Cutaneous aging translates drastic structural and functional alterations in the extracellular matrix (ECM). Multiple mechanisms are involved, including changes in protease levels. We investigated the age-related protein expression and activity of cysteine cathepsins and the expression of two endogenous protein inhibitors in young and aged Caucasian women skin epidermis. Immunofluorescence studies indicate that the expression of cathepsins K, S and V, as well as cystatins A and M/E within keratinocytes is reduced in photoprotected skin of aged women. Furthermore, the overall endopeptidase activity of cysteine cathepsins in epidermis lysates decreased with age. Albeit dermal elastic fiber and laminin expression is reduced in aged skin, staining of nidogen-1, a key protein in BM assembly that is sensitive to proteolysis by cysteine, metallo- and serine proteases, has a similar pattern in both young and aged skin. Since cathepsins contribute to the hydrolysis and turnover of ECM/basement membrane components, the abnormal protein degradation and deposition during aging process may be related in part to a decline of lysosomal/endosomal cathepsin K, S and V activity.

Human cysteine cathepsin S (catS) participates in distinct physiological and pathophysiological cellular processes and is considered as a valuable therapeutic target in autoimmune diseases, cancer, atherosclerosis, and asthma. We evaluated the capacity of negatively charged glycosaminoglycans (heparin, heparan sulfate, chondroitin 4/6-sulfates, dermatan sulfate, and hyaluronic acid) to modulate the activity of catS. Chondroitin 4-sulfate (C4-S) impaired the collagenolytic activity (type IV collagen) and inhibited the peptidase activity (Z-Phe-Arg-AMC) of catS at pH 5.5, obeying a mixed-type mechanism (estimated Ki = 16.5 ± 6 μM). Addition of NaCl restored catS activity, supporting the idea that electrostatic interactions are primarly involved. Furthermore, C4-S delayed in a dose-dependent manner the maturation of procatS at pH 4.0 by interfering with the intermolecular processing pathway. Binding of C4-S to catS was demonstrated by gel-filtration chromatography, and its affinity was measured by surface plasmon resonance (equilibrium dissociation constant Kd = 210 ± 40 nM). Moreover, C4-S induced subtle conformational changes in mature catS as observed by intrinsic fluorescence spectroscopy analysis. Molecular docking predicted three specific binding sites on catS for C4-S that are different from those found in the crystal structure of the cathepsin K-C4-S complex. Overall, these results describe a novel glycosaminoglycan-mediated mechanism of catS inhibition and suggest that C4-S may modulate the collagenase activity of catS in vivo.

Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional–translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.

Application of hormesis in aging research and interventions is becoming increasingly attractive and successful. The reason for this is the realization that mild stress-induced activation of one or more stress response (SR) pathways, and its consequent stimulation of repair mechanisms, is effective in reducing the age-related accumulation of molecular damage. For example, repeated heat stress-induced synthesis of heat shock proteins has been shown to have a variety of anti-aging effects on growth and other cellular and biochemical characteristics of normal human skin fibroblasts, keratinocytes and endothelial cells undergoing aging in vitro. Therefore, searching for potential hormetins - conditions and compounds eliciting SR-mediated hormesis - is drawing attention of not only the researchers but also the industry involved in developing healthcare products, including nutriceuticals, functional foods and cosmeceuticals. Here we present the example of a skin care cosmetic as one of the first successful product developments incorporating the ideas of hormesis. This was based on the studies to analyse the molecular effects of active ingredients extracted from the roots of the Chinese herb Sanchi (Panax notoginseng) on gene expression at the level of mRNAs and proteins in human skin cells. The results showed that the ginsenosides extracted from Sanchi induced the transcription of stress genes and increased the synthesis of stress proteins, especially the heat shock protein HSP1A1 or Hsp70, in normal human keratinocytes and dermal fibroblasts. Furthermore, this extract also has significant positive effects against facial wrinkles and other symptoms of facial skin aging as tested clinically, which may be due to its hormetic mode of action by stress-induced synthesis of chaperones involved in protein repair and removal of abnormal proteins. Acceptance of such a hormesis-based product by the wider public could be instrumental in the social recognition of the concept of hormesis as the beneficial effects of mild stress of choice, and will encourage the development of novel health care products with physical, nutritional and mental hormetins.

