Vascular Endothelial Growth Factor Gene Transfection Improves Endothelialization Of Polytetrafluoroethylene Vascular Graft: Experimental Study

Part 1Feasibility research of using polytetrafluoroethylene vascular graft materials as gene carrierObjective: To test a method of using polytetrafluoroethylene as gene carrier and investigate the feasibility of this method, to characterize the kinetics of release of gene plasmid carried by PTFE, to evaluate the transfection ability of vascular endothelial growth factor (VEGF) gene plasmid carried by PTFE vascular graft material.Methods: pCDI-hVEGF₁₂₁ and pCDI solution (1mg/ml) was mixed with equal volume fibronectin solution (lmg/ml), then were poured into PTFE grafts respectively. The two ends of grafts were clipped, then grafts were crushed until external walls appeared liquid bleeding, then grafts were dried in super clean bench, this procedure was repeated three times. The PTFE vascular graft materials carried pCDI-hVEGF₁₂₁/pCDI plasmid were clipped as strip (2 mm×10 mm) and were marked with ethidium bromide (EB) solution and observed under ultraviolet light. The strips were incubated in Tris-buffer solution (TBS) and the value of optical density of 260 nm at different time were measured, then the controlled release curve was made. The vascular graft materials carried pCDI-hVEGF₁₂₁ were implanted intothe left thigh muscles of rabbits and the vascular graft materials with pCDI were implanted into the right. The expression of VEGFi2imRNA versus P-actin mRNA in muscles around vascular graft materials was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of VEGF121 protein versus P-actin protein in muscle was determined by Western blot method.Results: The vascular graft materials carried pCDI-hVEGFi2i/ pCDI marked by EB were shining under ultraviolet light. The controlled release curve showed that the speed of gene release from PTFE materials was rapid within 8 hours, then slowed down and the gene released continuously even after 7 d, the total dose of plasmid that could be carried by PTFE strip (2 mmxlO mm) by our method was about 4 jxg. In VEGF group, RT-PCR and Western blot showed VEGF121 mRNA versus p-actin mRNA expressive level were 1.053±0.356, 1.718±0.404, 2.021±0.303, 1.872±0.231, 0.986±0.254 and 0.340±0.116 and VEGF121 protein expressive level were 0.328±0.088, 1.019±0.105, 2.249±0.203, 2.036±0.079, 1.670±0.132 and 0.636±0.107 corresponded to 1, 7, 14, 28, 42, and 56 days after implantation respectively. In control group, hVEGFm gene and protein could not be detected.Conclusion: The PTFE graft can be used as carrier of VEGF gene for gene therapy, VEGF gene can transfect skeletal muscle by this method.Part 2Proliferation of endothelial cell on polytetrafluoroethylene vascular graft materials carried VEGF gene plasmidObjective: To investigate whether vascular endothelial growth factor (VEGF)gene plasmid carried by polytetrafluoroethylene (PTFE) vascular graft material could transfect endothelial cells (EC) and promote their growth.Methods: PTFE vascular graft materials carried with pCDI-hVEGFm, pCDI or pEGFP were cut into small disks (6.5 mm in diameter) and incubated in Tris-buffer solution (TBS) and the value of optical density of 260 nm at different time were plotted, then the DNA controlled release curve was made. ECs were derived from human umbilical vein, the cells were identified by morphology, immunofluorescence stain and transmission electronic microscopy (TEM). ECs were seeded on the pCDI-hVEGFi2i/pCDI/pEGFP/Fibronectin-PTFE materials or tissue culture plates (TCPs), ECs numbers were counted and VEGF protein concentrations at 6, 24, 72 and 120 h were measured by enzyme-linked immunoadsorbent assay method. GFP expression in ECs on pEGFP-PTFE materials was examined with fluorescence microscopy and transfection efficiency was evaluated by flow cytometer (FCM). Scanning electronic microscopy (SEM) observed the cells' growth status on the PTFE materials.Results: The cultured human umbilical vein endothelial cells attached completely after 24 h in vitro, reached confluence in 5-6 