Localization, fate and interactions of Emilin-1 in human skin.

OBJECTIVE: Emilin-1 is a versatile protein abundant in tissues where resilience and elastic recoil are prominent and interacting with components of the extracellular matrix. Still, little is known about Emilin-1 in the skin. Therefore, we investigated Emilin-1 in the skin, its localization, its fate upon ageing, its interactions with other proteins and the effect of its knockdown. METHODS: Skin explants from young or old Caucasian women, immunofluorescently labelled by anti-Emilin-1, anti-Fibrillin-1 and anti-Elastin antibodies, were analysed using confocal microscopy. Skin explants subjected to UV-induced skin ageing were also analysed. Colocalization of Emilin-1 with Collagen IV, Fibrillin-1 and Elastin was studied by multiphoton microscopy and co-immunoprecipitation. Finally, the effect of Emilin-1 extinction was studied by producing small interfering RNA (siRNA) knockdown fibroblasts and by analysing the outcome on selected genes. RESULTS: In skin sections from young donors, Emilin-1 localizes similarly to Elastin and Fibrillin-1. In the papillary dermis, it shows clear and ramified structures, perpendicular to the dermo-epidermal junction that are reminiscent of the oxytalan fibres. In the reticular dermis, Emilin-1 signal appears identical to that of the elastic fibres network. Upon intrinsic or UV-induced ageing, the signal associated with Emilin-1 is drastically reduced and disorganized. Multiphoton microscopy study shows that, as expected, Emilin-1 colocalizes with Elastin. It also colocalizes with Collagen IV in the basement membrane and within dermal fibroblasts. Interaction of Emilin-1 with Elastin and Collagen IV was also found by co-immunoprecipitation. It also reveals interaction with Laminin-5. Finally, siRNA-mediated knockdown of EMILIN-1 show little effect on the expression level of the 61 genes we studied. The most striking change is a downregulation of fibroblast growth factor receptor 2 that show a decrease similar to that of EMILIN-1 itself and after 8 days a downregulation of COL6A1. CONCLUSION: In skin, Emilin-1 locates in the dermis, up to the basement membrane, interacting with components of the extracellular matrix but also with the anchoring complex. These interactions are important for cell adhesion, migration, proliferation and would suggest that Emilin-1 might be important for maintaining the 3D structure of the extracellular matrix.

OBJECTIF: Emilin-1 est une protéine polyvalente, abondante dans les tissus où résilience et élasticité sont importantes et qui interagit avec la matrice extracellulaire. Pourtant, la protéine Emilin-1 a été peu étudiée dans la peau. Nous avons donc étudié sa localisation, son devenir lors du vieillissement, ses interactions avec d'autres protéines et l'effet de son inhibition dans la peau. MÉTHODES: Des explants de peau de femmes caucasiennes jeunes ou âgées, marqués par immunofluorescence avec des anticorps anti-Emilin-1, anti-Fibrilline-1 et anti-Élastine, ont été analysés par microscopie confocale. Des explants cutanés soumis au soumis aux UV pour mimer un photo-vieillissement ont également été analysés. La co-localisation de l'Emilin-1 avec le collagène IV, la fribrilline-1 et l'élastine a été étudiée par microscopie multiphotonique et par co-immunoprécipitation. Enfin, l'effet de l'inhibition de l'expression de la protéine Emilin-1 par interférence ARN a été étudié sur 61 gènes. RÉSULTATS: Dans les coupes de peau de jeunes donneurs, Emilin-1 est localisée comme l'élastine et la fibrilline-1. Dans le derme papillaire, elle présente des structures claires et ramifiées, perpendiculaires à la jonction dermo-épidermique, qui font penser aux fibres d'oxytalane. Dans le derme réticulaire, le signal de l'Emilin-1 apparaît identique à celui du réseau de fibres élastiques. Lors du vieillissement intrinsèque ou induit par les UV, le signal associé à Emilin-1 est considérablement réduit et désorganisé. Une étude en microscopie multiphotonique montre que l'Emilin-1 est co-localisée avec de l'élastine. Elle est aussi co-localisée avec le collagène IV dans la membrane basale et dans les fibroblastes du derme. La co-immunoprécipitation montre l'existence d'interactions entre l'Emilin-1 et l'Elastine ou le collagène IV. Une interaction avec la laminine-5 a aussi été mise en évidence. Enfin, l'inhibition de l'expression de l'EMILIN-1 n'a que peu d'effet sur l'expression des 61 gènes étudiés. Le changement le plus frappant est une diminution de l'expression de FGFR2 (récepteur 2 du facteur de croissance des fibroblastes), diminution similaire à celle d'EMILIN-1, et, après 8 jours, une diminution de l'expression de COL6A1. CONCLUSION: Dans la peau, Emilin-1 se localise dans le derme, jusqu'à la membrane basale, et interagit avec les composants de la matrice extracellulaire mais aussi avec les complexes d'ancrage. Ces interactions sont importantes pour l'adhésion, la migration, la prolifération cellulaire et suggéreraient qu'Emilin-1 pourrait être important pour le maintien de la structure de la matrice extracellulaire.