Neurotrophins (NTs) belong to a family of growth factors that play a critical role in the control of skin homeostasis. NTs act through the low‐affinity receptor p75NTR and the high‐affinity receptors TrkA, TrkB, and TrkC. Here we show that dermal fibroblasts (DF) and myofibroblasts (DM) synthesize and secrete all NTs and express NT receptors. NTs induce differentiation of DF into DM, as shown by the expression of α‐SMA protein. The Trk inhibitor K252a, TrkA/Fc, TrkB/Fc, or TrkC/Fc chimera prevents DF and DM proliferation. In addition, p75NTR siRNA inhibits DF proliferation, indicating that both NT receptors mediate DF proliferation induced by endogenous NTs. Autocrine NTs also induce DF migration through p75NTR and Trk, as either silencing of p75NTR or Trk/Fc chimeras prevent this effect, in absence of exogenous NTs. Finally, NGF or BDNF statistically increase the tensile strength in a dose dependent manner, as measured in a collagen gel through the GlaSbox device. Taken together, these results indicate that NTs exert a critical role on fibroblast and could be involved in tissue re‐modeling and wound healing. J. Cell. Physiol. 227: 1017–1025, 2012. © 2011 Wiley Periodicals, Inc.

Cathepsin S (catS), which is expressed in normal human keratinocytes and localized close to the dermal-epidermal junction (DEJ) degrades some of major basement membrane (BM) constituents. Among them, catS readily hydrolyzed in a time and dose dependent manner human nidogen-1 (nid-1) and nidogen-2, which are key proteins in the BM structure. CatS preferentially cleaved nid-1 at both acid and neutral pH. Hydrolysis of nid-1 was hampered in murine ctss(-/-) spleen lysates pretreated with inhibitors of other classes of proteases. Nid-1 was cleaved within its G2 and G3 globular domains that are both involved in interactions with other BM components. Binding assays with soluble and immobilized ligands indicated that catS altered the formation of complexes between nid-1 and other BM components. Assuming that the cleavage of nid-1 impairs its ability to crosslink with BM partners and perturbs the viscoelastic properties of BM matrix, these data indicate that catS may participate in BM proteolysis, in addition to already identified proteases.

The process of facial skin aging differs between different ethnic populations. Standardized tools to objectively evaluate facial skin aging in clinical and epidemiological studies in different ethnic populations are needed. We designed and validated a photonumeric scale for assessment of facial aging in the Indian population. A total of 149 subjects were selected from an electronic photographic database of 300 women from the Mumbai region. An expert consensus panel selected nine subjects representing a spectrum of skin damage from none to severe, on the basis of wrinkling and tissue slackening. The nine-point scale, composed of one face-on and one oblique photo of the same subject per grade, was validated by nine independent judges in 99 subjects with a repetition two weeks later. Inter-observer reliability and intra-observer reproducibility were calculated using Kappa coefficients. Validation of the scale showed a high level of inter-observer reliability between the consensus and independent panels, and intra-observer reproducibility between the two evaluations performed by the independent judges. In conclusion, this nine-point facial aging scale is a reliable tool for clinical evaluations of skin damage in Indian women, suitable for use in clinical and epidemiological studies.

Transfer of the melanocyte-specific and lysosome-related organelle, the melanosome, from melanocytes to keratinocytes is crucial for the protection of the skin against harmful ultraviolet radiation (UVR)-our main physiological cutaneous stressor. However, this commonplace event remains a most enigmatic process despite several early hypotheses. Recently, we and others have proposed a role for filopodia in melanin transfer, although conclusive experimental proof remained elusive. Using known filopodial markers (MyoX/Cdc42) and the filopodial disrupter, low-dose cytochalasin-B, we demonstrate here a requirement for filopodia in melanosome transfer from melanocytes to keratinocytes and also, unexpectedly, between keratinocytes. Melanin distribution throughout the skin represents the key phenotypic event in skin pigmentation. Melanocyte filopodia were also necessary for UVR-stimulated melanosome transfer, as this was also inhibited by MyoX knockdown and low-dose cytochalasin-B. Knockdown of keratinocyte MyoX protein, in its capacity as a phagocytosis effector, resulted in the inhibition of melanin uptake by keratinocytes. This indicates a central role for phagocytosis by keratinocytes of melanocyte filopodia. In summary, we propose a new model for the regulation of pigmentation in human skin cells under both constitutive and facultative (post-UVR) conditions, which we call the "filopodial-phagocytosis model." This model also provides a unique and highly accessible way to study lysosome-related organelle movement between mammalian cells.-Singh, S. K., Kurfurst, R., Nizard, C., Schnebert, S., Perrier, E., Tobin, D. J. Melanin transfer in human skin cells is mediated by filopodia-a model for homotypic and heterotypic lysosomerelated organelle transfer. FASEB J. 24, 3756-3769 (2010). www.fasebj.org