d with the typical appearance of "stone of pave road", immunofluorescence stain showed positive expression of von Willebrand factor, TEM showed Weibel-palade body in cells. The controlled release curve showed that the speed of gene release from PTFE materials was rapid within 8 hours, then slowed down and that the gene released continuously even after 72 d, the total dose of plasmid that could be carried by one 6.5 mm diameter PTFE disk by our method was about 6 fig. At 24, 72 and 120 h, ECs number and proliferation rate of pCDI-hVEGF 121-PTFE materials were higher than that those of pCDI, pEGFP or Fibronectin-PTFE materials (P<0.05). VEGF protein concentration of pCDI-hVEGFm-PTFE materials were higher than that of pCDI, pEGFP or Fibronectin-PTFE materials at 6, 24, 72 and 120 h (P<0.01). GFPexpression in ECs on the pEGFP-PTFE materials could be detected by fluorescence microscopy. FCM showed, at 6, 24, 72 and 120 h, the proportions of GFP-positive cells (transfection efficiency) on pEGFP- PTFE materials were (1.19%±0.31%), (2.89%±0.42%), (4.78%±0.73%) and (7.14%±0.94%) respectively. SEM showed cell's gowth status on pCDI-hVEGFui-PTFE materials was better than that on fibronectin-PTFE materials.Conclusion: PTFE graft can be used as a carrier of VEGF gene plasmid, VEGF gene carried by PTFE can transfect ECs and promote ECs growth.Part 3The experimental study on the endothelialization of polytetrafluoroethylene vascular graft carried VEGF geneObjective: To investigate whether vascular endothelial growth factor (VEGF) gene plasmid carried by polytetrafluoroethylene (PTFE) vascular graft material could transfect vascular bed tissue cells, promote endothelium growth and inhibit thrombosis.Methods: The 4 mm-diameter PTFE vascular grafts carried pCDI-hVEGFm were implanted in the abdominal aorta of rabbits (VEGF group, n=36), vascular grafts with pCDI also were implanted into other rabbits (control gene, n=12). The implanted grafts with surrounding tissues were retrieved at 1 d, 1 w, 2 w, 4 w, 6 w and 8 w after implantation. The patency and mural thrombus of grafts were evaluated. The thick of neointima was measured under microscopy. The expression of VEGF121 mRNA versus (3-actin mRNA in transplanted vascular beds was evaluated by reversetranscriptase polymerase chain reaction (RT-PCR). The expression of VEGF 121 protein was determined by Western blot and immunohistochemical method. The analyses of endothelium in the grafts were completed under scanning electron microscopy (SEM).Results: All implanted vascular grafts were patent. Implanted vascular grafts were cut longitudinally, stoma suture and luminal surface of grafts had been covered by white, glossy and complete intima in the VEGF group, while control grafts did not have complete intima growth. The thrombus in the grafts was far less in the VEGF group than in the control group {4 w: (6.26±3.72)% vs (37.46±10.12)%;6 w: (7.11±3.78)% vs (56.54±15.88)%;8 w: (5.87±3.11)% vs (28.37±9.1)5%;P<0.01}. The thick of neointima of gene group was also less than that of control group {4 w: (82±24) Jim vs (164±36) urn;6 w: (94±28) urn vs (198±43) urn;8 w: (105±37) urn vs (256±58) urn;PO.01}. In the VEGF group, RT-PCR and Western blot showed VEGF121 mRNA versus P-actin mRNA expressive level were 1.053±0.356, 1.718±0.404, 2.021±0.303, 1.872±0.231, 0.986±0.254, 0.340±0.116 and VEGF121 protein expressive level were 0.328±0.088, 1.019±0.105, 2.249±0.203, 2.036±0.079, 1.670±0.132, 0.636±0.107 corresponded to 1 d, 1 w, 2 w, 4 w, 6 w and 8 w after implantation respectively, immunohistochemistry displayed the VEGF121 protein was highly expressed in the new endothelium, the pores of prosthetic grafts as well as the adventitia around the vascular grafts. SEM demonstrated that vascular endothelial cells grew tightly, regularly and in fusiform in the wall of grafts of 4 weeks after the implantation in the VEGF group.Conclusion: The PTFE graft can be used as carrier of pCDI-hVEGF^i for gene therapy with high efficiency of transfection and expression. VEGF gene transfer can accelerate the endothelialization of grafts.