Collateral artery enlargement is impaired in eNOS−/− mice due to a greater increase in oxidative stress in eNOS−/− than in wild‐type mice after induction of hindlimb ischemia and can not be reversed by eNOS gene transfer

Abstract

Nitric oxide (NO) derived from endothelial nitric oxide synthase (eNOS) is critical to vascular homeostasis. Oxidative stress increases the production of reactive oxygen species (ROS), causing local tissue damage and depleting tetrahydrobiopterin (BH4). Thus, eNOS activity and the resultant NO bioavailability depends on the redox state of the surrounding tissue. Using wild-type C57Bl/6 (WT) and eNOS−/− mice, we surgically induced hindlimb ischemia and attempted to increase NO bioavailability by adenoviral mediated eNOS gene transfer. AdeNOS injection significantly increased collateral artery enlargement in wild-type, but not eNOS−/− mice, in spite of successful eNOS gene transfer. We hypothesized that this failure to reverse the eNOS−/− phenotype was due to an increase in oxidative stress in these mice that inhibited normal eNOS activity. Thigh muscle protein extracts from either eNOS−/− or WT mice were added to positive controls in a NOS activity assay. Ischemic eNOS−/−, but not WT tissue significantly inhibited the NOS activity of these reactions. In addition, the Amplex Red assay for hydrogen peroxide in ischemic thigh muscle showed a greater increase in eNOS−/− mice than WT controls. These results show a greater increase in oxidative stress in eNOS−/− mice after induction of muscle ischemia that cannot be reversed by eNOS gene transfer, suggesting that endothelial dysfunction plays a critical role in collateral artery enlargement. RO1HL75353-01.