SpectraCam® : A new polarized hyperspectral imaging system for repeatable and reproducible in vivo skin quantification of melanin, total hemoglobin, and oxygen saturation.

BACKGROUND: An accurate way to determine skin pigmentation is to acquire the spectral reflectance of a skin sample and to quantify chromophores by reverse calculation from physical models of light propagation. Therefore, we tested a new hyperspectral imaging device and software suite, the SpectraCam® system, and evaluated its accuracy to quantify skin chromophores. METHODS: Validation of the SpectraCam® system was performed by, firstly, comparing the known and the acquired reflectance spectra of color phantoms. Repeatability and reproducibility were then evaluated by two operators who performed acquisitions at different time points and compared the acquired reflectance spectra. The specificity of the system was tested by quantitative analysis of single chromophore variation models: lentigo and pressure relief. Finally, we tested the ability of the SpectraCam® system to detect variations in chromophore in the eye region due to the daily application of a new anti-dark circle cosmetic product. RESULTS: The SpectraCam® system faithfully acquires the reflectance spectra of color phantoms (r2 >0.90). The skin reflectance spectra acquired by different operators at different times are highly repeatable (r2 >0.94) and reproducible (r2 >0.99). The SpectraCam® system can also produce qualitative maps that reveal local variations in skin chromophore or underlying structures such as blood vessels. The system is precise enough to detect melanin variation in lentigo or total hemoglobin and oxygen saturation variations upon pressure relief. It is also sensitive enough to detect a decrease in melanin in the eye region due to the application of an anti-dark circle cosmetic product. CONCLUSION: The SpectraCam® system proves to be rapid and produces high-resolution data encompassing a large field of view. It is a robust hyperspectral imaging system that quantifies melanin, total hemoglobin, and oxygen saturation and is well adapted to cosmetic research.

Identification of new morphological differences between Chinese and Caucasian faces and influence of BMI on these characteristics.

BACKGROUND: Concepts of beauty are more and more globalised leading to the homogenisation of the physical appearance. It is therefore important to identify morphological characteristics of ethnic groups. We compare faces from Chinese and Caucasian women, identify morphological differences that were not documented yet and study the influence of BMI on these differences. METHODS: The study was carried on groups of 60 women: a Chinese and a Caucasian group. Both included two equal sub-groups: normal BMI and higher BMI. Face widths were measured from individual pictures and from reconstructed average faces obtained using a new reconstruction algorithm. Cheek/chin and neck/chin angles were determined from individual pictures. Topography of the cheekbone and temple face was determined by fringe projection technique. Ultrasound analysis allows measurements of hypodermis thickness. RESULTS: Our innovative average face reconstruction algorithm produced images of a yet unequalled quality with width characteristics similar to those of individual pictures. Analysis shows that faces of Chinese women are larger and rounder. They present other differences that were so far unidentified. Finally, overweight impacts differently Chinese and Caucasian women faces and has greater influence on Chinese women faces.

Exposure to acute electromagnetic radiation of mobile phone exposure range alters transiently skin homeostasis of a model of pigmented reconstructed epidermis.

Exposure to electromagnetic radiations (EMR) produced by mobile phone concerns half the world's population and raises the problem of their impact on human health. In this study, we looked at the effects of mobile phone exposure (GSM basic, 900 MHz, SAR 2 mW g(-1) , 6 h) on a model of pigmented skin. We have analysed the expression and localization of various markers of keratinocyte and melanocyte differentiation 2, 6, 18 and 24 h after EMR exposure of reconstructed epidermis containing either only keratinocytes or a combination of keratinocytes and melanocytes grown on dead de-epidermized dermis, using histology, immunohistochemistry and Western blot. No changes were found in epidermal architecture, localization of epidermal markers, presence of apoptotic cells and the induction of p53 in both types of epidermis (with or without melanocytes) after exposure to EMR. In pigmented reconstructs, no change in the location and dendricity of melanocytes and in melanin transfer to neighbouring keratinocytes was detected after EMR exposure. Loricrin, cytokeratin 14 were significantly decreased at 6 h. The level of all markers increased at 24 h as compared to 6 h post-EMR exposure, associated with a significant decrease of the 20S proteasome activity. Our data indicate that exposure to 900 MHz frequency induces a transient alteration of epidermal homoeostasis, which may alter the protective capacity of the skin against external factors. Presence or absence of melanocytes did not modify the behaviour of reconstructs after EMR exposure.

Ultrastructural assessments of the melanosome distribution patterns and pigmentation features in human epidermal cells after UV irradiation and kojic acid treatment.

