Introduction: Aging is a known risk factor for poor wound healing, neovascularization, and tissue regeneration following an ischemic insult. The process of neovascularization is regulated by the transcription factor HIF-1α which is post-transcriptionally regulated through hydroxlase mediated degradation. We hypothesize that aging results in decreased HIF-1α stabilization resulting in reduced expression of the proangiogenic cytokines VEGF and SDF-1α leading to impaired wound healing and neovascularization.

Materials and Methods: Primary young and aged murine and human fibroblasts were exposed to normoxia and hypoxia and assessed for HIF-1α using Western blot. The downstream effectors VEGF and SDF-1α were analyzed using ELISA and real-time PCR. Prolyl hydroxylases (PHD1, 2, 3) and factor inhibiting HIF (FIH) were also analyzed using Western blot and real-time PCR. Excisional wounds and ischemic flaps were elevated on the dorsum of young and aged mice (n=4 per group) and analyzed for HIF-1α stabilization, VEGF expression, and neovascularization using CD31 staining. The known hydroxylase inhibitor, dimethyloxaloylglycine (DMOG) was administered in vitro and in vivo as rescue experiments.

Results: Aged murine and human fibroblasts demonstrated dramatically reduced HIF-1α stabilization in hypoxia compared to young controls. This correlated with decreased VEGF (25.30±5.30 versus 615.85±35.75 pg/mL, aged versus young, P<0.00001) and SDF-1α (fold induction: 1.90±0.21 versus 0.90±0.17, P<0.008) expression in response to hypoxia. Western blot and real-time PCR demonstrated significant upregulation of all PHDs and FIH in murine aged fibroblasts. Aged mice demonstrated significantly delayed wound healing and greater flap necrosis than young mice which corresponded with reduced HIF-1α stabilization, VEGF expression, and neovascularization. DMOG was able to increase HIF-1α stabilization and VEGF secretion in vitro, however, no differences were seen in PHD or FIH protein levels. In vivo, DMOG prevented flap necrosis in aged mice comparable to young controls with increased VEGF and SDF expression and neovascularization. DMOG also improved wound healing in aged mice.

Conclusions: Age associated defects in wound healing and the impaired response to ischemic injury result from increased HIF-1α degradation. Therapeutic strategies designed to augment the HIF response may represent a novel modality for treating wounds and ischemic damage in the aged population.