Abstract: The phosphorylated form of histone H2AX, gammaH2AX, is a component of the DNA repair system. Most studies have focused on the role of gammaH2AX during cell transformation and human cancer, but little is known about its role in keratinocytes and the skin during UV irradiation. We analyzed the response to UV irradiation focusing on the phosphorylation of histone H2AX both in vitro, in keratinocyte cultures and in artificial epidermis, and then in vivo, in human skin. Acute UVB irradiation of human keratinocytes increased the phosphorylation of H2AX in a dose-dependent manner; two types of gammaH2AX response were observed either in vitro or in vivo. After a low nonapoptotic UVB irradiation, cells contained phosphorylated H2AX and arrested their cell cycle to repair the DNA damages. For a stronger and proapoptotic UVB irradiation, keratinocytes dramatically increased the phosphorylation of H2AX and committed apoptosis. Our results indicate that gammaH2AX constitutes a highly sensitive marker relevant for studying subapoptotic doses as well as proapoptotic doses of UVB in human skin.
Abstract: Cell longevity is linked to sirtuins (silent information regulators), which belong to a family of enzymes implicated in gene silencing, apoptosis, fatty acid metabolism, and regulation of cellular life spans of organisms. Sirtuins are associated with genes that coordinate and optimize the functions of cells as cells struggle to survive in a stressful environment, as it is the case for skin cells. This study focuses on 1) yeast Kluyveromyces biopetides in stimulating the expression of sirtuin in human cutaneous cells and 2) the benefit for the skin of an active skin care product containing yeast Kluyveromyces biopetides.
Abstract: During chronic UV irradiation, which is part of the skin aging process, proteins are damaged by reactive oxygen species resulting in the accumulation of oxidatively modified protein. UV irradiation generates irreversible oxidation of the side chains of certain amino acids resulting in the formation of carbonyl groups on proteins. Nevertheless, certain amino acid oxidation products such as methionine sulfoxide can be reversed back to their reduced form within proteins by specific repair enzymes, the methionine sulfoxide reductases A and B. Using quantitative confocal microscopy, the amount of methionine sulfoxide reductase A was found significantly lower in sun-exposed skin as compared to sun-protected skin. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during aging and conditions of oxidative stress, the fate of this system was investigated after UVA irradiation of human normal keratinocytes. When keratinocytes are exposed to 15 J/cm(2) UVA, methionine sulfoxide reductase activity and content are decreased, indicating that the methionine sulfoxide reductase system is a sensitive target for UV-induced inactivation.
Abstract: BACKGROUND AND OBJECTIVES: Cell longevity is linked to sirtuins (silent information regulators), which belong to a family of enzymes implicated in gene silencing, apoptosis, fatty acid metabolism, and regulation of cellular life spans of organisms. Sirtuins are associated with genes that coordinate and optimize the functions of cells as cells struggle to survive in a stressful environment, as it is the case for skin cells. This study focuses on 1) yeast Kluyveromyces biopetides in stimulating the expression of sirtuin in human cutaneous cells and 2) the benefit for the skin of an active skin care product containing yeast Kluyveromyces biopetides. METHODS: Silent mating type information regulation 2 homolog 1 (SIRT1) was investigated by immunostaining, Westem blotting, and cytometry on normal human skin cells in culture and on healthy skin samples ex vivo. SIRT7 are mammalian versions of the yeast SIR2 gene. Cellular integrity and aging was followed by comet assays measuring DNA fragmentation and beta galactosidase activity (a marker of senescence). The test product was yeast Kluyveromyces biopeptides. Thirty-three female subjects aged 37 to 64 years (mean 51.6 years) enrolled in the study. Subjects applied a formulation enriched in 1% of the yeast biopeptides SIRT1 activator once daily to the face and neck for 4 weeks. Dermatologists used a graded scale (1-9) to score fine lines and wrinkles, hydration, pigment color intensity, complexion radiance, skin density, firmness, complexion homogeneity, and texture of the skin before and after the first application and again after 4 weeks of use. A Pixel Skin method, based on an analysis of the gray-level variance and surface of imperfections (age-related parameters) from numerical pictures of the faces, was used to objectively measure the skin care efficacy. RESULTS: The yeast Kluyveromyces biopeptides 1) significantly increased SIRT1 expression in normal human dermal skin fibroblasts in vitro (+172%) and in epidermal cells of healthy human skin ex vivo and 2) decreased cell senescence and DNA fragmentation induced by ultraviolet-B (UVB) stress. At the end of the study, facial improvements could be seen on fine lines and wrinkles, hydration, pigmented spot color intensity, complexion radiance, firmness, complexion homogeneity, and texture. Improvement in hydration was significant immediately after the first application. Skin-pixel measurement and analysis show a significant reduction of the gray variance linked to pixel heterogeneity (-4.2%) and a significant reduction of the surface of skin imperfections (-30.4%). All the indicators from clinical evaluation to the objective measurements of the skin show a significant improvement of the aged skin. CONCLUSION: These results demonstrate the efficacy of the yeast Kluyveromyces biopeptides in activating SIRT 1 of human skin cells, improving their DNA resistance and senescence, and of a formulation enriched in this ingredient in treating multiple skin aging signs.
