Intraluminal gene transfer of endothelial cell-nitric oxide synthase suppresses intimal hyperplasia of vein grafts in cholesterol-fed rabbit: A limited biological effect as a result of the loss of medial smooth muscle cells

Ex vivo electroporation as a potent new strategy for nonviral gene transfer into autologous vein grafts

“…A nuclear-targeted lacZ gene driven with the chicken ␤-actin promoter pAct-NLS-LacZ was prepared as previously described (28,29). The plasmid pUCCAGGS-ecNOS, expressing full-length bovine ecNOS cDNA (3.7 kb; kindly provided by T. Michael, Harvard Medical School) driven by the CAG promoter [human cytomegalovirus immediate early enhance/chicken, ␤-actin promoter, and rabbit ␤-globin poly (A)], was prepared as previously described (29,33). All the plasmids were purified by using Mega kits (Qiagen, Hilden, Germany).…”

“…The internal jugular vein, which had been isolated and filled with DNA solution, was sandwiched between the electrodes, and electric pulses were delivered to the vein wall. After electroporation and flushing of the vector solution with 20 ml of physiological saline containing heparin (5 U/ml), two procedures were performed: one involved declamping the clips to restore venous circulation (for luciferase gene transfer: total n ϭ 30; n ϭ 5 each at 0, 5, 10, 20, 30, and 50 V, respectively; for lacZ gene transfer: n ϭ 3), and the other involved excising the internal jugular vein for reversed vein graft and implantation into the arterial circulation of the ipsilateral common carotid artery (n ϭ 5 for luciferase gene transfer), described as follows (33).…”

“…Hypercholesterolemic rabbits (n ϭ 24) were fed 1% cholesterol chow for 4 wk after surgery (33). In this model, blood samples were analyzed at the time of graft implantation as well as at the time of graft harvest.…”

“…In the last decade, it has been hoped that gene therapy would overcome this problem; several experimental and clinical studies, including ours, have reported that gene transfer into the wall of the vein graft might have the potential to prevent late graft failure (11,13,25,29,33). A number of methods and techniques for in vivo gene transfer into the vasculature are available, including viral vector (6,22,27,33,35,37,41,43), lipofection (32), viral envelope-coated liposomes (45), and naked DNA delivery with or without hydrogels (24,38).…”

“…A number of methods and techniques for in vivo gene transfer into the vasculature are available, including viral vector (6,22,27,33,35,37,41,43), lipofection (32), viral envelope-coated liposomes (45), and naked DNA delivery with or without hydrogels (24,38). Viral vectors are the most widely accepted gene transfer method for cardiovascular tissues in vivo, inasmuch as the efficiency of gene transfer is usually sufficient (13,35).…”