The development of new therapeutic approaches to chronic wounds hasn’t lost its relevance. In non-healing diabetic ulcers (diabetic foot) auto-transplantation (a common treatment approach) is not effective due to impaired fibroblast functionality and shifts in synthesis of ECM proteins (COL1, COL3, FN) and certain growth factors. It was shown that HIF1 (hypoxia-induced factor) is a key regulator of ECM remodeling by fibroblasts through control of collagen prolyl hydroxylase (P4HA1, P4HA2 – both required for collagen deposition) and lysylhydroxylase (PLOD2 – modulates collagen fibers strength) expression. Therefore, the use of hypoxia-stimulated fibroblasts (sFbs) may represent a reasonable treatment alternative in the maintenance of chronic diabetic wounds. Here, we evaluated the expression levels of wound healing-related genes and proteins in sFbs after 24h, 48h and 72h exposure to 1% O2. After a short-term (24/48h) exposure to hypoxia, sFbs exhibited a marked increase in expression of a number of genes as compared to 21% O2 culture: Hif1a – 7.4 and 16.9 fold respectively; Vegf1a – 21/60; Egln1 – 9/9; Egln2 – 6.5/5; Egln3 – 53/110; COL1 – 2.4/3; Fn – 3.3/1.7; P4HAa1 - 5.8/7.8; P4ha2 – 6.6/8.5; and Plod2 - 9/25 fold. In contrast, with a longer exposure to hypoxia (72h), activation was significantly diminished (Hif1a – 2 fold; Vegf1a – 6; Egln3 – 2.4) or completely abrogated (Egln1, Egln2, COL1, Fn, P4ha1, P4ha2, and Plod2). Importantly, at protein level, we observed only COLI  (2 fold ) stimulation by hypoxia . The stimulation of COLIII and Fn synthesis in sFb was detected only after a 6-18h period of their re-oxygenation. Based on our results, we propose that short-term stimulation of dermal fibroblasts (for example, immobilized on a biopolymer scaffold) by hypoxia could be beneficial for chronic diabetic wound treatment. The absence of HLA-DR (one of the main MHC-II class proteins, responsible for graft rejection) on sFbs and certain similarity between Fbs and mesenchymal stromal cells may represent other advantages of this approach. (NRC Kurchatov Institute intramural grant supported research).

Hypoxia-pretreatment as a possible approach to stimulate wound healing potentials of dermal fibroblasts in treatment of chronic skin ulcers