>氧化蛋白聚集是细胞衰老的标志,也是导致细胞出现衰老的主要原因之一。引起氧化蛋白聚集的主要原因是持续性的蛋白氧化损伤及细胞对受损氧化蛋白清除能力的下降。细胞内氧化蛋白的清除机制通常依赖于蛋白酶体系统对氧化蛋白的降解;此外,蛋白内部还有一些其他修复机制修复受损的氧化蛋白,如蛋氨酸残基的氧化可通过蛋氨酸硫氧化

Several aquaporins (AQPs) are expressed in mammalian skin. Some are directly involved in water transport, such as AQP5, which is involved in sweat secretion. In contrast, the physiological role of skin aquaglyceroporins, which permeate both water and glycerol, appears more and more complex. AQP3 is the most abundant skin aquaglyceroporin. Both water and glycerol transport by AQP3 appear to play an important role in hydration of mammalian skin epidermis. In addition, recent data suggest that glycerol transport by AQP3 is involved in the metabolism of lipids in skin as well as in the regulation of proliferation and differentiation of keratinocytes. Finally, AQP3 is also believed to be important in wound healing, as a water channel by facilitating cell migration, and as a glycerol transporter by enhancing keratinocyte proliferation and differentiation.

BACKGROUND AND OBJECTIVES:Skin without significant dyschromia is an aesthetic goal of people worldwide. Current options for lightening skin could have significant drawbacks. The antisense strategy may be a viable alternative. The reactions in melanogenesis are catalyzed mainly by tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2. Activation of tyrosinase is associated with phosphorylation by protein kinase C-betaI (PKC-betaI) and formation of a complex between phosphorylated tyrosinase and TRP-1. The aim of this study was to use 2 antisense oligonucleotides to modulate the synthesis of the tyrosinase/TRP-1 complex, PKC-beta, or both by interacting with the targeted mRNA, thus whitening skin by interfering with melanogenesis at the translational level.METHODS/STUDY DESIGN:In the in vitro study, the effect of the antisense oligonucleotides was evaluated by measuring the rate at which dihydroxyphenylalanine (DOPA) oxidase transforms L-DOPA to DOPAchrome in the pathway for melanin biosynthesis. A reduction in the reaction rate compared to the controls corresponded to a decrease in the enzyme activity and, consequently, to a reduction of the formation of melanin pigments. To evaluate the in vivo lightening effect of the antisense oligonucleotides, 30 Asian women volunteers with pigmented spots on both hands applied the test product twice daily for 8 weeks. The test product was applied to 2 marked-off areas of the hand: a pigmented spot (to evaluate the effect of the test product on the color of the spot) and a nonpigmented spot area (to evaluate the effect of the test product on normal skin pigmentation). The lightening effect was evaluated by comparing chromametric and mexametric parameters before treatment, after 4 weeks, and after 8 weeks.RESULTS:In vitro DOPA-oxidase activity was inhibited by 13% in melanocytes treated with the antisense sequence for PKC-BI alone, by 16% with the antisense sequence for TRP-1 alone, and by 36% with the association of 2 sequences. The inhibiting effect with both sequences required the specific sequences with nonreversed polarities. In vivo clinical results showed statistically significant whitening in both pigmented spots and nonpigmented spots when the test product was applied twice daily for 8 weeks by up to 30 Asian women.CONCLUSIONS:The association of TRP-1 and PKC-betaI antisense molecules significantly increased the inhibition of tyrosinase activity on human melanocytes. Antisense oligonucleotides are a new generation of active cosmetic ingredients that offer unprecedented specificity, biological stability, and safety in lightening skin. This is the first report of positive results in a cosmetic based on the use of these new active agents.

