Connexin mimetic peptide Gap27 alters gene expression of actively migrating dermal fibroblasts in high glucose environments in vitro

Research output: Contribution to conferencePaper

Abstract

Background: Significant increases in skin wound healing rates occur by altering gap junction intercellular communication. We have previously shown that the connexin mimetic peptide, Gap27, targeted to Cx43 to block gap junction functionality, increases rates of dermal fibroblast migration across scrape wounds in vitro and has therapeutic potential. To begin to dissect the mechanism of connexin/peptide action we have analysed the impact of Gap27 on gene expression of extra-cellular matrix (ECM) and adhesion molecules during cell migration of dermal fibroblasts in normal and high (diabetic) levels of glucose.
Methods: Primary dermal fibroblasts were derived from child foreskins obtained with ethical approval and patient consent. Cells were grown in DMEM containing normal (5.5 mM) or high (25 mM) glucose and were treated for 24 hours with 100 µM Gap27, or 100 µM scrambled peptide (control). mRNA was harvested from actively migrating cells and subjected to PCR array analysis (SABiosciences) where fold changes in 84 ECM and adhesion genes were examined. Fold changes =2 in gene expression were considered regulatory. All array sets were repeated in triplicate.
Results: Differential gene expression was seen in fibroblasts grown in normal or high glucose in the presence or absence of Gap27. Thirty-four genes associated with cell adhesion and ECM were upregulated and 1 gene (hyaluronan synthase 1) was downregulated in normal glucose by Gap27. Collagen1a1, connective tissue growth factor (CTGF), integrinß1, MMP1 and C-type lectin domain family 3 member B were significantly upregulated (P < 0.05). By contrast, under high glucose conditions, one gene (collagen XII a1) was upregulated and 9 genes were downregulated by Gap27, including collagens, integrins, fibronectin-1 and versican. These gene expression changes are being confirmed by QRT-PCR.
Conclusion: Increasing cell migration by the application of Gap27 has differential effects on gene expression in normal and high (diabetic) levels of glucose in dermal fibroblasts. In euglycaemic conditions during migration Gap27 induces upregulation of genes associated with remodelling of ECM components involved in the migration process. In high glucose, these mechanisms appear impaired, and this may contribute to the dysfunctional cell migration and wound healing seen in diabetic skin. Whether this is due to connexin signalling events or a novel role in cell adhesion remains to be explored.
Original languageEnglish
Publication statusPublished - 2009

Keywords

  • connexin mimetic peptides
  • Gap 27

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