A hypoxia response element in the Vegfa promoter is required for basal Vegfa expression in skin and for optimal granulation tissue formation during wound healing in mice

PLoS One. 2017 Jul 7;12(7):e0180586. doi: 10.1371/journal.pone.0180586. eCollection 2017.

Abstract

Hypoxia in skin wounds is thought to contribute to healing through the induction of hypoxia inducible factor-1 (HIF-1). Although HIF-1 can regulate the expression of vascular endothelial growth factor A (Vegfa), whether hypoxia and HIF-1 are required to induce Vegfa expression in the context of wound healing is unknown. To test this hypothesis, we evaluated Vegfa expression and wound healing in mutant mice that lack a functional HIF-1 binding site in the Vegfa promoter. Full-thickness excisional wounds were made using a biopsy punch, left to heal by second intention, and granulation tissue isolated on a time course during healing. mRNA levels of Vegfa and its target genes platelet-derived growth factors B (Pdgfb) and stromal cell-derived factor-1 (Sdf1) were measured by RT-qPCR, and HIF-1alpha and VEGFA protein levels measured by immunoblotting. Lower levels of Vegfa, Pdgf1 and Sdf1 mRNA were found in intact skin of mutant mice relative to wild-type controls (n = 6 mice/genotype), whereas levels in granulation tissue during wound healing were unaltered. VEGFA protein levels were also lower in intact skin of the mutant versus the wild-type mice. Decreased Vegfa mRNA levels in skin of mutant mice could not be attributed to decreased HIF-1alpha protein expression, and were therefore a consequence of the loss of HIF-1 responsiveness of the Vegfa promoter. Comparative histologic analyses of healing wounds in mutant and wild-type mice (n = 8 mice/genotype) revealed significant defects in granulation tissue in the mutant mice, both in terms of quantity and capillary density, although epithelialization and healing rates were unaltered. We conclude that HIF-1 is not a major regulator of Vegfa expression during wound healing; rather, it serves to maintain basal levels of expression of Vegfa and its target genes in intact skin, which are required for optimal granulation tissue formation in response to wounding.

MeSH terms

  • Animals
  • Binding Sites
  • Chemokine CXCL12 / genetics
  • DNA-Binding Proteins / genetics
  • Disease Models, Animal
  • Gene Expression Regulation
  • Granulation Tissue / metabolism
  • Granulation Tissue / physiopathology
  • Humans
  • Hypoxia-Inducible Factor 1, alpha Subunit / biosynthesis
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics*
  • Lymphokines / genetics
  • Mice
  • Platelet-Derived Growth Factor / genetics
  • Promoter Regions, Genetic
  • Response Elements / genetics
  • Skin / metabolism*
  • Skin / physiopathology
  • Vascular Endothelial Growth Factor A / biosynthesis
  • Vascular Endothelial Growth Factor A / genetics*
  • Wound Healing / genetics*

Substances

  • Chemokine CXCL12
  • Cxcl12 protein, mouse
  • DNA-Binding Proteins
  • Hif1a protein, mouse
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • Lymphokines
  • Pdgfd protein, mouse
  • Platelet-Derived Growth Factor
  • Vascular Endothelial Growth Factor A
  • vascular endothelial growth factor A, mouse

Grants and funding

This work was supported by the Natural Sciences and Engineering Research Council of Canada (CT), grant #250231 (http://www.nserc-crsng.gc.ca). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.