Polymer-mediated Delivering Of IGF1R-shRNA Plasmid Inhibited Vascular Smooth Muscle Cell Proliferation And Migration In Vitro And Neointimal Hyperplasia In Rabbit Model

ObjectiveThat polymer potentiating gene transfection into target cells has been evaluated in various cancer cell lines. But the feasibility of polymer-based gene transfection to smooth muscle cells has never been systematically evaluated. IGF1/IGF-1R signal pathway which activated by hemodynamic mechanical stress in vein grafts mediates smooth muscle cell proliferation and intimal hyperplasia. In this study, we evaluated the transfection efficiency and therapeutic potential of polymer-based transfection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-IRs) in smooth muscle cells and in vein grafts.Methods:Cells grow from explants within one week. At two weeks, cell cultures became confluent in approximately75%. Aortic smooth cells were used between passages3and5. Plasmids expressing GFP and shRNA targeting IGF-1R (pGFPshIGF-lR) were constructed by corporation of Genepharma. Transfection in vitro was performed as the manufacturer’s protocol in a6-well plate. In vivo transfection. a total of0.2to0.3mL MegaTran1.0/DNA mixture was instilled into the isolated external jugular vein segment through internal jugular vein. After four weeks, rabbits were sacrificed and the vein grafts were fixed with10%formaldehyde solution, embedded in paraffin. Tissue sections were stained with hematoxylineosin, and morphometric analysis was performed using image analysis software. Vein graft thickening was defined as the area between the lumen and the adventitia and determined by subtracting the luminal area from the total vessel wall area.ResultsPolymer-based transfection provided a high efficiency in transgene expression in vitro. IGF-1R specific-shRNA transfected by polymer inhibited IGF-1R protein by49±2.0%. In vivo, the delivering efficiency of the pGFPshIGF-lR plasmids into the vein grafts was significantly high. In vivo, IGF-1R specific-shRNA inhibited IGF-1R protein by77.0±3.6%,65.6±4.9%, and78.0±4.3%, at24h,48h, and72h, respectively. Polymer-based delivering sh-IGFIR inhibited the migration and proliferation of VSMCs. Moreover, gene transfection with polymer resulted in a reduction of vein graft thickening (control:0.7±0.1mm2, shIGFIR:0.48±0.15mm2, P<0.02).The luminal area and ratio of lumen/total cross-sectional area of the vessels were significantly increased in the polymer group than that in the control group. And these effects were mediated via inhibiting PI3K/AKT pathway.ConclusionsOur findings indicated that polymer-based transfection may be a promising method that allows the targeting of gene therapy to prevent vein graft restenosis.