Small Interfering RNA To C-myc Inhibits Vein Graft Restenosis Both In Vitro And In Vivo

Autologous saphenous vein is one of the most commonly used vascular conduits for coronary heart disease. It was reported that the great saphenous vein are used in almost about80%of all the CABG However, the long-term treatment outcome is far from satisfactory due to the gradually diminished patency rate of the vein grafts over time. One year, and10years following CABG surgery, the patency rate was about85%and50-60%, respectively. It is benefit to improve the long-term patency rate of vein grafts.Three distinct phases of vein grafts failure have been recognized till now. Early graft occlusion relates to technical complications and thrombosis. Mid-term graft occlusion is due to the development of fibrotic intimal hyperplasia. And atherosclerotic degeneration contributes to the late time restenosis.Vascular intimal hyperplasia was found to be one of the major causes for occlusion of the vein grafts. It was reported that the smooth muscle cells play a central role in the vein grafts’ intimal hyperplasia. Evidences suggested that most of the smooth muscle cells come from the local vessel wall in the progress of vein graft’s intimal hyperplasia. After reperfusion, VSMCs of the vein grafts would gradually change from contractile to synthetic phenotype. The phenotypic switching of the VSMCs will make the cells immigrate to the tunica intima of the vein grafts, enter the cell cycle and produce extracellular matrix. These contribute to the vein grafts stenosis. While traditional pharmacotherapy has no benefits to intimal hyperplasia, gene therapy provides a possible treatment for inhibiting intimal hyperplasia.Many genes are involved in the progress of intimal hyperplasis. The progress of the intimal hyperplasia of the vein grafts was consistent with the expression of the c-myc gene. It is believed that the continuous high expression of the c-myc gene is partly responsible for the development of intimal hyperplasia.The use of synthetic siRNA has been established as a technique for gene silencing. It was reported that antisense oligonucleotides to c-myc gene could reduce c-myc gene expression and inhibit VSMCs growth. Here, we employed siRNA as a medium to silence c-myc gene expression in order to inhibit VSMCs proliferation and protect vein grafts from late restenosis in a rat autologous vein graft model.Part Ⅰ:Small interfering RNA to c-myc inhibits VSMCs proliferation in vitroObjective:Three c-myc siRNAs and one SCR were synthesized. The effects of siRNAs on VSMCs proliferation in vitro were examined both in cell proliferation and c-myc geng expression.Methods:VSMCs from Sprague-Dawley rat were isolated and harvested. Cells were identified by morphology and immunohistochemistry for α-actin, and were used for experiments at passage5. VSMCs were transfected with the different siRNAs by using Lipofectamine2000reagent (Invitrogen Life, Biotechnologies, Carlsbad, CA). MTT assay, WB and q-rt-PCR were used to detecte the inhibition efficiency in cell proliferation.Result:The sequence ACAUCAUCAUCCAGGACUGdTdT was selected as the most effective siRNA in our study. In vitro, at48hours after transfection, this c-myc siRNA inhibited cell proliferation by40%, reduced c-myc mRNA level by70%and c-myc protein level by50%as compared with the controls.Conclusions:The VSMCs proliferation in vitro could be reduced effectively by downregulation c-myc gene expression utilizing c-myc siRNA. Part Ⅱ:Small interfering RNA to c-myc inhibits vein graft restenosis in a rat vein graft modelObjective:In vitro study, the c-myc siRNA was selected. To investigate the inhibition efficiency of this c-myc siRNA on grafted veins’ intimal hyperplasis, a rat vein graft model was used in this study.Methods:Autologous jugular vein to carotid artery reverse interposition grafts were performed in male Sprague-Dawley rats.32rats were divided into four groups as follows:nontreated group, receiving no treatment (n=8); only gel group,200ul of25%pluronic F-127gel (sigma) in5%glucose solution was applied to the surface of the vein grafts immediately after arteriovenous anastomosis (n=8); scrambled siRNA treated group, treated with200ul of above gel containing50ug scrambled siRNA (n=8); c-myc siRNA treated group,200ul gel containing50ug c-myc siRNA was applied to the surface of the grafts(n=8). Rats were sacrificed3weeks after surgery. The intimal hyperplasis and the c-myc gene expression were detected. Stains for PCNA were also preformed.Result:In vivo, the c-myc siRNA treatment reduced intimal hyperplasia by75%. Compared to the controls, the c-myc gene expression was also reduced in c-myc siRNA treatment group as well as the PCNA expression.Conclusions:Small interfering RNA to c-myc can inhibit vein graft restenosis in a rat vein graft model.