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Adenoviral gene transfer of nitric oxide synthase: high level expression in human vascular cells
K M Channon1, M A Blazing, G A Shetty
1Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Objectives:
Nitric oxide synthases (NOS) generate nitric oxide (NO), a second messenger with key regulatory roles. In the cardiovascular system, deficient endothelial NO production is an early, persistent feature of atherosclerosis and vascular injury. Accordingly, the NOS isoforms represent attractive targets for vascular gene therapy. We aimed to generate and evaluate an adenoviral vector for gene transfer of an NOS isoform to vascular cells.
Methods:
We constructed a recombinant adenovirus, Ad.nNOS, for gene transfer of the neuronal isoform of NOS (nNOS) and characterized its expression in 293 cells, human vascular smooth muscle cells (hVSMC) and human umbilical vein endothelial cells (HUVEC). NOS expression was analyzed by Western immunoblotting, and NOS enzyme activity in response to receptor-dependent and receptor-independent agonists was determined by Griess assay or by NO chemiluminescence.
Results:
Ad.nNOS-infected 293 cells expressed high levels of functional nNOS enzyme, even higher than in 293.NOS cells (a cell line that expresses supraphysiologic levels of nNOS). In hVSMC, nNOS activity reached levels 50% of those seen in 293.NOS cells. nNOS expression and activity in hVSMC increased linearly with titer of Ad.nNOS. NO production in hVSMC was stimulated both by calcium ionophore and by physiologic agonists such as acetylcholine or bradykinin. In HUVEC, endogenous NOS activity was significantly augmented by Ad.nNOS infection. Supplementation with the tetrahydrobiopterin precursor sepiapterin enhanced NOS activity in all cells.
Conclusions:
Ad.nNOS, a novel adenoviral vector for gene transfer of NOS, generates high-level nNOS expression in a variety of vascular cell types. nNOS activity in hVSMC is physiologically regulated and of a magnitude comparable to native eNOS activity in HUVEC. Our findings demonstrate Ad.nNOS to be a versatile and efficient tool for nNOS gene transfer, with widespread potential applications in cell culture and for gene therapy.