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Gene transfer to vein gratis has therapeutic potential to prevent late graft failure; however, certain issues, including efficacy and safety, have hindered the clinical application of this treatment modality. Here, we report the successful and efficient gene transfer of plasmid DNA via ex vivo electroporation into veins as well as into vein grafts. Two approaches were used: one involved transluminal in situ gene transfer using a T-shaped electrode (the 'Lu' method), and the other was an adventitial ex vivo approach using an electroporation cuvette followed by vein grafting (the 'Ad' method). The Lu method was earned out at 10 V, with optimal gene transfer efficiency in the in situ jugular veins of rabbits, and transgene expression was observed primarily in endothelial cells. However, when these veins were grafted into the arterial circulation, no luciferase activity was detected; this effect was probably due to the elimination of the gene-transferred cells as a result of endothelial denudation. In contrast, optimal and satisfactory gene transfer was obtained with the vein grafts subjected to the Ad method at 30 V, and transgene expression was seen primarily in adventitial fibroblasts. Gene transfer of endothelial nitric oxide synthase cDNA to the vein graft via the Ad method successfully limited the extent of intimal hyperplasia, even under hyperlipidemic conditions, at 4 wk after grafting. We thus propose that the Ad method via ex vivo electroporation may provide a novel, safe, and clinically available technique for nonviral gene transfer to sufficiently prevent late graft failure. endothelial cell nitric oxide synthase; adventitia; endothelial cells; hyperlipidemia; plasmid

The externally fertilized, optically clear zebrafish embryo provides an unprecedented opportunity to visualize and examine the formation of embryonic blood vessels. We have studied the formation of the trunk intersomitic vessels using transgenic zebrafish expressing green fluorescent protein throughout the vasculature, permitting direct, real-time imaging of the dynamics of vessel formation in living embryos. The intersomitics are dorsal-ventrally aligned vessels at vertical myoseptal boundaries that form shortly after the primary axial vessels of the trunk (the dorsal aorta and posterior cardinal vein). We have found that intersomitic vessels form via a novel two-step process. A primary intersomitic vascular network forms first, sprouting only from the dorsal aorta. Upon completion of this network a second set of angiogenic vessels sprout from the posterior cardinal vein and quickly interconnect and merge with the primary network. We will present data comparing the role of flow dynamics versus genetic factors in the formation of these vessels. Our data show that intersomitic vein formation is temporally distinct from intersomitic artery formation and suggest a possible mechanistic basis for the anatomical association commonly found between arteries and veins.