Comparison of surface free energy between reconstructed human epidermis and in situ human skin

Background/aims: The objectives of this study were to characterise the Episkin^® model of reconstructed epidermis histologically and energetic, and to compare the results with the data obtained by Mavon (9, 10) on different areas of skin on the human body and on different skin types. Methods: Histologically, the two skin systems studied were stained using Goldner's trichome method. Surface free energy was determined by the measurement of contact angles, using the posed drop method. Results: Histologically, the two skin systems demonstrated the same cellular organisation in stratified layers. With regard to surface free energy, Episkin, skin from the forehead, normal and hyper‐seborrheic skins are hydrophilic surfaces, while skin from the forearm or of an oligo‐seborrheic type tends to be hydrophobic. Episkin and skin from the forehead exhibit similar surface free energy profiles. It is impossible to state whether Episkin has characteristics closer to those of normal or hyper‐seborrheic skin, but they differ significantly from those of oligo‐seborrheic skin. Conclusion: The results obtained during this study justify the use of Episkin in a range of tests where the use of in situ human skin raises problems.     

Topical ceramides neither enhance UVB‐induced apoptosis in normal human keratinocytes nor affect viability in UVB‐irradiated reconstructed human epidermis

Ceramides are the major lipid of lamellar sheets present in intercellular spaces of the stratum corneum contributing to epidermal barrier properties. Therefore, ceramides and their analogues have been studied for barrier enhancing and water‐holding properties for decades. In vitro studies have indicated cytotoxic potential for cell‐permeable ceramides thereby raising the question whether topical ceramide application might contribute to UVB‐induced apoptosis. Phytosphingosine, N‐hexanoyl‐phytosphingosine and N‐stearoylphytosphingosine (ceramide III) in concentrations ≤5 μm have been used for co‐stimulation with low (160 J/m²) or high (600 J/m²) UVB doses in subconfluent basal and confluent differentiating keratinocytes. Significantly, increased caspase‐3 activity was observed in basal keratinocytes irradiated with 600 J/m² UVB and in differentiating keratinocytes with both UVB doses. Co‐stimulation with the named ceramides did not further increase (i) caspase‐3 activity and (ii) nucleosomal fragmentation in differentiating keratinocytes. Moreover, co‐stimulation with 1‐mm ceramides did not further affect viability/lactate dehydrogenase release in UVB‐irradiated reconstructed human epidermis corroborating the safety of these ceramides.     

Carbohydrate expression and modification during keratinocyte differentiation in normal human and reconstructed epidermis

Using fluorescein isothiocyanate (FITC)-labeled lectins we were able to demonstrate the presence of specific carbohydrate moieties in normal human and reconstructed epidermis. Evidence is provided that in both cases the strongly reduced lectin staining at the level of the stratum corneum is the result of a hindered accessibility of the lectins in this lipid-rich hydrophobic environment. Isolated corneocytes and purified cornified envelopes (CEs) exhibited clearly glycosylated structures reacting with distinct lectins. The presence of glycosidase activity, particularly in the upper layers of the epidermis characterized by an acidic environment (pH 5.5), indicates that modifications of the sugar residues might be important in epidermal homeostasis, barrier behavior and desquamation. Absent or strongly reduced glycosidase activity in the stratum corneum of reconstructed epidermis with an impaired pH gradient could be in part responsible for the reduced barrier function and the lack of desquamation in this model.     

