Oral Tetrahydrobiopterin Improves the Beneficial Effect of Adenoviral-mediated eNOS Gene Transfer After Induction of Hindlimb Ischemia

Abstract

We tested the hypothesis that oral supplementation with the endothelial nitric oxide synthase (eNOS) cofactor tetrahydrobiopterin (BH(4)) improved the therapeutic efficacy of eNOS gene transfer in the ischemic rat hindlimb. BH(4) or vehicle were begun 1 week before induction of hindlimb ischemia, whereas recombinant adenovirus containing bovine eNOS cDNA (AdeNOS) or vehicle [phosphate-buffered saline (PBS)] was infused intra-arterially into the ischemic hindlimb 10 days after induction of ischemia. Rats receiving co-treatment with dietary BH(4) and eNOS gene transfer (the [eNOS, +BH(4)] group) had greater eNOS expression, phospho-eNOS expression (Ser(1177)), Ca(2+)-dependent NOS activity, and nitrite + nitrate concentrations in the ischemic gastrocnemius than did rats receiving AdeNOS alone. The [eNOS, +BH(4)] group demonstrated less nitrotyrosine and a higher ratio of reduced:oxidized glutathione (GSH:GSSG) in the ischemic gastrocnemius muscle than did rats receiving AdeNOS alone. The [eNOS, +BH(4)] group had greater flow recovery and a higher capillary:myocyte ratio in the ischemic hindlimb than did rats receiving AdeNOS alone. Finally, the [eNOS,+BH(4)] group had less necrosis of hindlimb muscles than rats given AdeNOS alone. We conclude that adjunctive dietary therapy with BH(4) increases the beneficial effects of eNOS gene transfer within the ischemic gastrocnemius muscle, as evidenced by increased nitric oxide (NO) production, diminished oxidative stress, enhanced flow recovery, and reduced necrosis.