In vivo adenoviral gene transfer of nitric oxide synthase augments or restores vascular no production
Channon KM., Blazing MA., Pawloski J., McMahon T., Olmez E., Shetty GA., Stamler JS., George SE.
Nitric oxide (NO) has key regulatory roles in the cardiovascular system. NO production is defective in injured or diseased vessels and restoration of NO production inhibits the sequelae of vessel injury. The nitric oxide synthase (NOS) isoforms are therefore potential therapeutic agents for gene transfer to the vessel wall. Using a recombinant adenoviral vector, Ad.nNOS, containing the neuronal isoform of NOS (nNOS), we carried out in vivo gene transfer to rabbit carotid arteries (CA), with and without balloon denudation. Methods: Rabbits underwent balloon denudation of the left CA (2F Fogarty). Recombinant adenovirus (5 × 109pfu/ml), either Ad.nNOS (n=4) or, as controls, Ad.βGal or no virus (n=6) was instilled into the lumen and onto the adventitia of both CA, for 15 minutes. Vessels were harvested after 3 days, and analysed as follows: (1) NOS protein analysis by Western blotting. (2) Staining of cryosections by NADPH diaphorase and nNOS immunohistochemistry (3) NOS activity determination in intact vessels by 3H-Arginine conversion. (4) Isometric tension studies in organ baths for determination of vasomotor responses. Results: Western blotting, diaphorase and nNOS immunostaining revealed nNOS protein expression in intact and denuded CA infected with Ad.nNOS, but not in control CA. NOS activities (Mean ± SD, in pmol NO/hr/mg) are shown in the table (* p<0.05, **p<0.01): Intact Denuded Ad.βGal/No Virus 35.8 ± 48.2 22.9 ± 20.6 Ad.nNOS 85.2 ± 10.8** 44.4 ± 7.2* Vasomotor responses to phenylephrine and nitroprusside were unaffected by gene transfer. After Ad.nNOS infection, acetylcholine relaxation was augmented in intact CA, and A23187-induced paradoxical contraction was reduced in denuded CA. Conclusions: In vivo adenoviral gene transfer of NOS restores or augments NO production in denuded or intact CA, respectively, with corresponding changes in vessel physiology. Gene transfer strategies using NOS may potentially provide novel investigative and therapeutic approaches to vascular diseases.