-thymidine incorporation, flow cytometry, modified Boyden's chamber and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL). 5. Immunocytochemical stain and RT-PCR were employed to observe the expression of p21, p27, proliferating cell nuclear antigen (PCNA), cyclin dependent kinase 2 (CDK2), connexin43(Cx43), matrix metalloprotease 2 (MMP2), osteopontin, Bax and Bcl-2 after gax gene was transferred to VSMCs.Results: 1. Shuttle plasmid pDC316-gax containing gax expression cassette was constructed by recombinant DNA technique, and cotransfected into 293 cells with adenovirus genomic plasmid pBHGlox AEl,3Cre for site specific recombination by liposome. The resulting replication-defective recombinant adenovirus AdCMV-gax containing gax expression cassette was amplified and purified. The titer of the virus stocks was 5x1010 plaque forming units per milliliter (pfu/ml), and the optimal multi-plicity of infection (MOI) was 50.2. Cultured rat aortic cells were idenfied as VSMCs by morphology and Immunocytochemical stain.3. When AdCMV-gax was transfected to VSMCs, the expression of gax protein increased significantly in VSMCs , and reached 80% in 24 hours. High expression of gax protein could keep for over 5 days.4. Before AdCMV-gax was transfected to VSMCs, gax mRNA and protein could be down- regulated by platelet-derived growth factor (PDGF) BB, and Gax protein was reduced to 22.83% from 36.42% (P0.05) even treated withPDGF-BB 2 ng/ml. This concentration was well within physiological range. The extent of gax down-regulation correlated with the doses of PDGF-BB. After AdCMV-gax was transfected to VSMCs, the level of gax expression was significantly higher than that before transfection, and no correlation with the stimulation of PDGF-BB.5. The optical density (OD) of MTT and 3H-thymidine incorporation decreased significantly in VSMCs transfected with AdCMV-gax compared with that in VSMCs non-transfe...