Construction Of A Adenoviral Vector Containing Human Vascular Endothelial Growth Factor 165 Gene And Its Expression In Myoblast

Background Vascular endothelial growth factor(VEGF)plays a key role in mediating physiological and pathological angiogenesis. Therapeutic angiogenesis by delivering VEGF holds promise as a medical treatment for patients with coronary and peripheral artery disease. However, the morphology and function of newly formed blood vessels depends on the microenvironmental amount of VEGF secreted. Continuous overexpression of VEGF can lead to deleterious effects such as edema and hemangioma formation. Controlling the timing, duration and dosage of VEGF expression could avoid deleterious effects while leading to stable and mature vessels. A tetracycline-inducible recombinant adenoviral expression system could be used to define basic parameters such as timing and level of VEGF gene expression necessary for the successful induction of mature vessels. Autologous skeletal myoblasts may restore function after myocardial infarction. Transplanted cells may also serve as vehicles for therapeutic gene delivery. In this study we attempted to generate a Tet-Off regulatable recombinant adenovirus expressing hVEGF165 (AdTRE.VEGF165),and to determine whether human umbilical vein endothelial cells and rat primary skeletal myoblasts can be infected with the AdTRE.VEGF165 secrete VEGF.Methods The construction of a Tet-off regulatable recombinant adenovirus was completed in three phases. First, a Tet-responsive mammalian expression cassette which could be regulated by tetracycline was made by subcloning the full-length cDNA encoding hVEGF165 into pTRE-Shuttle2 vector.Next, the expression cassette was subcloned into the BD Adeno-X? Viral DNA to form a adenoviral plasmid pAd.VEGF165. The recombinant plasmid was comfirmed using both of restriction analysis and PCR. Finally,the pAd.VEGF165 was packaged into infectious recombinant adenoviral particles by transfecting HEK 293 cells. skeletal myoblast cells of adult Spage-Dawley rats were primary cultured with pectoralis major explant. The cells were counted and observed by light microscopy combined with immunocytochemical identification. Human umbilical vein endothelial cells and primary rat skeletal myoblasts were infected with both AdTRE.VEGF165 and Tet-off regulatory virus. Expression was tested by Enzyme-Linked Immunosorbent Assay .Results we constructed a tetracycline regulatable recombinant adenoviruses vector AdTRE.VEGF165 containing the cDNA for human VEGF165, a secreted endothelial cell–specific angiogenic growth factor. The myoblasts culture was more than 85% pure population of cells as assessed by desmin staining. More than 95% primary skeletal myoblasts could be infected with adenovirus vector at a low M.O.I.(20 pfu/cell) measured by AdCMV.eGFP. The primary skeletal myoblasts infected with AdTRE.VEGF165 (20 pfu/cell) and Tet-off regulatory virus (20 pfu/cell) demonstrated higher VEGF165 protein secretion into the supernatant compared with uninfected group(14.80±1.15ng.ml-1·1×105cell-1·24h-1 vs. 1.39±0.36ng.ml-1·1×105cell-1·24h-1 ,p<0.01=, as measured by ELISA at 5 day after infection.Conclusions these studies demonstrated that (1) Tet-regulatable adenoviral expression systems could be used to deliver gene of VEGF into mammalian cells efficiently. (2) The primary skeletal myoblasts infected with AdTRE.VEGF165 secreted VEGF165 protein at high levels. (3)myoblasts may serve as as a platform for gene transfer of VEGF to enhance angiogenesis.