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Targeted Scavenging of Reactive Oxygen Species Improves Diabetic Wound Repair and Neovascularization
Oriana Cohen, BA, Manisha Patel, BS/BA, John Layliev, MD, Caroline Szpalski, MD, Meredith Wetterau, MD, Denis Knobel, MD, Stephen Warren, MD, Pierre Saadeh, MD, Daniel Ceradini, MD.
NYU Langone Medical Center, New York, NY, USA.

INTRODUCTION: Increased levels of reactive oxygen species (ROS) have been implicated in delayed diabetic wound healing. The transcription factor Nrf2 and its key repressor Keap1 play a critical role in the protection against oxidative stress. We hypothesized that Keap1 silencing would lead to activation of Nrf2, increased antioxidant scavenging of ROS, and improved wound healing in the hyperglycemic state.
METHODS: Silencing RNA (siRNA) to Keap1 was generated and assayed for knockdown in 3T3 fibroblasts by RT-PCR and Western blot. Activation of antioxidant gene NQO-1 expression was quantified by RT-PCR. Using a diabetic murine excisional wound model (db/db), siRNA to Keap1 was topically applied to stented wounds, which were assessed over 30 days for epithelialization, granulation tissue formation, neovascularization, and ultimately, wound closure. Gene expression was assessed by RT-PCR and Western blot. Paired t-test determined statistical significance (p<0.05).
RESULTS: Keap1 silencing in 3T3 cells resulted in 63.5 ± 0.36% knockdown of Keap1 mRNA compared to nonsense controls. This resulted in a 12.25 fold increase in antioxidant gene NQO-1 expression. In diabetic wounds, Keap1 silencing resulted in 37 ± 0.18% knockdown of Keap1 mRNA expression compared to controls, which corresponded with a 1.8 fold increase in NQO-1 expression at day 10. Silenced wounds were completely epithelialized at day 10 compared to nonsense treated wounds, which demonstrated 1328 ± 259µm of epithelial gap. The Keap1-treated wounds had a nearly four-fold increase in both granulation tissue area (197,148 ± 3,525.2 vs. 53,165 ± 1,360 µm2) and neovascularization (61 vs. 18 CD31+cells/hpf). Moreover, indicators of ROS end products were decreased compared to controls (7.64 vs. 13.16 ng/mL). This was ultimately associated with accelerated clinical wound closure of Keap1 silenced wounds (21 vs. 27 days).
CONCLUSION: This study demonstrates the effectiveness of targeted topical silencing of Keap1, a key regulatory element in the broad anti-oxidant gene expression pathway, in improving diabetic wound healing. This provides strong evidence that the altered redox balance inherent to the hyperglycemic state accounts for delayed wound healing in diabetes.


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