The connexin mimetic peptide Gap27 increases cell migration in ‘diabetic’ conditions in vitro and alters gene expression of dermal fibroblasts

Significant increases in skin wound healing rates occur by reducing connexin (Cx) mediated communication (CMC). We analysed the effects of Gap27, a connexin mimetic peptide directly targeted to the extracellular loop of Cx43 and known to reduce CMC, on CMC and cell migration in vitro during scrape-wound closure of human dermal fibroblasts in euglycaemia and euinsulinaemia or hyperglycaemia and hyperinsulinaemia; and the expression of extra-cellular matrix (ECM) and adhesion genes during dermal fibroblast migration in euglycaemic and hyperglycaemic conditions.
Cells were treated with 5.5 mM glucose and 1 nM insulin (‘normal’ levels) or 25 mM glucose and 10 nM insulin (‘high’ levels). Individual migrating fibroblasts were injected with Alexa594 and Alexa488 dyes and dye spread to other cells monitored. Migration studies examined rates of scrape-wound closure over 48 hours in fibroblasts in normal or high conditions in the presence or absence of 100 µM Gap27. Dermal fibroblasts were cultured in 5.5 mM or 25 mM glucose and treated for 24 hours with Gap27, or scrambled peptide (control) in triplicate. mRNA from migrating cells 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 glucose and insulin 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 both normal and high glucose and insulin. In euglycaemic migrating fibroblasts, Gap27 upregulated thirty-four genes and downregulated 1 gene. Collagen1a1, connective tissue growth factor, integrinß1 and matrix metalloproteinase 1 were significantly upregulated (P < 0.05). By contrast, under hyperglycaemia, Gap27 upregulated one gene and downregulated 9 genes, including collagens, integrins, fibronectin-1 and versican.
Gap27 blocked CMC and increased cell migration rates in both normal and high levels of glucose and insulin. In euglycaemic conditions during fibroblast migration Gap27 induced upregulation of genes associated with ECM remodelling, and in high glucose genes associated with producing ECM components. Connexin mimetic peptides can improve migration in ‘diabetic’ conditions in vitro and have therapeutic potential in healing chronic wounds.