Rainer Wolber

Anti-inflammatory efficacy of licochalcone a : correlation of clinical potency and in vitro effects

Licochalcone A (LicA), a major phenolic constituent of the licorice species Glycyrrhiza inflata, has recently been reported to have anti-inflammatory as well as anti-microbial effects. These anti-inflammatory properties might be exploited for topical applications of LicA. We conducted prospective randomized vehicle-controlled clinical trials to assess the anti-irritative efficacy of cosmetic formulations containing LicA in a post-shaving skin irritation model and on UV-induced erythema formation. The clinical trials were accompanied by a series of in vitro experiments to characterize anti-inflammatory properties of LicA on several dermatologically relevant cell types. Topical LicA causes a highly significant reduction in erythema relative to the vehicle control in both the shave- and UV-induced erythema tests, demonstrating the anti-irritative properties of LicA. Furthermore, LicA is a potent inhibitor of pro-inflammatory in vitro responses, including N-formyl-MET-LEU-PHE (fMLP)- or zymosan-induced oxidative burst of granulocytes, UVB-induced PGE2 release by keratinocytes, lipopolysaccharide (LPS)-induced PGE2 release by adult dermal fibroblasts, fMLP-induced LTB4 release by granulocytes, and LPS-induced IL-6/TNF-α secretion by monocyte-derived dendritic cells. The reported data suggest therapeutic skin care benefits from LicA when applied to sensitive or irritated skin.

The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin

Interactions between melanocytes and neighboring cells in the skin are important in regulating skin color in humans. We recently demonstrated that the less pigmented and thicker skin on the palms and soles is regulated by underlying fibroblasts in those areas, specifically via a secreted factor (DKK1) that modulates Wnt signaling. In this study, we tested the hypothesis that dermal fibroblasts regulate the constitutive skin color of individuals ranging from very light to very dark. We used microarray analysis to compare gene expression patterns in fibroblasts derived from lighter skin types compared to darker skin types, with a focus on secreted proteins. We identified a number of genes that differ dramatically in expression and, among the expressed proteins, neuregulin-1, which is secreted by fibroblasts derived from dark skin, effectively increases the pigmentation of melanocytes in tissue culture and in an artificial skin model and regulates their growth, suggesting that it is one of the major factors determining human skin color.

The deceptive nature of UVA tanning versus the modest protective effects of UVB tanning on human skin.

The relationship between human skin pigmentation and protection from ultraviolet (UV) radiation is an important element underlying differences in skin carcinogenesis rates. The association between UV damage and the risk of skin cancer is clear, yet a strategic balance in exposure to UV needs to be met. Dark skin is protected from UV‐induced DNA damage significantly more than light skin owing to the constitutively higher pigmentation, but an as yet unresolved and important question is what photoprotective benefit, if any, is afforded by facultative pigmentation (i.e. a tan induced by UV exposure). To address that and to compare the effects of various wavelengths of UV, we repetitively exposed human skin to suberythemal doses of UVA and/or UVB over 2 weeks after which a challenge dose of UVA and UVB was given. Although visual skin pigmentation (tanning) elicited by different UV exposure protocols was similar, the melanin content and UV‐protective effects against DNA damage in UVB‐tanned skin (but not in UVA‐tanned skin) were significantly higher. UVA‐induced tans seem to result from the photooxidation of existing melanin and its precursors with some redistribution of pigment granules, while UVB stimulates melanocytes to up‐regulate melanin synthesis and increases pigmentation coverage, effects that are synergistically stimulated in UVA and UVB‐exposed skin. Thus, UVA tanning contributes essentially no photoprotection, although all types of UV‐induced tanning result in DNA and cellular damage, which can eventually lead to photocarcinogenesis.

