564 Subpopulations of dermal fibroblasts produce distinct extracellular matrices

Abstract

A major problem of diabetic foot ulcers is ischemia contributing to delayed wound closure. The gap junction protein Connexin43 (Cx43) is differentially remodelled during 'normal' and 'chronic' wound healing events. The mechanisms by which this protein is involved remains unresolved. This work investigated the expression and post translational modification of Cx43 and Pannexin 1 (Panx1) in skin biopsies from patients undergoing arterial reconstruction or major limb amputation for critical limb ischaemia and in keratinocytes cultured in normoxic (N) and hypoxic (H) (1% oxygen, 5% carbon dioxide and 94 % nitrogen) conditions. Skin tissue biopsies (proximal and distal to the point of venous bypass surgery) from diabetic and non-diabetic patients were processed for immunohistochemistry and stained with antibodies specific to Cx43 and Cx43Ser 368. To model the events in vitro HaCaT cells were grown in 12 well plates, scrape wounded and cultured in N or H environments for up to 48 hours (h). Cell movement into the denuded area was recorded. Cells were fixed or protein harvested and expression of hypoxic inducible factor 1-a (HIF-1a), Cx43, Cx43Ser 368 , Panx1 and Ki67 determined by immunocytochemistry and Western blot analysis. Cx43 and Cx43Ser 368 expression was significantly increased in the epidermis of skin biopsies isolated from the distal position (i.e. locations of ischaemia) in both diabetic and non-diabetic tissue. Scratch wound assay determined delayed wound closure in hypoxic conditions, that was confirmed by induction of HIF-1a expression. Cx43 and Cx43 Ser368 staining showed translocation of the protein to the nucleus. No changes in Panx1 were observed. In conclusion, post translational phosphorylation of Cx43 occurs in chronically wounded diabetic and ischemic skin. Under H conditions, scrape wound closure is reduced and immunofluorescence suggests that Cx43 and Cx43Ser 368 translocate to the nucleus after 24 h hypoxia. Thus, hypoxia impacts on Cx expression but not Panx1 expression in the epidermis.