A simple reconstructed human epidermis: preparation of the culture model and utilization in in vitro studies

The preparation of a reconstructed human epidermis is described with examples of its utilization in in vitro studies. The model was obtained by culturing normal human keratinocytes at high cell density for 14 days in serum-free and high calcium (1.5 mM) medium on an inert polycarbonate filter at the air-liquid interface. These stratified cultures showed histological features similar to those observed in vivo in the epidermis: a proliferating basal layer and differentiating spinous, granular, and cornified layers. Electron microscopy illustrated lamellar bodies, junctions and keratohyalin granules. Immunofluorescent localization of epidermal markers (keratins 14 and 10, involucrin and filaggrin) revealed typical differentiation. This in vitro reconstructed tissue was used in studies of toxic effects of chemicals. The modelled tissue showed progressive cytotoxicity of a skin irritant (benzalkonium chloride) and a sensitizer (dinitrochlorobenzene) as assessed by MTT assay. Moreover, differential release of interleukin-1 and interleukin-8 were measured after 20 h of incubation allowing the irritant to be distinguished from the sensitizer. Permeation studies indicated efficient barrier function of the reconstructed epidermis, as well as metabolizing properties towards hormones. This model can be custom-made and is potentially useful for studies involving keratinocytes in the epidermis, in basic science, dermatology or toxicology.     

The lazaroid tirilazad is a new inhibitor of direct and indirect UVA-induced lipid peroxidation in human dermal fibroblasts

Lipid peroxidation caused by oxidative stress within the tissue leads to destruction and dysfunction of cellular membranes. Human dermal fibroblasts in the skin are subject to constant photooxidative stress caused mainly by deeply penetrating UVA irradiation. Therefore, the membrane damage caused by this photooxidative stress may be a major promoter of photoaging and photocarcinogenic processes initiated and promoted by long-term UVA exposure of the skin. The oxidative destruction is counterbalanced by a complex network of enzymatic and nonenzymatic antioxidants creating the skins line of defence against UVA-induced reactive oxygen species. The lazaroid tirilazad represents a new synthetic group of antioxidants with structural molecular similarity to glucocorticosteroids. We investigated the antioxidative capacity of tirilazad by determining its effects on the levels of malondialdehyde (MDA), as a marker of lipid peroxidation, induced directly or indirectly by UVA in human dermal fibroblasts. In a time- and dose-dependent kinetic, we demonstrated that fibroblasts incubated with tirilazad are well protected against subsequent UVA irradiation and show no increase in MDA levels similar to the unirradiated controls. This was also observed when lipid peroxidation was caused chemically by incubation of human dermal fibroblasts with 200 M Fe³⁺-citrate and 1 mM ascorbyl phosphate as a model of indirect UVA-induced skin damage. Lysates of fibroblasts treated this way showed a tenfold increase in MDA levels, whereas preincubation with tirilazad resulted in a significantly lower increase in MDA levels. Furthermore, in a comparison with the well-established radical scavenger Trolox, an -tocopherol analogue, tirilazad offered better protection to the membranes. Our results demonstrate for the first time that the lazaroid tirilazad is an effective inhibitor of direct and indirect UVA-induced increases in MDA as a marker of lipid peroxidation in human dermal fibroblasts.     

Xenobiotic metabolizing enzymes in human skin and SkinEthic reconstructed human skin models

Skin metabolism is becoming a major consideration in the development of new cosmetic ingredients, skin being the first organ exposed to them. In order to replace limited samples of Excised human skin (EHS), in vitro engineered human skins have been developed. 3D models are daily used to develop and evaluate new cosmetic ingredients and have to be characterized and compared with EHS in terms of metabolic capabilities. This work presents the determination of apparent catalytic parameters (apparent Vmax, Km and the ratio Vmax/Km) in 3D models compared with EHS for cytochrome P450 dependent monooxygenase isoforms involved in drug metabolism, esterases, alcohol dehydrogenases, aldehyde dehydrogenases, peroxidases, glutathione S‐transferases, N‐acetyl transferases, uridinyl diphosphate glucuronyl transferases and sulfotransferases. Results show that all these enzymes involved in the metabolism of xenobiotics are expressed and functional in the EHS and 3D models. Also, the Vmax/Km ratios (estimating the intrinsic metabolic clearances) show that the metabolic abilities are the most often comparable between the skin models and EHS. These results indicate that the 3D models can substitute themselves for EHS to select cosmetic ingredients on the basis of their metabolism, efficacy or/and safety.     