It is well-known that skin pigmentation depends, among others, on number, aggregation and distribution of melanosomes in the epidermis. Here we describe a correlative microscopy-based ultrastructural approach that investigates the spatial distribution and pigmentation features of the melanosomes within melanocytes and keratinocytes. Data obtained from control skin, ultraviolet (UV)-stimulated tissue and kojic acid-treated UV-irradiated explants are compared. We introduce original parameters for the evaluation of the aggregation and pigmentation features of the melanosomes: the aggregation and pigmentation indexes. The aggregation index evaluates the presence of clustered melanosomes when the pigmentation index expresses the electron-density level of the pigment granules. The present study demonstrates that the last parameters clearly express histological effects induced by UVB irradiation. Results indicate that UV light did not change the number of melanosomes within either melanocytes or keratinocytes, but it definitely modified the distribution patterns of the pigment granules in both cell types. It also enhanced the pigmentation state of the epidermal cells. Moreover, statistical analysis concerning keratinocytes discloses a significant decrease in the mean pigmentation index when explants exposed to UV light were treated with kojic acid. Obviously, the present numerical findings point out the relevance of the introduced parameters to characterize the pigmentation state of skin.

Modulating effects of oatmeal extracts in the sodium lauryl sulfate skin irritancy model.

The aim of the present study was to assess the anti-inflammatory activity of two topically applied oatmeal extracts, i.e. Avena sativa and Avena Rhealba, using the sodium lauryl sulfate (SLS) irritation model. At baseline, test areas on the volar surface of the upper arms of 12 healthy individuals were pretreated with the two extracts and their vehicle (petrolatum ointment) under occlusion for 2 h, and one site was left untreated. Then a patch with a 1% SLS solution was applied to the test sites for 24 h. Irritation was determined at each period by measuring by chromametry and laser-Doppler. In a dose-ranging study with the Avena Rhealba extract alone, the 20 and 30% concentrations exerted a slight inhibition of the a* parameter increase and a marked reduction of the blood flow increase (p < 0.05, compared to vehicle). Then, the effects of the two extracts at the concentration of 20% were compared. All extracts displayed a statistically significant counteracting effect on both parameters (p < 0.05), but no statistically significant difference between treatment groups could be demonstrated. In conclusion, this study demonstrates the preventive effects of oatmeal extracts on skin irritation in the SLS model.

Tolerance profile of a sterile moisturizer and moisturizing cleanser in irritated and sensitive skin.

BACKGROUND: This study evaluates the tolerance of preservative free sterile cleanser and sterile moisturizer in irritated and sensitive face skin. MATERIALS AND METHODS: An exploratory, open-label study using the cleanser and the moisturizer in combination was performed with 98 patients with a documented history of allergic contact dermatitis. The 2 products could each be used once or twice daily for 28 days. The assessment parameters at baseline and end of treatment (day 28) included the intensity of erythema, dryness/scaling by the investigator and subjective signs (burning, pruritus and stinging), according to a defined 4-point scale (absent to severe). In addition, a global assessment of the change from baseline and the overall tolerance of the products were performed by the investigator at the end of treatment. RESULTS: Ninety-four patients were included for the efficacy analysis and 96 patients for the safety analysis. At baseline, a majority of patients expressed some degree of erythema (63%), and dryness/scaling (56%). Fewer patients experienced subjective signs at baseline (44%). At the end of treatment, the results showed a statistically significant improvement of all the objective signs of irritated skin (P = .0001, Mac Nemar test), as well as the subjective signs of sensitive skin (P < 0.02). This was confirmed by the overall investigator assessment, showing an excellent or good response in 90% of the patients. In the safety analysis, 1 patient developed contact allergy to 1 ingredient of the test products (carbomer), and 3 patients exacerbated their dermatitis. CONCLUSION: Taken together, these results suggest that adequately formulated cosmetics might reduce both irritated and sensitive skin, with clinical improvement of dryness, erythema and stinging.

A low-salt medical water reduces irritancy of retinoic acid in facial acne.

Although effective medications are available for the treatment of acne, tolerance problems may preclude adequate treatment regimens such as topical retinoic acid, and reduce patient compliance. The present study was conducted to evaluate whether a medical water (Avène) in conjunction with retinoic acid may improve local tolerance in acne. A controlled, open, randomised, multicentric study was completed after 28 days of treatment in 69 acne patients, 34 applying a retinoic acid preparation alone, and 35 applying retinoic acid in association with the water spray used ad libitum. Topical retinoic acid treatment induced prominent signs of irritation in both groups. However, a statistically significant reduction between the two treatment groups could be demonstrated on scaling at all assessment visits (p< or =0.02, Wilcoxon test). No significant water effect on erythema, burning and itching was shown during the treatment period. The overall tolerance assessed by the investigator was significantly improved with the water (p = 0.04, Wilcoxon). Taken together, water with a low mineral content appears to be a promising adjunctive treatment for improving the tolerance of topical retinoids in acne.

Cytotoxicity tests of antibacterial agents on human fibroblasts cultures.

The authors tried to determine and compare the cytotoxicity of several primary substances used in cosmetic or pharmaceutic industry as antimicrobial agents, on single-layer cultures of human fibroblasts. The cytotoxic effect of Germall 115*, Kathon CG* and Pentonium* was pointed out using the colorimetric method with MTT (3-[4-5-dimethyl thiazol 2-yl]-2,5 diphenyl tetrazolium bromide). For each one of these substances, we tested different concentrations with variable contact times. These trials allowed us to classify these products by increasing toxicity as follows: Kathon CG* and Pentonium*. In our experimental conditions, Germall 115* was not cytotoxic.