Abstract: During chronic UV irradiation, which is part of the skin aging process, proteins are damaged by reactive oxygen species resulting in the accumulation of oxidatively modified protein. UV irradiation generates irreversible oxidation of the side chains of certain amino acids resulting in the formation of carbonyl groups on proteins. Nevertheless, certain amino acid oxidation products such as methionine sulfoxide can be reversed back to their reduced form within proteins by specific repair enzymes, the methionine sulfoxide reductases A and B. Using quantitative confocal microscopy, the amount of methionine sulfoxide reductase A was found significantly lower in sun-exposed skin as compared to sun-protected skin. Due to the importance of the methionine sulfoxide reductase system in the maintenance of protein structure and function during aging and conditions of oxidative stress, the fate of this system was investigated after UVA irradiation of human normal keratinocytes. When keratinocytes are exposed to 15 J/cm(2) UVA, methionine sulfoxide reductase activity and content are decreased, indicating that the methionine sulfoxide reductase system is a sensitive target for UV-induced inactivation.
Abstract: Aquaporins (AQPs) are proteins that facilitate the transport of water across cell membranes. AQP3 expression is related to the expressions of other epidermal proteins involved in water maintenance (ie, CD44, claudin-1, and filaggrin). The expressions of AQP3 water channels are strongly affected by age and chronic sun exposure, and a defective osmotic equilibrium could occur in the epidermis, which would account for the skin dryness observed in older people and skin areas most exposed to sunlight. We investigated active ingredients that are able to increase AQP3 levels in order to improve hydration in human skin keratinocytes. We selected an ethanolic/water (70/30 v/v) extract of Ajuga turkestanica, a plant from Central Asia, as the hydrating agent. After 17 days of treatment every 2 days with this extract (2.5 microg/mL) in vitro, AQP3 expression measured at the protein level in human reconstructed epidermis was significantly increased. Water transport through both aquaporins and aquaglyceroporins and glycerol transport through aquaglyceroporins alone are important to skin hydration. The distribution and the variability of aquaporins in human skin cells suggest that these channels may have important roles in skin physiology. AQPs appear to be key protein targets to improve the resistance and quality of the skin surface as well as to improve aging and sun exposure-induced dryness as shown by their roles in 1) hydrating the living layers of the epidermis where the keratinocyte differentiation takes place and 2) barrier formation and recovery.
Abstract: Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.