Gene expression profiles of three different models of reconstructed human epidermis and classical cultures of keratinocytes using cDNA arrays

The gene expression profiles of three different models of reconstructed human epidermis were analyzed in a comparative study using cDNA array technology. The study also included normal human subconfluent keratinocytes cultured on plastic. Arrays were custom-made and comprised 504 known genes related to cutaneous biology. The gene expression profiles of the three reconstructed epidermis models shared 86% similarity; only 22 of the 504 examined genes showed a different expression level. A comparison of the 3D models with keratinocyte cultures on plastic dishes revealed a set of six genes with a considerably higher expression in the 3D models. These genes were keratin 1, corneodesmosin, filaggrin, loricrin, calmodulin-like skin protein and caspase 14, all related to keratinocyte terminal differentiation. The reported data may contribute to a better understanding and characterization of reconstructed epidermal models and may also serve as established references for investigations related to epidermal differentiation and proliferation.     

Adaptive cellular protection against UVA-1-induced lipid peroxidation in human dermal fibroblasts shows donor-to-donor variability and is glutathione dependent

Photo-oxidative stress and subsequent lipid peroxidation (LPO) is one of the major mechanisms of UVA-related skin pathology. The skin's protection system against photo-oxidative stress involves low molecular scavengers as well as highly specialised antioxidant enzymes like glutathione peroxidase (GPX). Against repetitive UVA-1 exposures in vitro it is partly adaptive, as recent studies have shown exemplarily for antioxidant enzymes. We now investigated in vitro by repetitively irradiating human dermal fibroblasts with UVA-1 whether this adaptive response might reflect itself in reduced cellular membrane damage, that is, LPO. Our experiments show that the degree of cellular protection against LPO and the adaptive potential of the cells against a repetitive UVA-1 exposure varies from donor-to-donor and depends highly on glutathione.     

Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data

Background: Efforts to replace the rabbit skin irritation test have been underway for many years, encouraged by the EU Cosmetics Directive and REACH. Recently various in vitro tests have been developed, evaluated and validated. Objective: A key difficulty in confirming the validity of in vitro methods is that animal data are scarce and of limited utility for prediction of human effects, which adversely impacts their acceptance. This study examines whether in vivo or in vitro data most accurately predicted human effects. Methods: Using the 4‐hr human patch test (HPT) we examined a number of chemicals whose EU classification of skin irritancy is known to be borderline, or where in vitro methods provided conflicting results. Results: Of the 16 chemicals classified as irritants in the rabbit, only five substances were found to be significantly irritating to human skin. Concordance of the rabbit test with the 4‐hr HPT was only 56%, whereas concordance of human epidermis models with human data was 76% (EpiDerm) and 70% (EPISKIN). Conclusions: The results confirm observations that rabbits overpredict skin effects in humans. Therefore, when validating in vitro methods, all available information, including human data, should be taken into account before making conclusions about their predictive capacity.     

Inhibitory effect of the water-soluble polymer-wrapped derivative of fullerene on UVA-induced melanogenesis via downregulation of tyrosinase expression in human melanocytes and skin tissues

