One very promising gene therapy regimen involves the delivery of nitric oxide synthase (NOS) to the vasculature as a means of augmenting local nitric oxide levels. Nitric oxide is a diffusable molecule that, once synthesized, can target a whole population of cells. Nitric oxide has been shown to play an important role in many processes throughout the cardiovascular system, including normal maintenance functions, atherosclerosis, hypertension, vasospasm, and ischemia-reperfusion.49 Nitric oxide inhibits platelet aggregation, leukocyte chemotaxis, and smooth muscle cell proliferation and migration and promotes endothelial cell regeneration. Because of these vasoprotective properties, methods of delivering nitric oxide to sites of vascular injury were devised, including supplementing dietary L-arginine, the substrate for NOS activity, and using nitric oxide donor compounds. These methods all resulted in reduced IH in rodent models of vascular injury. In 1995, von der Leyen et al22 transduced endothelial NOS (eNOS) into rat carotid arteries following balloon injury using hemagglutinating virus of Japan modified liposomes and reported a 70% reduction in the intima-media ratio compared with control arteries at 14 days. These results have been reproduced by other investigators using eNOS gene transfer by either liposomal or adenoviral gene delivery methods; eNOS was a natural candidate for these studies because it is believed that vascular injury results in the loss of eNOS function and, therefore, nitric oxide. This enzyme produces nitric oxide in low levels in response to mechanical and agonist stimulation. In contrast, the inducible NOS (iNOS) is expressed following cellular stress and is capable of producing significantly greater quantities of nitric oxide. A theoretical advantage of iNOS for gene therapy is its ability to produce adequate local concentrations of nitric oxide in the vasculature with lower gene transfer efficiencies. This is an important consideration in the coronary circulatory system, where even short periods of blood flow disruption can result in myocardial injury. In 1996, Tzeng et al50 performed ex vivo delivery of retroviral human iNOS to balloon-injured pig arterial segments and essentially abolished myointimal thickening, despite a very low (1%) gene transfer efficiency. Subsequently, adenoviral delivery of iNOS to balloon-injured rat carotid arteries using low titers of virus resulted in a 97% inhibition of IH compared with controls at 2 weeks.51 This therapy was similarly effective in a porcine model of iliac artery injury where iNOS gene transfer reduced IH by more than 50%. Finally, iNOS gene transfer into jugular vein grafts implanted into the pig carotid circulation also protected against graft IH by 35%. These studies suggest the great potential utility of NOS gene therapy for the prevention of IH and have fueled the approval by the National Institutes of Health (Bethesda, Md) and the Food and Drug Administration (Rockville, Md) of iNOS gene transfer in a clinical trial to treat in-stent stenoses following coronary angioplasty or stenting.