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Short Hairpin RNA Interference Therapy for Diabetic Murine Wound Closure and Hindlimb Ischemia
Kevin J. Paik, A.B.1, Robert Rennert, B.S.1, Michael T. Chung, B.S.1, Michael Sorkin, M.D.1, Dominik Duscher, M.D.1, David Atashroo, M.D.1, Hsin-Han Chen, M.D.1, Shane D. Morrison, M.S.1, Andrew Zimmermann, B.S.1, Allison Nauta, M.D.1, Sae-Hee Ko, M.D.1, Ruth Tevlin, M.D.1, Elizabeth Zielins, M.D.1, Michael S. Hu, M.D.1, Adrian McArdle, M.D.1, Graham Walmsley, B.S.1, Kshemendra Senarath-Yapa, M.D.1, Wan Xing Hong, M.S.1, Rebecca M. Garza, M.D.1, Christopher Duldulao, B.S.1, Taylor Wearda, B.S.1, Arash Momeni, M.D.1, Joseph C. Wu, M.D., Ph.D.2, Geoffrey C. Gurtner, M.D.1, Michael T. Longaker, M.D., M.B.A.1, Derrick C. Wan, M.D.1.
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford, CA, USA, 2Department of Radiology and Department of Medicine, Division of Cardiology, Stanford, CA, USA.
The transcription factor hypoxia-inducible factor 1-alpha (HIF1A) is responsible for the downstream expression of over 60 genes that affect cell survival and metabolism in hypoxic conditions, including angiogenic growth factors. However, under normoxic circumstances, HIF1A is hydroxylated by prolyl hydroxylase 2 (PHD2), ubiquitinated, and degraded with a biological half-life of only 5 minutes. The present study investigated the therapeutic potential of inhibiting HIF1A degradation through short hairpin RNA (shRNA) knockdown of PHD2 for the treatment of diabetic wounds and ischemic hindlimbs in a murine model.
PHD2 and control shRNAs were used to transfect mouse fibroblasts in vitro. Protein and RNA were harvested for Western blot and for qRT-PCR to evaluate PHD2 knockdown. qRT-PCR was also used to measure subsequent expression of downstream angiogenic genes. For assessment of plasmid function in vivo, six-millimeter full thickness wounds were created on the dorsa of diabetic db/db mice. Wounds were then injected with either PHD2 shRNA or control shRNA. Sample protein and RNA were collected from wounds for Western and qRT-PCR confirmation of in vivo PHD2 knockdown. Wound healing was then monitored and measured photometrically every two days till closure. Finally, ischemic hindlimbs were perfused with PHD2 or control shRNAs. Muscle ischemia was analyzed histologically and distal digit tip necrosis was evaluated.
PHD2 knockdown in diabetic mouse fibroblasts resulted in enhanced expression of multiple angiogenic factors compared to control shRNA-treated fibroblasts. Diabetic skin wounds treated with PHD2 shRNA were observed to close within 14 ±0.47 days while control shRNA-treated wounds closed at 18 ±1.36 days. Finally, perfusion of ischemic hindlimbs with PHD2 shRNA resulted in decreased muscle necrosis histologically and no distal digit tip necrosis.
Nonviral shRNA treatment holds significant promise as a future avenue for gene therapy. This study demonstrates that knockdown of regulatory factors involved in angiogenesis, as explored here with PHD2, might present new opportunities for wound healing treatments.
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