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Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs
Kevin J. Paik, AB1; Zeshaan Maan, MD1; Shane D. Morrison, MS1; Hsin-Han Chen, MD1; Robert Rennert, BS1; Michael Sorkin, MD1; Dominik Duscher, MD1; Michael T. Chung, BS1; David Atashroo, MD1; Michael S. Hu, MD1; Shuli Li, MD, PhD1; Kshemendra Senarath-Yapa, MD1; Adrian McArdle, MD1; Elizabeth A. Brett, MS1; Anna Luan, MS1; Ruth Tevlin, MD1; Elizabeth R. Zielins, MD1; Wan Xing Hong, MS1; Graham Walmsley, BS1; Christopher Duldulao, BS1; Taylor Wearda, BS1; Owen Marecic, BS1; Joseph C. Wu, MD PhD2,3; Geoffrey C. Gurtner, MD1; Michael T. Longaker, MD, MBA1,4; Derrick C. Wan, MD1
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, 2Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA, 3Department of Radiology, Stanford University School of Medicine, Stanford, CA, 4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA
The transcription factor hypoxia-inducible factor 1-alpha (HIF-1a) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1a is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. The present study investigated the therapeutic potential of inhibiting HIF-1a degradation through short hairpin RNA (shRNA) knockdown of PHD-2 for the treatment of diabetic wounds and ischemic hindlimbs in a mouse model.
PHD-2 and non-targeting scramble shRNAs were used to transfect diabetic mouse fibroblasts in vitro. In vivo, ischemic hindlimbs were also treated with PHD-2 shRNA or scramble shRNA. Perfusion was measured with laser doppler, distal digit tip necrosis was evaluated, surviving muscle bulk was analyzed histologically, and CD31 staining was performed on gastrocnemius muscles. Additionally, 6 mm full thickness wounds were created on the dorsa of diabetic db/db mice. Wounds were injected with either shPHD-2 or shScr. Wound healing was monitored and measured photometrically every two days till closure, and CD-31 staining was performed.
Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR and western blot, higher levels of HIF-1a as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFa, as measured by qRT-PCR and western blot. In vivo, shPHD-2 resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Delivery of shPHD-2 also accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control (14.33 ± 0.45 days v. 19 ± 0.33 days), and was associated with an increased vascular density.
Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1a and upregulation of pro-angiogenic genes downstream of HIF-1a, and may represent a viable, non-viral gene therapy for ischemia related applications.
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