The C60-fullerene derivatives are expected, as novel and potent anti-oxidants, to more effectively protect skin cells against oxidative stress. UVA-induced oxidative stress is considered to promote melanogenesis and serious skin damage. The effect of any fullerene derivatives on UVA-induced melanogenesis is still unknown. Here, we evaluated effects of a water-soluble polyvinylpyrrolidone (PVP)-wrapped fullerene derivative (named "Radical Radical Sponge" because of its anti-oxidant ability) on melanogenesis, which was promoted by UVA-irradiation to human melanocytes and skin tissues. Radical Sponge markedly scavenged UVA-induced reactive oxygen species (ROS) inside human melanocytes as shown by fluorometry using the redox indicator CDCFH-DA. After treatment with Radical Sponge or other agents, human melanocytes and skin tissues were irradiated by UVA. Then, cellular melanin content, tyrosinase activity and the ultrastructural change of skin melanosomes were examined. Radical Sponge showed to significantly inhibit UVA-promoted melanogenesis in normal human epidermis melanocytes (NHEM) and human melanoma HMV-II cells within a non-cytotoxicity dose range. As compared with two whitening agents, arbutin and L-ascorbic acid, Radical Sponge demonstrated the stronger anti-melanogenic potential according to spectrophotometric quantification for extracted melanin. In human skin cultures also, UVA-promoted melanin contents were repressed by Radical Sponge according to Fontana-Masson stain, suggesting its ability to repress UVA-induced tanning. Transmission electron microscopic ultrastructural images also proved that UVA-increased melanosomes in human skin tissue were obviously reduced by Radical Sponge. The UVA-enhanced tyrosinase enzymatic activity in NHEM melanocytes was inhibited by Radical Sponge more markedly than by arbutin and L-ascorbic acid. The UVA-enhanced tyrosinase protein expression, together with cell-size fatness and dendrite-formation, was also inhibited more markedly by Radical Sponge according to immunostain and flow cytometry using anti-tyrosinase antibody. Thus the depigmentating action of Radical Sponge might be due to its down-regulating effect on the tyrosinase expression, which is initiated by UVA-caused ROS generation.     

The induction of ornithine decarboxylase in human epidermis is independent of lipoxygenase and cyclo-oxygenase pathways.

In vivo studies in rodents suggest that prostaglandins and/or leukotrienes are involved in the epidermal induction of ornithine decarboxylase (ODC). Recently, we have shown that, in human epidermis, prostaglandins are not involved in this process. Here we report the role of leukotrienes in epidermal ODC induction in human skin. Topical flufenamic acid (Dignodolin), vehicle, or nothing was applied under plastic occlusion to three sites on the backs of healthy volunteers. This was followed 1 h later by Sellotape stripping. After renewed application and occlusion for 8 h, biopsies were carried out for the estimation of ODC levels. There were no significant differences in the levels of ODC between the flufenamic acid treated and control sites. To confirm this finding, test sites were irradiated with 3 MED of UVB. This was immediately followed by the application of flufenamic acid, vehicle, or nothing to the three irradiated sites. After 8 h, biopsies were taken, and the levels of ODC were again similar in the flufenamic acid- and the vehicle-treated sites. The data indicate that, following Sellotape stripping or UVB irradiation, neither lipoxygenase not cyclooxygenase products contribute to the in vivo induction of ODC in human epidermis.     

Identification of pancreatic type I secreted phospholipase A2 in human epidermis and its determination by tape stripping

Phospholipases A2 (PLA2) catalyse the release of fatty acids from the sn-2 position of phospholipids and have been suggested to play a key part in permeability barrier homeostasis. Using a sensitive and versatile fluorometric method, significant PLA2 activity has been detected in both human skin homogenates and tape strippings of stratum corneum. Based on various properties (resistance to heat and sulphuric acid treatment, neutral optimal pH, absolute requirement for millimolar calcium concentrations, inhibition by dithiothreitol and p-bromophenacyl bromide, and resistance to a trifluoromethyl ketone derivative of arachidonic acid, AACOCF3, a specific inhibitor of cytosolic PLA2), this enzyme was characterized as a secretory PLA2 (sPLA2). Immunohistochemistry revealed strong labelling of type I pancreatic sPLA2 at the stratum corneum-stratum granulosum junction, type II sPLA2 being undetectable. An increase in PLA2 activity in tape-stripped material from the deepest level of the stratum corneum was correlated with partial morphological disappearance of type I sPLA2 immunolabelling. Our data thus provide the first convincing evidence that pancreatic sPLA2 is significantly expressed in human epidermis, where it might participate in the accumulation of free fatty acids contributing to the permeability barrier. In addition, our method for determining PLA2 activity in easily available tape strippings should allow further clinical studies aimed to explore possible PLA2 abnormalities in various dermatoses.     