BACKGROUND AND OBJECTIVES:Ultraviolet (UV) light produces reactive oxygen species (ROS) in skin, which accelerate aging by damaging DNA, proteins, lipids, and other cellular constituents. The aims of this study were to 1) evaluate the antioxidant properties of a Vitis vinifera shoot extract on cultured normal human keratinocytes, 2) compare the in vivo antioxidant of this extract in combination with a biotechnological extract (Ronacare Hydroine), and 3) evaluate the efficacy on photoaging skin of a serum based on a combination (Vitis vinifera shoot extract in hydroglycolic solution, or Sarmentine, and Ronacare Hydroine) after a 4-week application, and to quantify the additional improvement given by applying a cream with the serum.METHODS/STUDY DESIGN:An in vitro study was conducted to evaluate the antioxidant properties of Vitis vinifera shoot extract added to cultured normal human keratinocytes. A fluorescent probe was used to quantify cytoplasmic endogenous species formed in response to oxidative stress induced by H2O2. The antioxidant activity of Vitis vinifera shoot extract was compared to that of a solvent control and 2 positive controls, vitamin E and vitamin C. In the first in vivo study, 2 test products were included in a comparative, randomized, single-blind trial in which 27 subjects acted as their own (untreated) controls. Products were applied 4 times to randomized areas of the inner surface of the forearm for one day. The following day, treated and untreated (control) areas of stratum corneum were sampled for fluorimetric analysis. A decrease in fluorescence compared with untreated control reflected a decrease in the level of ROS, in which case the product had a scavenging effect. The 2 products contained a combination of Sarmentine and Ronacare Hydroine, whose antioxidant properties were under investigation. Other products were known antioxidants. In the second in vivo study, 60 female subjects applied either serum or serum plus cream twice daily for 28 days for clinical evaluation. Overall improvement was rated on a quartile scale (0%-25%, 26%-50%, 51%-75%, 76%-100%) and changes in firmness, radiant glow, evenness, smoothness, wrinkles, fine lines, hydration, texture, and softness were rated on a negative to positive scale (-5=worse to +5=greatly improved).RESULTS AND CONCLUSIONS:Vitis vinifera shoot extract appears to have significantly stronger in vitro antioxidant capacity than vitamin C or vitamin E. In the same vehicle (placebo emulsion), ascorbic acid (0.5%), Sarmentine (1%), and the Sarmentine (1%) plus Ronacare Hydroine (1%) combination had a significant in vivo antioxidant effect versus a nontreated area. The combination Sarmentine (1%) plus Ronacare Hydroine (1%) showed a higher efficacy than Sarmentine alone. The dermatologic evaluation showed that a 4-week twice-daily application of a serum containing the combination improved the main clinical signs of photoaged skin. The addition of the cream with the serum appears to enhance the serum-induced improvement of most of the skin characteristics.

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.

BACKGROUND/OBJECTIVES:[corrected] The signs of aging may originate from natural processes or from exposure to the sun, wind, or other environmental factors. To evaluate the anti-aging effects of potential agents researchers must first identify and be able to quantify epidermal markers that change with aging. This paper summarizes the results of studies conducted to evaluate the transcriptional effects of an Aframomum angustifolium seed extract and Malva Sylvestris extract, and the antiaging efficacy of a skin care product containing the Aframomum angustifolium seed extract.METHODS:The transcriptional effect of an Aframomum angustifolium seed extract on normal human keratinocytes (NHKs) and normal human fibroblasts (NHF) was evaluated in vitro with the use of a low-density DNA array technology. The Malva Sylvestris extract was studied with a commercial DNA macroarray and by a real-time quantitative reverse transcriptase-polymerase chain reaction. The in vitro anti-aging activities of the Malva sylvestris extract were compared with those of all-trans retinoic acid (RA), a well-established topical therapy for photodamage and wrinkles. The genes studied were known to be modified by RA. The anti-aging efficacy of a facial skin care product containing Aframomum angustifolium seed extract was evaluated in a single-center study using image processing analysis and in a 2-center study by evaluation of the photographs by the investigator, independent evaluators, and subjects.RESULTS:In general, the Aframomum angustifolium seed extract strongly modified the gene expression profiles of NHKs and weakly modified the gene expression profiles of NHFs. After incubation with Aframomum angustifolium seed extract, the expressions of 3 antioxidant genes (metallothionein 1, metallothionein 2, and thioredoxin) were increased in NHKs, while expressions of 1 antioxidant gene (glutathione peroxidase) was increased in NHFs. Concerning the Malva sylvestris extract, a cDNA macro-array technology experiment with the reconstructed human epidermis model showed that some genes modulated by treatment with the Malva sylvestris extract are also regulated by RA treatment indicating a similar activity at the mRNA level. In the single-center study, a facial skin care product containing the Aframomum angustifolium seed extract significantly improved the homogeneity of the skin. The areas of the detected objects (skin imperfections) decreased significantly on each studied area of the face and the variance decreased significantly over the entire face. In the 2-center study, 28% percent of the subjects reported a greater than 50% overall global improvement in their skin by the end of the study compared to 11% of the subjects after 4 weeks of treatment. Seventy-six percent of subjects said they would purchase the cream.CONCLUSIONS:The authors developed a low-density DNA chip method that permitted the study of the transcriptional effect of Malva Sylvestris extract and of Aframomum angustrifolium seed extract. The gene expression profiles obtained demonstrate the anti-aging properties of these compounds. An in vivo single-center study, performed and analyzed with an assay based on image processing analysis, demonstrated the antiwrinkle activity of a formulation containing the Aframomum angustifolium seed extract. The data obtained in the 2-center study suggests that the cosmeceutical containing Aframomum angustifolium seed extract produces a global rejuvenation effect in terms of redness, pigmentation, and fine lines similar to that noted utilizing an intense pulse light source.