Abstract
Manipulation of connexin mediated communication networks can modify wound healing rates by altering epidermal keratinocyte and/or fibroblast behaviour. Agents that directly target specific connexins and their mechanisms of action have great therapeutic potential, thus we analysed their effects on cell-cell communication and cell proliferation responses during wound healing.
HeLa cells stably transfected with Cx43, Cx26 or Cx40 were cultured as monolayers with DMEM. Primary human skin fibroblasts, derived from child foreskins obtained with ethical approval and patient consent, were cultured similarly. Following treatment with connexin mimetic peptides (Gap26, Gap26M and Gap27) targeted to Cx43 to block protein functionality, the cells were microinjected with Alexa 488 or 594 fluorescent dyes, and the dye spread between cells measured by confocal microscopy. RTPCR determined the connexin expression profile for fibroblasts, and migration studies compared rates of wound closure with and without peptide treatment, which was replaced every 12 hours.
Gap26 and Gap26M dose-dependently inhibited dye transfer in the HeLa cell model in a non-connexin-specific manner (ANOVA: P < 0.01), whereas Gap27 was more effective in HeLa-Cx43 cells than HeLa-Cx26 and HeLa-Cx40 cells at low concentrations (P < 0.01). Gap26M significantly reduced dye transfer between fibroblasts at all concentrations studied (P < 0.01). Fibroblasts were found to express Cx43, Cx40 and Cx45 mRNA; and treatment with Gap 26M did not alter expression levels. In migration assays, 100 ¿M Gap26M increased wound closure rates.
In conclusion, Gap26 and Gap26M are non-connexin-specific peptides, whereas Gap27 is Cx43 specific. Gap26M and Gap27 were also effective at low doses (10 – 50 ¿M). That Gap26M did not alter fibroblast connexin expression confirms that connexin mimetic peptides act functionally. Gap26M effectively blocked cell-cell communication in primary cells and increased wound closure rates, exemplifying the huge potential of peptides as therapeutic agents.
HeLa cells stably transfected with Cx43, Cx26 or Cx40 were cultured as monolayers with DMEM. Primary human skin fibroblasts, derived from child foreskins obtained with ethical approval and patient consent, were cultured similarly. Following treatment with connexin mimetic peptides (Gap26, Gap26M and Gap27) targeted to Cx43 to block protein functionality, the cells were microinjected with Alexa 488 or 594 fluorescent dyes, and the dye spread between cells measured by confocal microscopy. RTPCR determined the connexin expression profile for fibroblasts, and migration studies compared rates of wound closure with and without peptide treatment, which was replaced every 12 hours.
Gap26 and Gap26M dose-dependently inhibited dye transfer in the HeLa cell model in a non-connexin-specific manner (ANOVA: P < 0.01), whereas Gap27 was more effective in HeLa-Cx43 cells than HeLa-Cx26 and HeLa-Cx40 cells at low concentrations (P < 0.01). Gap26M significantly reduced dye transfer between fibroblasts at all concentrations studied (P < 0.01). Fibroblasts were found to express Cx43, Cx40 and Cx45 mRNA; and treatment with Gap 26M did not alter expression levels. In migration assays, 100 ¿M Gap26M increased wound closure rates.
In conclusion, Gap26 and Gap26M are non-connexin-specific peptides, whereas Gap27 is Cx43 specific. Gap26M and Gap27 were also effective at low doses (10 – 50 ¿M). That Gap26M did not alter fibroblast connexin expression confirms that connexin mimetic peptides act functionally. Gap26M effectively blocked cell-cell communication in primary cells and increased wound closure rates, exemplifying the huge potential of peptides as therapeutic agents.
| Original language | English |
|---|---|
| Publication status | Published - 2006 |
Keywords
- connexin mimetic peptides
- wound closure
- connexin specificity