Abstract: Senile lentigo is a common component of photoaged skin. It is characterized by hyperpigmented macules which affect chronically irradiated skin mostly after the age of 50. This study was undertaken to assess the morphology of senile lentigo on the dorsum of the hands. A systematic comparison between lesional and perilesional skin using histology and transmission electron microscopy was done to determine whether melanocytes or keratinocytes are affected in the evolution of lesions and which tissue structure is modified. The histology study showed that lesional skin is characterized by a hyperpigmented basal layer and an elongation of the rete ridges, which seem to drive deeply into the dermis. The epidermis contained clusters of keratinocytes, which retained and accumulated the melanin pigment. Electron microscopy studies showed important modifications in the lesional skin ultrastructure in comparison with perilesional skin. In melanocytes from perilesional and lesional skin, we observed normal size melanosomes at all stages of maturation in the cytoplasm and in migration within dendrites. No pigment accumulation was observed. However, the morphology of melanocytes in lesional skin revealed an activated status with numerous mitochondria and a well-developed endoplasmic reticulum, which could reflect intense protein synthesis. In basal keratinocytes from lesional skin, we observed numerous melanosome complexes called polymelanosomes, which formed massive caps on the nuclei. Observations in colored semi-thin sections also revealed perturbed structures in the basal layer region, which could explain the skin perturbation. Indeed, we observed keratinocytes that presented important microinvaginations and pendulum melanocytes, which sank into the dermis, beneath the basal layer of keratinocytes. These cell modifications seemed to be due to a perturbation of the dermal-epidermal junction, which appeared disorganized and disrupted and could directly disturb the basal support of the cells.
Abstract: Senile lentigo is a common component of photoaged skin. It is characterized by hyperpigmented macules which affect chronically irradiated skin mostly after the age of 50. This study was undertaken to assess the morphology of senile lentigo on the dorsum of the hands. A systematic comparison between lesional and perilesional skin using histology and transmission electron microscopy was done to determine whether melanocytes or keratinocytes are affected in the evolution of lesions and which tissue structure is modified. The histology study showed that lesional skin is characterized by a hyperpigmented basal layer and an elongation of the rete ridges, which seem to drive deeply into the dermis. The epidermis contained clusters of keratinocytes, which retained and accumulated the melanin pigment. Electron microscopy studies showed important modifications in the lesional skin ultrastructure in comparison with perilesional skin. In melanocytes from perilesional and lesional skin, we observed normal size melanosomes at all stages of maturation in the cytoplasm and in migration within dendrites. No pigment accumulation was observed. However, the morphology of melanocytes in lesional skin revealed an activated status with numerous mitochondria and a well-developed endoplasmic reticulum, which could reflect intense protein synthesis. In basal keratinocytes from lesional skin, we observed numerous melanosome complexes called polymelanosomes, which formed massive caps on the nuclei. Observations in colored semi-thin sections also revealed perturbed structures in the basal layer region, which could explain the skin perturbation. Indeed, we observed keratinocytes that presented important microinvaginations and pendulum melanocytes, which sank into the dermis, beneath the basal layer of keratinocytes. These cell modifications seemed to be due to a perturbation of the dermal-epidermal junction, which appeared disorganized and disrupted and could directly disturb the basal support of the cells.
Abstract: More than other tissues, skin is exposed to numerous external stresses generating ROS that, in addition to endogenous oxygen radicals, cause keratinocyte alterations and contribute in part to photocarcinogenesis and aging. Recent evidence suggests a differentiation-dependent susceptibility of keratinocytes to apoptosis. We explored hydrogen peroxide-induced cell death in normal human keratinocytes according to their differentiation. On H(2)O(2)-exposed skin explants, caspase-3 was strongly activated in basal keratinocytes double stained with beta(1) integrin, whereas DNA fragmentation occurred in suprabasal cells only without caspase-3 activation. In addition, isolated basal keratinocytes, selected by adhesion to type IV collagen, were more sensitive than nonadherent cells to H(2)O(2)-induced apoptosis with regard to mitochondrial transmembrane potential (Deltapsi(mt)) collapse and membrane integrity. Similarly, necrotic/late apoptotic cells were present at low levels only in the adherent epidermal population. Furthermore, in primary cultures of undifferentiated keratinocytes H(2)O(2)-induced cell death appeared via a mitochondrial failure. Deltapsi(mt) collapse was associated with a strong early activation of the initiatory caspase-8, then the executive caspase-3, and, to a lesser extent, the inflammatory caspase-1. Finally, undifferentiated basal cells possess a higher sensitivity than differentiated suprabasal cells to H(2)O(2)-induced cell death, and apoptosis in human keratinocytes occurs via different pathways depending on the cell's differentiation state.
Abstract: Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.
Abstract: Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.
