In vivo pulmonary artery adenoviral gene transfer of nitric oxide synthase enhances pulmonary nitric oxide production
Channon KM., Kypson AP., Daggett CW., Blazing MA., Koch WJ., George SE.
Nitric oxide (NO), generated by nitric oxide synthase (NOS), has key regulatory roles in the normal pulmonary circulation, and also inhibits pathological processes such as platelet aggregation, neutrophil adhesion and smooth muscle cell proliferation. NO production is defective in disease states such as pulmonary hypertension. Restoring or enhancing NO production, by gene transfer of NOS, may therefore offer a potential therapeutic strategy. We used a recombinant adenoviral vector, Ad.nNOS, containing the neuronal isoform of NOS (nNOS), to carry out in vivo gene transfer to the pulmonary vasculature in the rabbit. Methods: Rabbits underwent left thoracotomy to expose the left pulmonary artery (PA). Recombinant adenovirus, either Ad.nNOS (n=6) or, as a control, Ad.βGal (n=3) was diluted in 4 ml saline and injected rapidly into the left PA, which was occluded for 5 minutes. Lungs were harvested after 5 days. The left and right lungs were processed for NOS protein analysis by Western blotting and for NOS activity determination by 3H-Arginine conversion. The left lung was also divided for analysis into left upper (LUL) and lower (LLL) lobes. Results: Immunoblotting demonstrated high-level nNOS protein expression in LLL and LUL, but barely detectable in RL. No nNOS protein was seen in Ad.βGal-infected lungs. NOS activities (Mean ± SD, in pmol NO/min/mg protein) are shown in the table (* p < 0.05): Left Lung Left UL Left LL Right Lung Ad.βGal 6.3 ± 2.7 5.05 ± 2.7 7.5 ± 2.6 6.1 ± 3.3 Ad.nNOS 18.6 ± 12.8* 14.0 ± 14.5 23.3 ± 10.2* 8.5 ± 3.4 Specificity of NOS activity was demonstrated by inhibition in the presence of N-methyl-L-arginine (1 mM), a specific NOS inhibitor. Conclusions: In vivo adenoviral gene transfer of NOS via the pulmonary artery results in efficient recombinant protein expression and significantly augments NO production in the lung, by 3-fold. These findings highlight the investigative and therapeutic potential of NOS gene transfer strategies in pulmonary vascular diseases.