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Hossein Elbadawy, Mohit Parekh, Carlo Cagini, Diego Ponzin, Stefano Ferrari; Connexin 43 mimetic peptide Gap27 promote healing of human corneal epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):702.
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© ARVO (1962-2015); The Authors (2016-present)
Gap junctions are channels between adjacent cells. Down regulation of the most widely expressed gap junction protein, connexin 43, accelerates cell migration and wound closure. Here, we examined the effects of the Cx43 mimetic peptide Gap27 in wound healing of human corneal epithelium cells (HCEC) in in vitro and ex vivo models.
Extracted HCEC from biopsies were co-cultured with lethally irradiated 3T3 cells in tissue culture inserts. Confluent cultures were treated with either Gap27 or scrambled Gap27 (scGap27) for 1 hour and wound closure rates were measured. In human corneas ex vivo, a 500µm wound was introduced to the epithelium of corneas pre-treated with Gap27 or scGap27 using an AlgerBrush II. Corneas were fixed in 4% paraformaldehyde at each time point (pre-treatment, post-treatment, 6 hours, day 1, 3, 5 and 7) and immune-staining was performed on corneal sections (15µm). Antibodies were used against several markers; Cytokeratin 12, Ki67, P63, F4/80, MPO and VEGF.
In HCEC, Gap27 was able to accelerate the healing of monolayer cultures in vitro by 27.27%, as compared to scGap27 treatment. In human corneas, the expression profiles of Cx43, ki67 and VEGF did not show any patterns. The expression of the epithelialisation marker Cytokeratin 12 was seen more to the outermost layer of the epithelium in corneas treated with Gap27 until day 7 when no differences were noted. Gap27 reduced the expression of the neutrophils marker MPO on day 3, with even more evident reduction after 5 days. Marker of macrophages (F4/80) was less expressed in the limbus of Gap27 treated corneas. The expression peaked after 6 hours then reduced by day 3 as compared to controls (scGap27). Limbal stem cells marker P63 was more expressed in the epithelium of Gap27 treated corneas, and after 3 and 7 days in incubation. As compared to controls, formation of a well-developed multi-layered epithelium occured earlier in Gap27 than in scGAP27 treated corneas.
Connexin 43 mimetic peptide, Gap27, accelerated the wound healing process of HCEC in vitro and ex vivo. Gap27 also accelerated and improved re-epithelialisation of the ocular surface. Additionally, more P63 positive cells were observed and the migration of inflammation mediators towards the injury site was reversed faster in Gap27 treated corneas, as compared to those treated with scGap27.
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