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.
Highlights
Hypoxia within skin wounds arises primarily in response to traumatic destruction of the cutaneous vasculature and augmentation of cellular oxygen consumption rates due to temporary increases in cellular density and metabolic activity [1,2,3,4]
While our experimental goal was achieved, the outcome of our study demonstrated that, contrary to prediction, hypoxia inducible factor-1 (HIF-1) is required for basal expression of vascular endothelial growth factor A (Vegfa) in intact skin, rather than in granulation tissue during wound healing
Hypoxia associated with cutaneous wounding triggers the expression of a number of genes required for the healing process, including the key angiogenic factor vascular endothelial growth factor (VEGFA) [4, 22]
Summary
Hypoxia within skin wounds arises primarily in response to traumatic destruction of the cutaneous vasculature and augmentation of cellular oxygen consumption rates due to temporary increases in cellular density and metabolic activity [1,2,3,4]. Acute hypoxia favors wound healing [1, 2] because low oxygen tension levels promote angiogenesis [5], fibroplasia [1, 2, 4, 5], epithelialization [1, 4], and extracellular matrix (ECM) synthesis [1, 4] These effects are regulated, at least in part, by hypoxia-induced increases in the expression of various growth factors including transforming growth factor (TGF)-ß1 [1, 2, 4], platelet-derived growth factor (PDGF) [4] and vascular endothelial growth factor (VEGFA) [2, 4]. It exerts its transcriptional regulatory effects by binding a site named the hypoxia-response element (HRE) found within the promoters of various genes including VEGFA, angiopoietin (ANGPT), ANGPT2, PDGFB, glucose transporter (GLUT)-1 and stromal cell-derived factor (SDF)-1 [3, 6,7,8]
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