Modulation of Cx43 increases dermal fibroblast migration in ‘diabetic’ scrape wounds in vitro

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Abstract

Significant increases in skin wound healing rates occur by reducing connexin (Cx) mediated communication (CMC). We analysed effects of Gap27, a connexin mimetic peptide that reduces Cx43 CMC, on CMC and cell migration in vitro of human dermal fibroblasts in euglycaemia and euinsulinaemia or hyperglycaemia and hyperinsulinaemia; and expression of extra-cellular matrix (ECM) and adhesion genes in migrating fibroblasts in euglycaemia and hyperglycaemia.
Cells were treated with 5.5 mM glucose and 1 nM insulin (‘normal’) or 25 mM glucose and 10 nM insulin (‘high’). Individual migrating fibroblasts were injected with Alexa594 and Alexa488 dyes and dye spread monitored. Migration studies examined rates of scrape-wound closure in fibroblasts in normal or high conditions with or without 100 µM Gap27. Migrating fibroblasts were cultured in 5.5 or 25 mM glucose and treated with Gap27 or scrambled peptide (control) in triplicate. mRNA was subjected to PCR array analysis (SABiosciences), with gene expression fold changes = 2 considered regulatory.
Cells in high glucose and insulin showed reduced CMC compared to those in normal levels (mean cells receiving dye ± SEM: 2.3 ± 0.8 v 6.6 ± 1.8). Gap27 increased cell migration rates of fibroblasts (P < 0.01) in normal and high glucose and insulin. In euglycaemic migrating fibroblasts, Gap27 upregulated thirty-four genes and downregulated 1 gene: connective tissue growth factor and matrix metalloproteinase 1 were significantly upregulated (P < 0.05). By contrast, under hyperglycaemia, Gap27 upregulated one gene and downregulated 9 genes, including collagens and fibronectin-1.
Gap27 blocked CMC and increased cell migration rates in both normal and high levels of glucose and insulin. In euglycaemic conditions Gap27 induced upregulation of fibroblast genes associated with ECM remodelling, and in hyperglycaemia genes associated with producing ECM components. Connexin mimetic peptides improve migration in ‘diabetic’ conditions in vitro and have therapeutic potential in healing chronic wounds.
Original languageEnglish
Publication statusPublished - 2010

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Keywords

  • Cx34
  • dermal fibroblast migration
  • diabetic
  • scrape wound
  • in vitro

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