Prevention Of Vein Graft Restenosis With Widl-type P53 Gene Transfection And Laser Irradiation

Objective: Autologous sapheneous vein coronary artery bypass graft surgery is complicated by late graft failure due to neointima formation and subsequent atherosclerosis. According to former studies, the cellular events that account for the late vein restenosis include migration and proliferation of the vascular smooth muscle cells (VSMC), while the off-balance of proliferation and apoptosis leads to the hyperplasia and remodeling of the neointima. Effective therapy for treatment of late vein graft failure is still lacking. Here, we investigated the effects of transfecting wild-type p53 gene and low energy laser irradiation on the proliferation and apoptosis of the VSMC in the veingraft, to evaluate the possibility of using trans-gene therapy and laser therapy for treatment of late vein graft failure, hoping to provide new strategy for further experimental study and clinical application. Methods: Recombinant adenovirus carrying human wild type p53 gene (Ad-p53) and recombinant adenovirus carrying green fluorescent protein gene (Ad- GFP), were amplified in 293 cells on a large scale, and purified by ultra- centrifugation in CsC1 step gradient solution. The viral particles were determined by OD26Onm and purity was evaluated by the ratio of OD26Onm/OD28Onm. The viral titers were determined by plaque assays. In the rabbit jugular veins grafted into the carotid arteries models, experiments were done according to the groups randomly divided as below. 1. P53 gene transduced group: the vein graft was infused with Ad- p53 for 30 minutes before grafting; 2. GFP gene transduced group: the vein graft was infused with Ad-GFP for 30 minutes before grafting; 3. Low energy laser irradiated group: the vein graft was irradiated from the outside of the luminal by 51Onm copper vapor laser with power density of 500mW/cm~2 for 1000 seconds after the grafting; 4. P53 gene transduced and low energy laser irradiated group: the vein graft was treated with the combined P53 gene transduction and low energy laser irradiation as group 1 and group 3. Four weeks after the operation, p53 protein and proliferating cell nuclear antigen (PCNA) were localized by immunohistochemical procedure, apoptosis was evaluated through terminal d-UTP mck end-labeling (TUNEL). The area of neointima and medial were calculated from sections stained with HE, Masson and Victoria blue by computer image analysis. Results: 1. After the adenovirus delivery of the wild type p53 gene, p53 protein can be over-expressed in the VSMC of the vein graft. Four weeks after the operation, compared with the control group, the proliferation rate of the VSMC decreased 61%, while the apoptosis rate increased 25%. The thickness of the intima and media decreased 60% and 33% respectively, the ratio of intima thickness / media thickness (IIM) decreased 37%. However, 4 weeks after the adenovirus delivery and over-expression of GFP gene in the vein graft by the same way, compared with the control group, there is no significant difference in the proliferation and apoptosis rate of the VSMC, while the thickness of the intima and media differed little either. 2. In the low energy laser irradiated group, 4 weeks after the operation, compared with the control group, the proliferation rate of the VSMC decreased 42.5%, the apoptosis rate increased 40.9%, the thickness of the intima and media decreased 5 8.5% and 18% respectively, the I/M ratio decreased 47.2%. 3. In the vein graft treated with wild-type P53 gene transducation and low energy laser irradiation, 4 weeks after...