Protective and determining factors for the overall lipid peroxidation in ultraviolet A1-irradiated fibroblasts: in vitro and in vivo investigations

BACKGROUND: Lipid peroxidation (LPO) is one major effector mechanism by which ultraviolet (UV) A contributes to photoageing and the promotion of skin cancer. It is a fingerprint of photo-oxidative stress within the skin, and is initiated by several pathways, with different reactive oxygen species (ROS) and iron ions being involved. OBJECTIVES: To elucidate factors involved in UVA1-induced LPO in human dermal fibroblasts and mouse dermis, and the role of antioxidant enzymes in protecting cells against LPO. METHODS: Using a highly sensitive high-performance liquid chromatography procedure, we measured malondialdehyde (MDA), a specific metabolic tracer molecule for LPO, to determine the overall LPO produced by a given UVA1 dose in vitro and in vivo. By using the iron chelator desferrioxamine (DFO), the hydroxyl radical scavenger dimethylsulphoxide (DMSO) and fibroblasts that specifically overexpress single antioxidant enzymes, we further indirectly assessed the protective effect of manganese superoxide dismutase (MnSOD), catalase and phospholipid hydroperoxide glutathione peroxidase (PHGPx) as well as the relative importance of different ROS and the role of transitional iron for the total amount of LPO induced by a distinct UVA dose. RESULTS: UVA1 irradiation resulted in a time- and dose-dependent increase in MDA levels in vitro, and the in vitro results were shown to have in vivo relevance. Fibroblasts incubated with DFO or DMSO produced lower levels of MDA than controls, as did fibroblasts overexpressing MnSOD, catalase or PHGPx. CONCLUSIONS: The cellular iron pool and hydroxyl radicals were the most important determining factors for the total amount of MDA produced after a given UVA1 dose, and PHGPx overexpression had the greatest protective effect against LPO.     

Strong induction of AIM2 expression in human epidermis in acute and chronic inflammatory skin conditions

Absent in melanoma 2 (AIM2) is a double‐stranded DNA receptor, and its activation initiates an interleukin‐1 beta processing inflammasome. AIM2 is implicated in host defense against several pathogens, but could hypothetically also contribute to autoinflammatory or autoimmune diseases, such as is the case for NLRP3. Using thoroughly characterised antibodies, we analysed AIM2 expression in human tissues and primary cells. A strong epidermal upregulation of AIM2 protein expression was observed in several acute and chronic inflammatory skin disorders, such as psoriasis, atopic dermatitis, venous ulcera, contact dermatitis, and experimental wounds. We also found AIM2 induction by interferon‐gamma in submerged and three‐dimensional in vitro models of human epidermis. Our data highlight the dynamics of epidermal AIM2 expression, showing Langerhans cell and melanocyte‐restricted expression in normal epidermis but a pronounced induction in subpopulations of epidermal keratinocytes under inflammatory conditions.     

Topical glucocorticosteroids modulate the expression of CRABP I and II in human skin differently

Abstract Epidermal cells express two retinotic acid-binding proteins (CRABP I and II). Because CRABP II protein is strongly induced by topical retinoic acid, the respective roles of the two proteins in the pharmacological activity and toxicity of topical retinoids deserve particular attention. Since topical steroids diminish the irritation induced by retinoic acid (RA), whereas retinoic acid may counteract the atrophogenic effects of steroids, the possible interplay of both compounds in the expression of CRABP I and II appeared worth studying. We have analyzed the effects of topical application of triamcinolone acetonide (TA) on the retinoic acid-induced altered expression of CRABP I and II in normal human skin, at the protein and mRNA levels. We found that CRABP II protein and mRNA were strongly increased upon retinoic acid application: this induction was significantly inhibited by concomitant application of triam-cinolone acetonide; a more potent steroid, dilluocortolone valerate, was also found to dimish normal endogenous expression of CRABP II. In contrast, CRABP I protein was decreased by topical retinoic acid, and the down modulating effect of retinoic acid was counteracted by triamcinolone acetonide.