Connexin mimetic peptides enhance wound closure rates in human fibroblasts and keratinocytes in vitro

Recent studies suggest significant increases in skin wound healing rates occur by altering gap junction intercellular communication (GJIC). Agents that directly target specific gap junction subunits, or connexins (Cx), such as connexin mimetic peptides, have great therapeutic potential. To this end we analysed their effect on cell-cell communication and cell migration responses during wound healing.
Primary human skin fibroblasts and keratinocytes, derived from child foreskins obtained with ethical approval and patient consent, were cultured as monolayers with DMEM or Epilife respectively. Cells were treated with two connexin mimetic peptides targeted to Cx43 to block protein functionality: Gap26M which is non-connexin specific, and Gap27 which is Cx43-specific. Following this, cells were microinjected with fluorescent dyes, and dye spread between cells assessed. RTPCR determined connexin expression profiles, and migration studies compared rates of wound closure with and without peptide treatment, which was replaced every 12 hours.
Keratinocytes and fibroblasts expressed a variety of connexins, with Cx43 predominating, which did not alter with peptide treatment. Gap26M significantly reduced dye transfer between fibroblasts at 10-100 µM (e.g. mean ± SEM transfer to 5-10 cells: 4.1 ± 1.9%, 50 µM Gap26M v 20.2 ± 5.3%, control; P < 0.01), whilst Gap27 was more effectual (50 µM, transfer to 2-3 cells: 15.3 ± 4.7% v 38.2 ± 4.7%, P < 0.05) than Gap26M in keratinocytes. 100 µM Gap26M and Gap27 significantly increased wound closure rates in both keratinocytes and fibroblasts (P < 0.01), where Gap27 closed wounds faster than Gap26M in fibroblasts, the contrary in keratinocytes.
Connexin mimetic peptides blocked gap junction functionality in human skin cells and increased wound closure rates, although effects differed in static and migrating cells. Results suggest that connexin mimetic peptides can thus directly improve wound closure and have therapeutic potential in wound healing.