Modelling the human epidermis in vitro: tools for basic and applied research

Culture models of tissues and organs are valuable tools developed by basic research that help investigation of the body functions. Modelling is aimed at simplifying experimental procedures in order to better understand biological phenomena, and consequently, when sufficiently characterized, culture models can also be utilized with high potential in applied research. In skin biology and pathology, the development of cultures of keratinocytes as monolayers has allowed the elucidation of most functional and structural characteristics of the cell type. Beside the multiple great successes that have been obtained with this type of culture, this review draws attention on several neglected characteristics of monolayer cultures. The more sophisticated models created in order to reconstruct the fully differentiated epidermis have followed the monolayers. The epidermal reconstruction produces all typical layers found in vivo and thus makes the model much less simple, but only this kind of model allows the study of full differentiation in keratinocyte and production of the cornified barrier. In addition to its interest in basic research, the reconstructed epidermis is currently gaining a lot of interest for applied research, particularly as an alternative to laboratory animals in the chemical and cosmetic industry. Today several commercial providers propose reconstructed skin or epidermis, but in vitro assays on these materials are still under development. In order to be beneficial at long term, the validation of assays must be performed on a material whose availability will not be interrupted. We warn here providers and customers that the longevity of in vitro assays will be guaranteed only if these assays are done with well-described models, prepared according to published procedures, and must consider having a minimum of two independent simultaneous producers of similar material

Theophylline exerts complex anti‐ageing and anti‐cytotoxicity effects in human skin ex vivo

Objective Theophylline is a phosphodiesterase inhibitor that is being used clinically for asthma therapy. In addition, it is recognized as a cosmetic agent with possible anti‐ageing and anti‐oxidative properties. Nevertheless, how it affects human skin is still poorly examined. Methods Theophylline (10 or 100 µM) was administered to the culture medium of full‐thickness human skin ex vivo for 24 or 72 h. Results Theophylline stimulated protein expression of the anti‐oxidant metallothionein‐1 and mRNA levels of collagen I and III. Assessment of fibrillin‐1 immunohistology revealed enhanced structural stability of dermal microfibrils. Theophylline also exerted extracellular matrix‐protective effects by decreasing MMP‐2 and MMP‐9 mRNA levels, partially antagonizing the effects of menadione, the potent, toxic ROS donor. In addition, it decreased menadione‐stimulated epidermal keratinocytes apoptosis. Interestingly, theophylline also increased the level of intracutaneously produced melatonin, that is the most potent ROS‐protective and DNA damage repair neuromediator, and tendentially increased protein expression of MT1, the melatonin receptor. Theophylline also increased the expression of keratin 15, the stem cell marker, in the epidermal basal layer but did not change mitochondrial activity or epidermal pigmentation. Conclusion This ex vivo pilot study in human skin shows that theophylline possesses several interesting complex skin‐protective properties. It encourages further examination of theophylline as a topical candidate for anti‐ageing treatment. Résumé Objectif la théophylline est un inhibiteur de la phosphodiestérase actuellement utilisée en clinique pour le traitement de l’asthme. En outre, elle est reconnue comme étant un agent cosmétique ayant des propriétés potentiellement anti‐âge et antioxydantes. Cependant, la manière dont elle affecte la peau chez l’homme est encore très peu étudiée. Méthodes de la théophylline (10 ou 100 μM) a été ajoutée dans le milieu de culture d’un échantillon de peau humaine d’épaisseur totale ex vivo pendant 24 ou 72 h. Résultats la théophylline a stimulé l’expression de la métallothionéine‐1, une protéine antioxydante, et les taux d’ARNm du collagène I et III. L’évaluation immunohistologique de la fibrilline‐1 a révélé une meilleure stabilité structurale des microfibrilles du derme. La théophylline a également exercé des effets protecteurs sur la matrice extracellulaire en diminuant les taux d’ARNm des métalloprotéinases matricielles MMP‐2 et MMP‐9, neutralisant en partie les effets de la ménadione, puissant donneur d’espèces réactives de l'oxygène (ROS) toxiques. En outre, elle a diminué l’apoptose des kératinocytes épidermiques stimulés par la ménadione. Fait intéressant, la théophylline a également augmenté le taux de mélatonine produite de manière intra‐cutanée, la mélatonine étant le plus puissant neuromédiateur protecteur contre les ROS et réparateur des lésions de l’ADN. Elle a augmenté de façon tendancielle l’expression de la protéine MT1, récepteur de la mélatonine. La théophylline a également augmenté l’expression de la kératine 15, marqueur de cellules souches, dans la couche basale épidermique, mais n’a pas modifié l’activité mitochondriale ou la pigmentation épidermique. Conclusion cette étude pilote ex vivo réalisée sur de la peau humaine montre que la théophylline a plusieurs propriétés protectrices de la peau complexes et intéressantes. Ces résultats encouragent à poursuivre l’étude de la théophylline en tant que candidat à un traitement local anti‐âge. Theophylline is a phosphodiesterase inhibitor that is being used clinically for asthma therapy and recognized as a cosmetic agent. This pilot study in organ‐cultured middle‐aged skin shows that theophylline possesses several interesting and surprisingly complex skin‐protective, anti‐ageing and anti‐oxidative properties. This encourages further systematic examination of theophylline as a topical candidate for anti‐ageing

Lysosomal membrane permeabilization in cell death

18 páginas, 3 figuras, 2 tablas -- PAGS nros. 6434-6451Mitochondrial outer membrane permeabilization (MOMP) constitutes one of the major checkpoint(s) of apoptotic and necrotic cell death. Recently, the permeabilization of yet another organelle, the lysosome, has been shown to initiate a cell death pathway, in specific circumstances. Lysosomal membrane permeabilization (LMP) causes the release of cathepsins and other hydrolases from the lysosomal lumen to the cytosol. LMP is induced by a plethora of distinct stimuli including reactive oxygen species, lysosomotropic compounds with detergent activity, as well as some endogenous cell death effectors such as Bax. LMP is a potentially lethal event because the ectopic presence of lysosomal proteases in the cytosol causes digestion of vital proteins and the activation of additional hydrolases including caspases. This latter process is usually mediated indirectly, through a cascade in which LMP causes the proteolytic activation of Bid (which is cleaved by the two lysosomal cathepsins B and D), which then induces MOMP, resulting in cytochrome c release and apoptosome-dependent caspase activation. However, massive LMP often results in cell death without caspase activation; this cell death may adopt a subapoptotic or necrotic appearance. The regulation of LMP is perturbed in cancer cells, suggesting that specific strategies for LMP induction might lead to novel therapeutic avenuesResearch in our labs is supported by grants from Ministry of Science (BFU-2006-00508) and from Fundación La Caixa (BM06-125-1) to PB and Ligue Nationale contre le Cancer (Equipe labellisée), European Commission (Active p53, Apo-Sys, RIGHT, TransDeath, ChemoRes, DeathTrain), Agence Nationale pour la Recherche, Institut National contre le Cancer, Cancéropôle Ile-de-France and Fondation pour la Recherche Médicale to GKPeer reviewe