Hypoxia and Foxn1 alter the proteomic signature of dermal fibroblasts to redirect scarless wound healing to scar-forming skin wound healing in Foxn1 mice.

Background: Foxn1 deficient mice are a rare model of regenerative skin wound healing among mammals. In wounded skin, the transcription factor Foxn1 interacting with hypoxia-regulated factors affects re-epithelialization, epithelial-mesenchymal transition (EMT) and dermal white adipose tissue (dWAT) reestablishment and is thus a factor regulating scar-forming/reparative healing. Here, we hypothesized that transcriptional crosstalk between Foxn1 and Hif-1α controls the switch from scarless (regenerative) to scar-present (reparative) skin wound healing. To verify this hypothesis, we examined (i) the effect of hypoxia/normoxia and Foxn1 signalling on the proteomic signature of Foxn1 (regenerative) dermal fibroblasts (DFs) and then (ii) explored the effect of Hif-1α or Foxn1/Hif-1α introduced by a lentiviral (LV) delivery vector to injured skin of regenerative Foxn1 mice with particular attention to the remodelling phase of healing.

Results: We showed that hypoxic conditions and Foxn1 stimulation modified the proteome of Foxn1 DFs. Hypoxic conditions upregulated DF protein profiles, particularly those related to extracellular matrix (ECM) composition: plasminogen activator inhibitor-1 (Pai-1), Sdc4, Plod2, Plod1, Lox, Loxl2, Itga2, Vldlr, Ftl1, Vegfa, Hmox1, Fth1, and F3. We found that Pai-1 was stimulated by hypoxic conditions in regenerative Foxn1 DFs but was released by DFs to the culture media exclusively upon hypoxia and Foxn1 stimulation. We also found higher levels of Pai-1 protein in DFs isolated from Foxn1 mice (reparative/scar-forming) than in DFs isolated from Foxn1 (regenerative/scarless) mice and triggered by injury increase in Foxn1 and Pai-1 protein in the skin of mice with active Foxn1 (Foxn1 mice). Then, we demonstrated that the introduction of Foxn1 and Hif-1α via lentiviral injection into the wounded skin of regenerative Foxn1 mice activates reparative/scar-forming healing by increasing the wounded skin area and decreasing hyaluronic acid deposition and the collagen type III to I ratio. We also identified a stimulatory effect of LV-Foxn1 + LV-Hif-1α injection in the wounded skin of Foxn1 mice on Pai-1 protein levels.

Conclusions: The present data highlight the effect of hypoxia and Foxn1 on the protein profile and functionality of regenerative Foxn1 DFs and demonstrate that the introduction of Foxn1 and Hif-1α into the wounded skin of regenerative Foxn1 mice activates reparative/scar-forming healing.