Abstract: BACKGROUND: Senile lentigo (SL) is a common component of photoaged skin. It is characterized by hyperpigmented macules which affect chronically irradiated skin mostly after 50 years of age. This study was undertaken to assess the basic morphology of SL on dorsum of hands. METHODS: A systematic comparison between lesional vs. perilesional skin using immunohistochemistry and electron microscopy was done to detect precursor lesions of SL and to determine whether melanocytes or keratinocytes were first affected in the evolution of lesions. RESULTS: In 12 cases studied, the main findings show that clusters of perilesional keratinocytes accumulate melanin in large melanosomial complexes, and that melanocytes counts are increased respective to total length of section in lesional skin, but the increment is probably due to the development of characteristic epidermal rete ridges. Melanocytes had overall a normal ultrastructure, with mostly quiescent features in perilesional skin and melanosomial transport seeming more active in lesional skin. CONCLUSIONS: Our data indicate that SL may represent a loss of epidermal melanin unit homeostasis due to chronic irradiation, where keratinocytic changes predominate over melanocytic changes. We hypothesize that abnormal pigment retention in keratinocytes is the primary defect in SL, which may partly explain the therapeutic effect of retinoids.
Abstract: Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.
Abstract: HSP 47 is a specific chaperone of pro-collagen. This heat shock protein is responsible for the correct 3-dimentional organization of pro-collagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP47 in aged, photoaged and senescent fibroblasts and its modulation by a plant extract (Salix Alba).
We measured the level of HSP47 and/or pro-collagen expression in fibroblasts by real time RT-PCR (mRNA transcripts) and by flow cytometry (immuno-chemistry technique for measurement of arbitrary fluorescence intensity). We used immuno-chemistry techniques and confocal microscopy to vizualized the cellular localization of HSP 47 and pro-collagen. These parameters were compared with different age-donors, non senescent and senescent fibroblasts. Fibroblasts were irradiated by a non cytotoxic dose of UVA (6 J/cm2 ) and HSP 47 level was evaluated. Salix Alba extract was tested for its capacity to modulate HSP 47 expression.
We showed colocalization of HSP47 and procollagen by confocal microscopy, indicating that HSP47 could play a role of procollagen molecular chaperone in our cellular model. We showed that HSP47 level is decreased in old-donor cells, senescent and irradiated cells. This decrease can be modulated by a salix alba extract (polyphenols rich) in a dose dependent manner.
The evaluation of HSP 47 expression in our experimental conditions can lead to a new approach of aging and photoaging pointing out the implication of this chaperone in these pathophysiologic phenomenons. Modulation of HSP 47 expression by molecules family could be of cosmetic and/or dermatologic interest.
Abstract: HSP 47 is a specific chaperone of pro-collagen. This heat shock protein is responsible for the correct 3-dimentional organization of pro-collagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP47 in aged, photoaged and senescent fibroblasts and its modulation by a plant extract (Salix Alba).
We measured the level of HSP47 and/or pro-collagen expression in fibroblasts by real time RT-PCR (mRNA transcripts) and by flow cytometry (immuno-chemistry technique for measurement of arbitrary fluorescence intensity). We used immuno-chemistry techniques and confocal microscopy to vizualized the cellular localization of HSP 47 and pro-collagen.
These parameters were compared with different age-donors, non senescent and senescent fibroblasts. Fibroblasts were irradiated by a non cytotoxic dose of UVA (6 J/cm2 ) and HSP 47 level was evaluated. Salix Alba extract was tested for its capacity to modulate HSP 47 expression.
We showed colocalization of HSP47 and procollagen by confocal microscopy, indicating that HSP47 could play a role of procollagen molecular chaperone in our cellular model. We showed that HSP47 level is decreased in old-donor cells, senescent and irradiated cells. This decrease can be modulated by a salix alba extract (polyphenols rich) in a dose dependent manner.
The evaluation of HSP 47 expression in our experimental conditions can lead to a new approach of aging and photoaging pointing out the implication of this chaperone in these pathophysiologic phenomenons. Modulation of HSP 47 expression by molecules family could be of cosmetic and/or dermatologic interest.
