Epidermal or dermal specific knockout of PHD-2 enhances wound healing and minimizes ischemic injury.
BACKGROUND
Hypoxia-inducible factor (HIF)-1α, part of the heterodimeric transcription factor that mediates the cellular response to hypoxia, is critical for the expression of multiple angiogenic growth factors, cell motility, and the recruitment of endothelial progenitor cells. Inhibition of the oxygen-dependent negative regulator of HIF-1α, prolyl hydroxylase domain-2 (PHD-2), leads to increased HIF-1α and mimics various cellular and physiological responses to hypoxia. The roles of PHD-2 in the epidermis and dermis have not been clearly defined in wound healing.


METHODS
Epidermal and dermal specific PHD-2 knockout (KO) mice were developed in a C57BL/6J (wild type) background by crossing homozygous floxed PHD-2 mice with heterozygous K14-Cre mice and heterozygous Col1A2-Cre-ER mice to get homozygous floxed PHD-2/heterozygous K14-Cre and homozygous floxed PHD-2/heterozygous floxed Col1A2-Cre-ER mice, respectively. Ten to twelve-week-old PHD-2 KO and wild type (WT) mice were subjected to wounding and ischemic pedicle flap model. The amount of healing was grossly quantified with ImageJ software. Western blot and qRT-PCR was run on protein and RNA from primary cells cultured in vitro.


RESULTS
qRT-PCR demonstrated a significant decrease of PHD-2 in keratinocytes and fibroblasts derived from tissue specific KO mice relative to control mice (*p<0.05). Western blot analysis showed a significant increase in HIF-1α and VEGF protein levels in PHD-2 KO mice relative to control mice (*p<0.05). PHD-2 KO mice showed significantly accelerated wound closure relative to WT (*p<0.05). When ischemia was analyzed at day nine post-surgery in a flap model, the PHD-2 tissue specific knockout mice showed significantly more viable flaps than WT (*p<0.05).


CONCLUSIONS
PHD-2 plays a significant role in the rates of wound healing and response to ischemic insult in mice. Further exploration shows PHD-2 KO increases cellular levels of HIF-1α and this increase leads to the transcription of downstream angiogenic factors such as VEGF.
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Andrew S
AS
Zimmermann
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Shane D
SD
Morrison
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America; Department of Surgery, John A. Burns School of Medicine, University of Hawai'i, Honolulu, Hawai'i, United States of America.
Michael S
MS
Hu
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Shuli
S
Li
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Allison
A
Nauta
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Michael
M
Sorkin
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Nathaniel P
NP
Meyer
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, United States of America.
Graham G
GG
Walmsley
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Zeshaan N
ZN
Maan
Department of Radiation Oncology, University of California San Francisco, San Francisco, California, United States of America.
Denise A
DA
Chan
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America.
Geoffrey C
GC
Gurtner
Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California, United States of America.
Amato J
AJ
Giaccia
Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, Stanford, California, United States of America; Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, United States of America.
Michael T
MT
Longaker
D000818:Animals; D020405:Dermis / Q000378:metabolism* / Q000503:physiopathology*; D004817:Epidermis / Q000378:metabolism* / Q000503:physiopathology*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000378:metabolism*; D007511:Ischemia / Q000378:metabolism / Q000503:physiopathology*; D015603:Keratinocytes / Q000378:metabolism / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D014945:Wound Healing / Q000502:physiology*
C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C490189:Egln1 protein, mouse; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases