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Background and Hypothesis: Impaired wound healing is a serious complication of diabetes and accounts for over 70,000 amputations annually in the United States. Accumulating evidence suggests that wounds with poor healing process display persistently elevated protease activity. Here we tested the hypothesis that genetic knockout or pharmacological inhibition of cathepsin K, a cysteine protease with potent collagenolytic and elastolytic activity, accelerates wound closure in diabetic mice. Methods: Diabetes was induced in cathepsin K knockout (Ctsk-/-) and wild type litter-mates by intraperitoneal injection of streptozotocin (STZ, 120 mg/Kg). Two weeks following STZ injection, a full thickness 8-mm excisional wound was created on the dorsum of the mice. In a separate set of experiments, excisional skin wounds were created in genetically diabetic (db+/db+) and their normoglycemic litter-mates. The wounds were treated with cathepsin K inhibitor II (0.35μg/d on days 0, 3, and 5), or vehicle, given as intradermal injections along the circumference of the wound. Wound healing was evaluated by measuring the wound closure rate, CD31 expression, and histological analysis. Results: In the STZ-induced diabetic model, wounds in the cathepsin K-knockout mice closed significantly earlier than the vehicle-treated wounds. Similarly, pharmacological inhibition of cathepsin K in db/db mice resulted in near complete resolution of the wound in 22 days compared to about 29 days in vehicle-treated mice. Histological evaluations demonstrated that topical delivery of cathepsin K inhibition resulted in higher levels of collagen deposition and increased angiogenesis. In an in vitro scratch-assay using cultured human fibroblasts, cell migration, which was significantly impaired by high-glucose concentrations, was restored by cathepsin K inhibitor-II. Conclusion: Cathepsin K may represent a potential treatment-target to facilitate diabetic wound healing. Disclosure R. Guo: None. L. Zhang: None. A. Thakar: None. T.E. Brown: None. J. Ren: None. C. Zgheib: None. J. Xu: None. K.W. Liechty: Consultant; Spouse/Partner; Cell research. S. Nair: None. Funding National Institute of General Medical Sciences (2P20GM1034320); University of Wyoming

The impairment in diabetic wound healing represents a significant clinical problem. Chronic inflammation is thought to play a central role in the pathogenesis of this impairment. We have previously shown that treatment of diabetic murine wounds with mesenchymal stem cells (MSCs) can improve healing, but the mechanisms are not completely defined. MicroRNA-146a (miR-146a) has been implicated in regulation of the immune and inflammatory responses. We hypothesized that abnormal miRNA-146a expression may contribute to the chronic inflammation. To test this hypothesis, we examined the expression of miRNA-146a and its target genes in diabetic and nondiabetic mice at baseline and after injury. MiR-146a expression was significantly downregulated in diabetic mouse wounds. Decreased miR-146a levels also closely correlated with increased gene expression of its proinflammatory target genes. Furthermore, the correction of the diabetic wound-healing impairment with MSC treatment was associated with a significant increase in the miR-146a expression level and decreased gene expression of its proinflammatory target genes. These results provide the first evidence that decreased expression of miR-146a in diabetic wounds in response to injury may, in part, be responsible for the abnormal inflammatory response seen in diabetic wounds and may contribute to wound-healing impairment.