Functional testing of topical skin formulations using an optimised ex vivo skin organ culture model.

A number of equivalent-skin models are available for investigation of the ex vivo effect of topical application of drugs and cosmaceuticals onto skin, however many have their drawbacks. With the March 2013 ban on animal models for cosmetic testing of products or ingredients for sale in the EU, their utility for testing toxicity and effect on skin becomes more relevant. The aim of this study was to demonstrate proof of principle that altered expression of key gene and protein markers could be quantified in an optimised whole tissue biopsy culture model. Topical formulations containing green tea catechins (GTC) were investigated in a skin biopsy culture model (n = 11). Punch biopsies were harvested at 3, 7 and 10 days, and analysed using qRT-PCR, histology and HPLC to determine gene and protein expression, and transdermal delivery of compounds of interest. Reduced gene expression of α-SMA, fibronectin, mast cell tryptase, mast cell chymase, TGF-β1, CTGF and PAI-1 was observed after 7 and 10 days compared with treated controls (p < 0.05). Histological analysis indicated a reduction in mast cell tryptase and chymase positive cell numbers in treated biopsies compared with untreated controls at day 7 and day 10 (p < 0.05). Determination of transdermal uptake indicated that GTCs were detected in the biopsies. This model could be adapted to study a range of different topical formulations in both normal and diseased skin, negating the requirement for animal models in this context, prior to study in a clinical trial environment

A comprehensive evidence-based review on the role of topicals and dressings in the management of skin scarring

Wound healing after dermal injury is an imperfect process, inevitably leading to scar formation as the skin re-establishes its integrity. The resulting scars have different characteristics to normal skin, ranging from fine-line asymptomatic scars to problematic scarring including hypertrophic and keloid scars. Scars appear as a different colour to the surrounding skin and can be flat, stretched, depressed or raised, manifesting a range of symptoms including inflammation, erythema, dryness and pruritus, which can result in significant psychosocial impact on patients and their quality of life. In this paper, a comprehensive literature review coupled with an analysis of levels of evidence (LOE) for each published treatment type was conducted. Topical treatments identified include imiquimod, mitomycin C and plant extracts such as onion extract, green tea, Aloe vera, vitamin E and D, applied to healing wounds, mature scar tissue or fibrotic scars following revision surgery, or in combination with other more established treatments such as steroid injections and silicone. In total, 39 articles were included, involving 1703 patients. There was limited clinical evidence to support their efficacy; the majority of articles (n = 23) were ranked as category 4 LOE, being of limited quality with individual flaws, including low patient numbers, poor randomisation, blinding, and short follow-up periods. As trials were performed in different settings, they were difficult to compare. In conclusion, there is an unmet clinical need for effective solutions to skin scarring, more robust long-term randomised trials and a consensus on a standardised treatment regime to address all aspects of scarring

Small Molecule Glycomimetics Inhibit Vascular Calcification via c-Met/Notch3/HES1 Signalling

© Copyright by the Author(s). Published by Cell Physiol Biochem Press. BACKGROUND/AIMS: Vascular calcification represents a huge clinical problem contributing to adverse cardiovascular events, with no effective treatment currently available. Upregulation of hepatocyte growth factor has been linked with vascular calcification, and thus, represent a potential target in the development of a novel therapeutic strategy. Glycomimetics have been shown to interrupt HGF-receptor signalling, therefore this study investigated the effect of novel glycomimetics on osteogenic signalling and vascular calcification in vitro. METHODS: Primary human vascular smooth muscle cells (HVSMCs) were induced by β-glycerophosphate (β-GP) and treated with 4 glycomimetic compounds (C1-C4). The effect of β-GP and C1-C4 on alkaline phosphatase (ALP), osteogenic markers and c-Met/Notch3/HES1 signalling was determined using colorimetric assays, qRT-PCR and western blotting respectively. RESULTS: C1-C4 significantly attenuated β-GP-induced calcification, as shown by Alizarin Red S staining and calcium content by day 14. In addition, C1-C4 reduced ALP activity and prevented upregulation of the osteogenic markers, BMP-2, Runx2, Msx2 and OPN. Furthermore, β-GP increased c-Met phosphorylation at day 21, an effect ameliorated by C2 and C4 and the c-Met inhibitor, crizotinib. We next interrogated the effects of the Notch inhibitor DAPT and confirmed an inhibition of β-GP up-regulated Notch3 protein by C2, DAPT and crizotinib compared to controls. Hes-1 protein upregulation by β-GP, was also significantly downregulated by C2 and DAPT. GOLD docking analysis identified a potential binding interaction of C1-C4 to HGF which will be investigated further. CONCLUSION: These findings demonstrate that glycomimetics have potent anti-calcification properties acting via HGF/c-Met and Notch signalling

Strategies for Inhibiting Advanced Glycation Endproduct (Age) Induced Vascular Calcification in a Smooth Muscle Cell Culture Model

Vascular calcification is implicated in a range of cardiovascular disease mechanisms, leading to an associated increase in morbidity and mortality. One such trigger are advanced glycation endproducts (AGEs), the tissue accumulation of which increases with age and is more prevalent in diabetic subjects due to oxidative stress and poor glycaemic control. The aim of this study was to investigate the osteogenic potential of AGEs and elucidate mechanisms of inhibiting these processes in a smooth muscle cell (SMC) culture model. Osteogenic differentiation of SMCs was induced using β-glycerophosphate (β-GP), carboxymethyllysine (CML), carboxyethyllysine (CEL) methylglyoxal (MGO) and glycated low density lipoprotein (gly-LDL). The cells were subsequently treated with aminoguanidine (AG), an inhibitor of AGE formation, and novel glycomimetic compounds in order to determine their anti-calcification potential in vitro using qPCR, ELISA, Alkaline phosphatase (ALP) activity and Alizarin red staining. Gly-LDL (10 µg/ml) and CML (2.5nM) increased the level of calcification observed compared to the β-GP (5 mM) positive control after 21 days (p < 0.05), with gly-LDL induced calcification apparent after 14 days. Both AG (250 µM) and the novel glycomimetic compounds reduced the level of mineralisation observed at 21 days compared with osteogenic treatments (p < 0.05). CEL (2.5 nM) and MGO (0.1 mM) both induced calcification, however mineralization was not as extensive as with β-GP. When compared to the structure of CML, the side-chain of CEL contains an extra methyl group, suggesting this group impacts RAGE receptor binding. It was also shown that β-GP combined with increased glucose concentration induced more extensive calcification unlike low glucose levels and β-GP alone. ALP activity, when stimulated with β-GP, CML and gly-LDL was greater at day 4 than at day 7, with AG reducing ALP activity measurements at day 4. Gly-LDL increases gene expression of OCN at day 4 compared with β-GP and CML, however this was reduced at day 7, corresponding with an increased expression of OPN and OPG. NOTCH-3 gene expression was also reduced at day 7. Gene expression of OPN, OPG and NOTCH-3 were reduced at both day 4 and day 7 compared with osteogenic treatments (β-GP, CML and gly-LDL). In summary, we conclude that gly-LDL and CML are potent inducers of calcification compared with β-GP, and that their osteogenic potential can be modulated by both AG and novel glycomimetic compounds