Connexin mimetic peptides: connexin specificity and effects on human epidermal fibroblast wound closure

Non-healing cutaneous wounds (ulcers) are a major health problem, especially in an ageing population. Manipulation of gap junction mediated communication networks can modify wound healing rates by altering epidermal keratinocyte and or fibroblast behaviour. Gap junctions, formed of connexin proteins, make channels for the direct exchange of small molecules among connected cells. Agents that directly target specific connexins have great therapeutic potential. Thus we analysed the connexin (Cx) specificity and effects of three connexin mimetic peptides, targeted to the extracellular loops of Cx43, on cell-cell communication and cell proliferation and migration responses during fibroblast wound healing, with a view to elucidate their therapeutic potential in regulating wound healing events.
To determine peptide specificity HeLa cells stably transfected to express Cx43, Cx26 or Cx40 were used. Primary human skin fibroblasts were also cultured as monolayers and used in cell migration and dye transfer assays. Following treatment with connexin mimetic peptides (Gap26, Gap26M and Gap27) cells were microinjected with Alexa488 (MW 570 Da) or Alexa594 (MW 795 Da), and the extent of dye spread between neighbouring cells was monitored. RTPCR determined the expression profile of the fibroblasts, and migration assays compared the rate of fibroblast wound closure with and without the peptide.
Gap26 and Gap26M dose-dependently inhibited dye transfer in all three HeLa cell model systems i.e. in a non-connexin specific manner (ANOVA n = 250, P < 0.01), whereas Gap27 was more effective in HeLa-Cx43 cells at low concentrations (50 ¿M) than in those expressing Cx26 or Cx40. Gap26M significantly reduced dye transfer between fibroblasts at 10–100 ¿M (n = 250, P < 0.01). Fibroblasts were found to express Cx43, Cx40 and Cx45, and treatment with Gap26M did not alter Cx expression levels. In migration assays, 100 ¿M Gap26M increased wound closure rates of fibroblasts.
In conclusion, Gap26 and Gap26M are non-connexin specific, whereas Gap27 is Cx43 specific. Gap26M and Gap27 are also effective at low doses (10-50 ¿M). Gap26M effectively blocked cell-cell communication in primary fibroblast cells and increased wound closure rates, exemplifying the potential of connexin mimetic peptides as therapeutic agents.