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(Circulation. 2004;110:2484-2493.)
© 2004 American Heart Association, Inc.

Molecular Cardiology

Gene Therapy of Endothelial Nitric Oxide Synthase and Manganese Superoxide Dismutase Restores Delayed Wound Healing in Type 1 Diabetic Mice

Jian-Dong Luo, MD, PhD; Ying-Ying Wang, MD; Wei-Ling Fu, MD, PhD; Jun Wu, MD, PhD; Alex F. Chen, MD, PhD

From the Departments of Pharmacology and Neurology and the Neuroscience Program (J.-D.L., Y.-Y.W., A.F.C.), Michigan State University, East Lansing; Department of Pharmacology, Guangzhou Medical College, Guangzhou, China (J.-D.L.); and Southwestern Hospital, Third Military Medical University, Chongqing, China (Y.-Y.W., W.-L.F., J.W., A.F.C.).

Correspondence to Alex F. Chen, MD, PhD, Departments of Pharmacology and Neurology and Neuroscience Program, College of Human Medicine, B403 Life Sciences Bldg, Michigan State University, East Lansing, MI 48824-1317. E-mail chenal{at}msu.edu

Received December 11, 2002; de novo received June 3, 2004; accepted June 14, 2004.

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.

Key Words: wound healing • nitric oxide • superoxides • diabetes mellitus • gene therapy

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