VEGF₁₆₅Gene Transfection Prevents The Restenosis Of Vein Grafts

Objective: To study the effect of VEGF165 (vascular endothelial growth factor 165) gene transfection by adenoviral-mediated to prevent the restenosis of vein grafts.Methods: The adenoviral shuttle plasmid pUC19-hVEGF was cotransfected together with the vector pDC315 into HEK293 cells by liposome-mediated. Human umbilical vein endothelial cells (ECV304) were cultured and infected by adenovirus pUC19-hVEGF. The efficiency of transfection and expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The effect of adenovirus on ECV304 proliferation was examinedby methyl thiazoly tetrazolium (MTT) assay. The models of carotid-jugular bypass were established in swine. Vein grafts were placed into the high titer (1 X 109 pfu/ml) adenovirus solution for VEGF165 gene incubation transfection under 37C temperature for 1 hour. In different postoperative time, the vein grafts were harvested. The expression of VEGF165 was examined by RT-PCR and immunohistochemistry. Thickness of neointima formation was assessed by histopathologic analysis.Results: The recombinant adenovirus pUC19-hVEGF was successfully constructed. PCR amplified both the fragment of VEGF165 (576 bp) and the special fragment of adenoviral backbone genome (860 bp) ; Restriction maps showed that VEGFies cDNA was inserted into the downstream of CMV(cytomegalovirus) promoter; It was confirmed that the sequence of VEGFies cDNA was identical to that reported in Genbank by gene sequensing. The adenovirus titer reached 1.6 X1012 pfu/ml(plaque forming unit per milliliter) and purity was 1.48. In the infected ECV304 cells, RT-PCR amplified a258 bp band representing VEGF165 mRNA and the expression of VEGF165 mRNA depended on the multiplicity of infection (MOI). The positive rate of VEGF165 protein rose when MOI increased. When M0I= 128 pfu/ml, The positive rate reached the peak, 30. 6%. VEGF165 protein was detected in the supernatants of infected ECV304 cells. The amount of product depended on time. MTT assay showed that infected ECV304 cells grew obviously quickly. After 2-3 days, ECV304 cells rapidly proliferated into logarithm growth period and cell doubling time shorten to around 3. 5 days. The result of RT-PCR suggested that VEGF165 mRNA expressed in the vein grafts transferred by adenovirus pUC19-hVEGF. The level rapidly increased and went up to the highest in 9 days postoperatively. There were positive staining granules for VEGF165 protein in intima and adventitia of the vein grafts transferred by adenovirus pUC19-hVEGF. The positive expression of VEGF165 protein remarkably increased after 10 days and kept on to 4 weeks later. Histopathologic images demonstrated that pUC19-hVEGF stimulated endothelial cells proliferation to repair theinner layer and the complete endothelialization was established in 10 to 14 days. Morphometric analysis showed that the thickness of neointima was significantly less for VEGF165-transferred group than control group. Conclusion: VEGF165 gene transfection stimulates endothelial cells proliferation, inhibits intimal hyperplasia and prevents the restenosis of vein grafts.