Wound closure rates in human monolayer and organotypic skin models are increased by connexin mimetic peptides

Significant increases in skin wound healing occur by altering gap junction intercellular communication (GJIC). Agents that directly target specific gap junction subunits (connexins (Cx)) such as connexin mimetic peptides, have great therapeutic promise. Thus we analysed their effect on cell migration 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. Organotypic cultures were created by seeding keratinocytes onto Transwell inserts following culture of fibroblasts on their undersides. Cells were cultured to confluence, and exposed to the air-liquid interface forming living skin equivalents. RT-PCR and immunocytochemistry determined connexin and differentiation marker expression profiles. Cells were treated with two connexin mimetic peptides targeted to Cx43 to block protein functionality: non-connexin specific Gap26M, and Cx43-specific Gap27. Migration assays compared rates of wound closure with and without 100 microM Gap26M or Gap27.
Keratinocytes and fibroblasts expressed a range of connexins, with Cx43 predominating. 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. A variety of differentiation markers including loricrin were expressed by stratified keratinocytes in the organotypic model. Cx43 was expressed in basal proliferating cells with patchy Cx26 predominantly in the differentiated upper layers showing limited Cx43 co-localisation. Gap26M and Gap27 significantly increased wound closure rates (P < 0.01), Gap27 closing wounds faster than Gap26M.
Connexin mimetic peptides blocked gap junction functionality in human skin cells and increased wound closure rates in monolayers and organotypic cultures, which were shown to be representative of human skin. This suggests that connexin mimetic peptides can thus directly improve wound closure and have therapeutic potential in wound healing.