Background— Nitric oxide (NO) deficiency contributes to diabetic wound healing impairment. The present study tested the hypothesis that increased cutaneous superoxide (O₂⁻) levels in type 1 diabetic mice cause NO deficiency and delayed wound healing.

Methods and Results— Wound healing was markedly delayed in streptozotocin-induced type 1 diabetic mice compared with the normal controls. There were significantly reduced levels of endothelial NO synthase (eNOS) protein and constitutive NOS activity in diabetic wounds, whereas O₂⁻ levels were markedly increased. A single regimen of cutaneous gene therapy of eNOS or manganese superoxide dismutase (MnSOD) restored such healing delay, with a concomitant suppression of wound O₂⁻ levels and augmentation of both eNOS protein and constitutive NOS activity. Gene therapy of MnSOD also increased cutaneous MnSOD activity. Cutaneous O₂⁻ levels were also increased in Ins2^Akita diabetic mice. In vitro glucose treatment of cutaneous tissues from normal mice for 24 hours increased O₂⁻ levels in a concentration-dependent manner. The enhanced cutaneous O₂⁻ levels induced by high glucose in both normal and diabetic mice were abolished by the NADPH oxidase inhibitor apocynin and the protein kinase C inhibitor chelerythrine. Furthermore, ex vivo gene transfer of dominant-negative HA-tagged N17Rac1, which inhibits NADPH oxidase subunit Rac1, significantly inhibited cutaneous O₂⁻ formation induced by high glucose in both normal and Ins2^Akita diabetic mice.

Conclusions— These results indicate that hyperglycemia augments cutaneous O₂⁻ levels, at least in part, via NADPH oxidase and protein kinase C pathways, resulting in impaired wound healing in type 1 diabetic mice. Gene therapy strategies aimed at restoring cutaneous NO bioavailability may provide an effective means to ameliorate delayed diabetic wound healing.