MESENCHYMAL STEM CELL TREATMENT IMPROVES DIABETIC WOUND HEALING THROUGH MODULATION OF MICRORNA EXPRESSION

Junwang Xu, Carlos Zgheib, Junyi Hu and Kenneth W. Liechty

University of Colorado Denver School of Medicine, Department of Surgery, Aurora, CO, USA

Abstract

INTRODUCTION
Impaired wound healing following injury in diabetics represents a major clinical problem, resulting in prolonged hospitalizations and significant healthcare expenditures. Diabetic wounds have been shown to have an abnormal inflammatory response, decreased angiogenesis and collagen deposition. MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate the translation of target mRNAs at the post-transcriptional level, and have been shown to play pivotal roles in the regulation of inflammation, angiogenesis, and collagen production. We have shown that Mesenchymal stem cell (MSC) treatment can improve healing in diabetic mice. However, the role of microRNA in this correction remain unclear. We hypothesize that MSC treatment improves diabetic wound healing, in part, by correcting abnormal miRNA gene expression.

METHODS
To test our hypothesis, we created 8 mm wounds on the back of diabetic (Db/Db) and non-diabetic (Db/+) mice. A subset of wounds were treated with 10⁶ MSC. The wounds were harvested at 0, 1, 3, 7 and 14 days and total cellular RNA isolated. Real-time PCR was performed for miRNAs and their target genes expression.

RESULTS
Treatment of diabetic wounds with MSC resulted in a significant improvement in wound closure. At day 7 after wounding, MSC treated wounds showed significantly reduced wound size, a significant reduction in the number of CD45+ inflammatory cells, a significant induction of CD31+ cells, and increasing production of collagen content. We found miR-146a, an anti-inflammation molecule, was significantly down-regulated in diabetic wounds. Decreased miR- 146a levels closely correlated with increased gene expression of its pro-inflammatory target genes in NF-κB signaling pathway. More interestingly, the correction of the diabetic wound healing impairment with MSC treatment was associated with a significant increase in miR-146a expression level and decreased gene expression of its proinflammatory target genes. MiR-15b, an anti-angiogenic molecule, was found significantly increased in diabetic wounds and associated with decreased expression of its target VEGFα through BCL2/HIF1/VEGF signaling pathway. MSC treatment resulted in significantly decreased expression of miR-15b, and increased expression of VEGFα. MiR-29a has been shown to repress type I collagen (Col1a1 and Col1a2) and type III collagen (Col3a1) mRNA and we found that miR-29a expression was significantly increased in murine diabetic wounds. Diabetic wounds treated with MSC demonstrated a significant decrease in miR-29a expression compared to controls and this was associated with a significant increase in collagen protein content.

CONCLUSION
These findings demonstrate that stem cell correction miRNA plays a role in the diabetic wound healing impairment and MSC mediated correction of the diabetic wound healing impairment is due, in part, to modulation of microRNA expression. These data also provides a new layer of regulatory mechanisms that could be targeted for potential therapeutic intervention in diabetic wounds.