In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-β1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100 nM–100 μM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM–100 μM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-β1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration.

The integumentary system comprises the skin and its appendages, which includes hair, nails, feathers, sebaceous and eccrine glands. In this review, we focus on the expression profile of connexins and pannexins throughout the integumentary system in mammals, birds and fish. We provide a picture of the complexity of the connexin/pannexin network illustrating functional importance of these proteins in maintaining the integrity of the epidermal barrier. The differential regulation and expression of connexins and pannexins during skin renewal, together with a number of epidermal, hair and nail abnormalities associated with mutations in connexins, emphasize that the correct balance of connexin and pannexin expression is critical for maintenance of the skin and its appendages with both channel and non-channel functions playing profound roles. Changes in connexin expression during both hair and feather regeneration provide suggestions of specialized communication compartments. Finally, we discuss the potential use of zebrafish as a model for connexin skin biology, where evidence mounts that differential connexin expression is involved in skin patterning and pigmentation.

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We present a topical drug delivery mechanism through the ear canal to the middle and inner ear using liposomal nanoparticles without disrupting the integrity of the tympanic membrane. The current delivery method provides a noninvasive and safer alternative to transtympanic membrane injections, ear tubes followed by ear drops administration, and systemic drug formulations. We investigate the capability of liposomal NPs, particularly transfersomes (TLipo), used as drug delivery vesicles to penetrate the tympanic membrane (TM) and round window membrane (RWM) with high affinity, specificity, and retention time. The TLipo is applied to the ear canal and found to pass through the tympanic membrane quickly in 3 h post drug administration. They are identified in the middle ear cavity 6 h and in the inner ear 24 h after drug administration. We performed cytotoxicity in vitro and ototoxicity in vivo studies. Cell viability shows no significant difference between the applied TLipo concentration and control. Furthermore, auditory brainstem response (ABR) reveals no hearing loss in 1 week and 1 month post-administration. Immunohistochemistry results demonstrate no evidence of hair cell loss in the cochlea at 1 month following TLipo administration. Together, the data suggested that TLipo can be used as a vehicle for topical drug delivery to the middle ear and inner ear.

A major problem of diabetic foot ulcers is ischemia contributing to delayed wound closure. The gap junction protein Connexin43 (Cx43) is differentially remodelled during 'normal' and 'chronic' wound healing events. The mechanisms by which this protein is involved remains unresolved. This work investigated the expression and post translational modification of Cx43 and Pannexin 1 (Panx1) in skin biopsies from patients undergoing arterial reconstruction or major limb amputation for critical limb ischaemia and in keratinocytes cultured in normoxic (N) and hypoxic (H) (1% oxygen, 5% carbon dioxide and 94 % nitrogen) conditions. Skin tissue biopsies (proximal and distal to the point of venous bypass surgery) from diabetic and non-diabetic patients were processed for immunohistochemistry and stained with antibodies specific to Cx43 and Cx43Ser 368. To model the events in vitro HaCaT cells were grown in 12 well plates, scrape wounded and cultured in N or H environments for up to 48 hours (h). Cell movement into the denuded area was recorded. Cells were fixed or protein harvested and expression of hypoxic inducible factor 1-a (HIF-1a), Cx43, Cx43Ser 368 , Panx1 and Ki67 determined by immunocytochemistry and Western blot analysis. Cx43 and Cx43Ser 368 expression was significantly increased in the epidermis of skin biopsies isolated from the distal position (i.e. locations of ischaemia) in both diabetic and non-diabetic tissue. Scratch wound assay determined delayed wound closure in hypoxic conditions, that was confirmed by induction of HIF-1a expression. Cx43 and Cx43 Ser368 staining showed translocation of the protein to the nucleus. No changes in Panx1 were observed. In conclusion, post translational phosphorylation of Cx43 occurs in chronically wounded diabetic and ischemic skin. Under H conditions, scrape wound closure is reduced and immunofluorescence suggests that Cx43 and Cx43Ser 368 translocate to the nucleus after 24 h hypoxia. Thus, hypoxia impacts on Cx expression but not Panx1 expression in the epidermis.