5,6-Dihydroxyindole eumelanin content in human skin with varying degrees of constitutive pigmentation.

Human skin contains two distinct components: brown to black, insoluble eumelanin and light colored, alkaline-soluble pheomelanin. Eumelanin consists of 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) moieties, while pheomelanin consists of benzothiazine (BT) and benzothiazole (BZ) moieties. These melanin monomer units can be quantitatively analyzed through specific degradation products by high-performance liquid chromatography (HPLC). Alkaline hydrogen peroxide oxidation (AHPO) of eumelanin gives rise to pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) as specific degradation products of the DHICA and DHI moieties, respectively. BZ moiety in pheomelanin can be analyzed as thiazole-2,4,5-tricarboxylic acid (TTCA). By reductive hydrolysis with hydroiodic acid, BT moieties in pheomelanin can be analyzed as 4-amino-3-hydroxyphenylalanine (4-AHP). As a recently improved AHPO-HPLC method enabled a better characterization of PDCA, this prompted us to address the question of DHI to DHICA ratio in human skin samples with varying degrees of constitutive pigmentation ranging from very light to dark. Results showed for the first time the ratio of 4 moieties: DHI 35%, DHICA 41%, BZ 20%, and BT 4%. The ratio is constant regardless of the degree of pigmentation. The high content of DHICA moiety may impart an antioxidant property to the epidermis melanin.

Chemical analysis of constitutive pigmentation of human epidermis reveals constant eumelanin to pheomelanin ratio.

The skin constitutive pigmentation is given by the amount of melanin pigment, its relative composition (eu/pheomelanin) and distribution within the epidermis, and is largely responsible for the sensitivity to UV exposure. Nevertheless, a precise knowledge of melanins in human skin is lacking. We characterized the melanin content of human breast skin samples with variable pigmentations rigorously classified through the Individual Typology Angle (ITA) by image analysis, spectrophotometry after solubilization with Soluene-350 and high-performance liquid chromatography (HPLC) after chemical degradation. ITA and total melanin content were found correlated, ITA and PTCA (degradation product of DHICA melanin), and TTCA (degradation product of benzothiazole-type pheomelanin) as well but not 4-AHP (degradation product of benzothiazine-type pheomelanin). Results revealed that human epidermis comprises approximately 74% of eumelanin and 26% pheomelanin, regardless of the degree of pigmentation. They also confirm the low content of photoprotective eumelanin among lighter skins thereby explaining the higher sensitivity toward UV exposure.

Different oxidative stress response in keratinocytes and fibroblasts of reconstructed skin exposed to non extreme daily-ultraviolet radiation.

Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as "daily UV radiation" (DUVR) with a higher UVA (320-400 nm) to UVB (280-320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure.

Improvement of the dermal epidermal junction in human reconstructed skin by a new c-xylopyranoside derivative.

Skin aging entails drastic changes in the extracellular dermal matrix (ECM) and dermal-epidermal junction (DEJ). These biological alterations are reflected in the clinical signs of aged skin. A new C-xylopyranoside derivative, C-beta-D-xylopyranoside-2-hydroxy-propane (C-Xyloside) has been shown to induce neo-synthesis of matrix proteins such as glycosaminoglycans and heparan sulfate proteoglycans. The aim of this study was to assess the effects of C-Xyloside on markers of the dermal epidermal junction. Basement membrane components, collagen IV, collagen VII and laminin 5 as well as sub-epidermal dermal markers, pro-collagen I and fibrillin 1 were analysed using immunohistochemistry in a reconstructed skin model, including a dermal equivalent populated with living fibroblasts. Levels of mRNA of collagen VII alpha1 and collagen IV alpha1 were evaluated in dermal fibroblasts using RT-PCR. The results showed that C-Xyloside significantly induced a higher deposition of basement membrane and DEJ proteins in the reconstructed skin model and increased collagen VII gene expression. These findings indicate that, in addition to stimulating glycosaminoglycan and heparan sulfate proteoglycan expression, C-Xyloside improves the morphogenesis of the whole DEJ, and strongly suggests beneficial effects in aged skin from restoring DEJ integrity.

Morphogenesis of dermal-epidermal junction in a model of reconstructed skin: beneficial effects of vitamin C.

In skin, cohesion between the dermis and the epidermis is ensured by the dermal-epidermal junction which is also required for control of epidermal growth and differentiation. Here we showed that addition of vitamin C optimized the formation of the dermal-epidermal junction in an in vitro human reconstructed skin model leading to a structure closer to that of normal human skin. Compared with controls, vitamin C treatment led to a better organization of basal keratinocytes, an increase in fibroblast number and a faster formation of the dermal-epidermal junction. Vitamin C also accelerated deposition of several basement membrane proteins, like type IV and VII collagens, nidogen, laminin 10/11, procollagens I and III, tenascin C and fibrillin-1 at the dermal-epidermal junction. The mechanism of action of vitamin C was investigated by quantitative polymerase chain reaction in fibroblasts and keratinocytes respectively. Vitamin C effects passed in part through an increase in col I alpha1, col III alpha1 and fibrillin-1 mRNA levels. Effects on the other markers appeared to happen at the translational and/or post-translational level, as illustrated for tenascin C, col IV alpha2 and col VII alpha1 mRNA levels which were reduced by vitamin C in both cell types.

Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin.

De novo dermal epidermal junction morphogenesis was studied in a skin model including dermal fibroblasts and epidermal keratinocytes. Sequential gene expression, protein deposition, and localization of basement membrane zone components were studied during 15 days. The morphogenesis of dermal epidermal junction is characterized by an implementation of the different components and then a subsequent plateau phase occurring at day 11. Three groups of genes were identified depending on cellular origin and expression profile: 1/genes of fibroblastic origin (col I alpha1, col III alpha1, nidogen, and fibrillin 1); 2/genes expressed in fibroblasts and keratinocytes with symmetrical expression pattern between both cell types (col IV alpha1, col VII alpha1, and tenascin C); 3/laminin beta3 only expressed in keratinocytes. Use of modified organotypic models excluding one cell type revealed a tight interplay between fibroblasts and keratinocytes for synthesis and localization of the components of dermal epidermal junction. Keratinocytes downregulated mRNA and proteins of fibroblastic origin, upregulated col VII in fibroblasts and were absolutely required for dermal-epidermal junction localization of fibroblastic proteins. Fibroblasts downregulated mRNA of keratinocytes and were needed for extracellular secretion and correct localization of type VII collagen and laminin 5.