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.
Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL‐6, IL‐8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real‐time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa β (NF‐κβ) was blocked with BAY 11‐7082, CX‐channel function was determined by adenosine 5′‐triphosphate (ATP) release assays. Enzyme‐linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL‐6, IL‐8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN‐induced CX26 and IL‐6 expression were also aborted by TLR2‐KD and inhibition of NF‐κβ. ATP and IL‐6 release were stimulated following 15 min and 1–24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX‐channel blockers, CX26‐, CX43‐ and TLR2‐KD. The IL‐6 response was also reduced by purinergic blockers. CX‐signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF‐κβ, directly activates CX26 and IL‐6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.
Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.
Epithelial tissue responds rapidly to environmental triggers and is constantly renewed. This tissue is also highly accessible for therapeutic targeting. This review highlights the role of connexin mediated communication in avascular epithelial tissue. These proteins form communication conduits with the extracellular space (hemichannels) and between neighboring cells (gap junctions). Regulated exchange of small metabolites less than 1kDa aide the co-ordination of cellular activities and in spatial communication compartments segregating tissue networks. Dysregulation of connexin expression and function has profound impact on physiological processes in epithelial tissue including wound healing. Connexin 26, one of the smallest connexins, is expressed in diverse epithelial tissue and mutations in this protein are associated with hearing loss, skin and eye conditions of differing severity. The functional consequences of dysregulated connexin activity is discussed and the development of connexin targeted therapeutic strategies highlighted.
Peroperative antegrade colonic lavage is often performed before primary anastomosis in emergency colonic surgery. The influence of colonic lavage on bacterial translocation from the obstructed colon was determined. Forty female Wistar rats were studied in four groups: (1) control; (2) non-obstructed with lavage; (3) obstructed; and (4) obstructed with lavage. Ligature obstruction of the rectum was performed in groups 3 and 4. Some 4 days later 35S-radiolabelled Escherichia coli was inoculated into the colon of all animals. Groups 2 and 4 underwent colonic lavage. Lavage in the group 4 animals with left-sided colonic obstruction significantly increased the levels of E. coli in regional nodes, liver, spleen, lung, kidney and blood (as assessed by organ culture and scintillation counting) compared with those in groups 1, 2 and 3 (P < 0.05). These results suggest that peroperative lavage of the obstructed colon significantly increases the level of bacterial translocation.
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