title: Short Hairpin RNA Silencing of PHD-2 Improves Neovascularization and Functional Outcomes in Diabetic Wounds and Ischemic Limbs.
abstract: The transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) 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-1α is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1α degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1α as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFα, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 ± 0.45 days vs. 19 ± 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1α and upregulation of pro-angiogenic genes downstream of HIF-1α, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Paik
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Zeshaan N
authors.initials: ZN
authors.lastname: Maan
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Zielins
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Dominik
authors.initials: D
authors.lastname: Duscher
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Whittam
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Shane D
authors.initials: SD
authors.lastname: Morrison
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Brett
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: Ransom
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Hu
authors.affiliation: Department of Medicine, Division of Cardiology, Stanford University School of Medicine, Stanford, CA, United States of America.|Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Geoffrey C
authors.initials: GC
authors.lastname: Gurtner
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.|Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Longaker
authors.affiliation: Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Plastic and Reconstructive Surgery Division, Stanford University School of Medicine, Stanford, CA, United States of America.
authors.forename: Derrick C
authors.initials: DC
authors.lastname: Wan
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D048909:Diabetes Complications / Q000473:pathology / Q000628:therapy*; D004195:Disease Models, Animal; D005121:Extremities / Q000098:blood supply*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015316:Genetic Therapy; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000037:antagonists & inhibitors* / Q000235:genetics; D007511:Ischemia / Q000628:therapy*; D051379:Mice; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000494:pharmacology; D014945:Wound Healing
chemical_list: C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases