ADENOVIRAL GENE-TRANSFER OF NITRIC-OXIDE SYNTHASE - HIGH-LEVEL EXPRESSION IN HUMAN VASCULAR CELLS

Objectives: Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular sys tem, deficient endothelial NO production is an early, persistent featu re of atherosclerosis and vascular injury. Accordingly, the NOS isofor ms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NO S isoform to vascular cells. Methods: We constructed a recombinant ade novirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nN OS) and characterized its expression in 293 cells, human vascular smoo th muscle cells (hVSMC) and human umbilical vein endothelial cells (HU VEC). NOS expression was analyzed by Western immunoblotting, and NOS e nzyme activity in response to receptor-dependent and receptor-independ ent agonists was determined by Griess assay or by NO chemiluminescence . Results: Ad.nNOS-infected 293 cells expressed high levels of functio nal nNOS enzyme, even higher than in 293.NOS cells (a cell line that e xpresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity rea ched levels 50% of those seen in 293.NOS cells. nNOS expression and ac tivity in hVSMC increased linearly with titer of Ad.nNOS. NO productio n in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. in HUVEC, endogenous NO S activity was significantly augmented by Ad.nNOS infection. Supplemen tation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells, Conclusions: Ad.nNOS, a novel adenoviral vecto r for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types, nNOS activity in hVSMC is physiologica lly regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficie nt tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.