Regulation of Human Skin Pigmentation in situ by Repetitive UV Exposure: Molecular Characterization of Responses to UVA and/or UVB

UV radiation is a major environmental factor that affects pigmentation in human skin and can eventually result in various types of UV-induced skin cancers. The effects of various wavelengths of UV on melanocytes and other types of skin cells in culture have been studied, but little is known about gene expression patterns in situ following in situ exposure of human skin to different types of UV (UVA and/or UVB). Paracrine factors expressed by keratinocytes and/or fibroblasts that affect skin pigmentation might be regulated differently by UV, as might their corresponding receptors expressed on melanocytes. To test the hypothesis that different mechanisms are involved in the pigmentary responses of the skin to different types of UV, we used immunohistochemical and whole human genome microarray analyses to characterize human skin in situ to examine how melanocyte-specific proteins and paracrine melanogenic factors are regulated by repetitive exposure to different types of UV compared with unexposed skin as a control. The results show that gene expression patterns induced by UVA or UVB are distinct-UVB eliciting dramatic increases in a large number of genes involved in pigmentation as well as in other cellular functions, whereas UVA had little or no effect on these. The expression patterns characterize the distinct responses of the skin to UVA or UVB, and identify several potential previously unidentified factors involved in UV-induced responses of human skin.

Microarray analysis sheds light on the dedifferentiating role of agouti signal protein in murine melanocytes via the Mc1r

The melanocortin-1 receptor (MC1R) is a key regulator of pigmentation in mammals and is tightly linked to an increased risk of skin cancers, including melanoma, in humans. Physiologically activated by α-melanocyte stimulating hormone (αMSH), MC1R function can be antagonized by a secreted factor, agouti signal protein (ASP), which is responsible for the lighter phenotypes in mammals (including humans), and is also associated with increased risk of skin cancer. It is therefore of great interest to characterize the molecular effects elicited by those MC1R ligands. In this study, we determined the gene expression profiles of murine melan-a melanocytes treated with ASP or αMSH over a 4-day time course using genome-wide oligonucleotide microarrays. As expected, there were significant reductions in expression of numerous melanogenic proteins elicited by ASP, which correlates with its inhibition of pigmentation. ASP also unexpectedly modulated the expression of genes involved in various other cellular pathways, including glutathione synthesis and redox metabolism. Many genes up-regulated by ASP are involved in morphogenesis (especially in nervous system development), cell adhesion, and extracellular matrix-receptor interactions. Concomitantly, ASP enhanced the migratory potential and the invasiveness of melanocytic cells in vitro. These results demonstrate the role of ASP in the dedifferentiation of melanocytes, identify pigment-related genes targeted by ASP and by αMSH, and provide insights into the pleiotropic molecular effects of MC1R signaling that may function during development and may affect skin cancer risk.

Pigmentation effects of solar simulated radiation as compared with UVA and UVB radiation

Different wavelengths of ultraviolet (UV) radiation elicit different responses in the skin. UVA induces immediate tanning and persistent pigment darkening through oxidation of pre‐existing melanin or melanogenic precursors, while UVB induces delayed tanning which takes several days or longer to develop and requires activation of melanocytes. We compared the effects of a 2‐week repetitive exposure of human skin to solar‐simulated radiation (SSR), UVA or UVB at doses eliciting comparable levels of visible tanning and measured levels of melanins and melanin‐related metabolites. Levels of eumelanin and pheomelanin were significantly higher in the order of SSR, UVB, UVA or unexposed control skin. Levels of free 5‐S‐cysteinyldopa (5SCD) were elevated about 4‐fold in SSR‐ or UVB‐exposed skin compared with UVA‐exposed or control skin. Levels of protein‐bound form of 5SCD tended to be higher in SSR‐ or UVB‐exposed skin than in UVA‐exposed or control skin. Total levels of 5‐hydroxy‐6‐methoxyindole‐2‐carboxylic acid (5H6MI2C) and 6H5MI2C were higher in SSR‐ than in UVB‐exposed or control skin. These results show that SSR is more effective in promoting delayed tanning than UVB radiation alone, suggesting a synergistic effect of UVA radiation. Furthermore, free 5SCD may serve as a good marker of the effect of SSR and UVB.

Regulation of eumelanin/pheomelanin synthesis and visible pigmentation in melanocytes by ligands of the melanocortin 1 receptor.

The production of melanin in the hair and skin is tightly regulated by the melanocortin 1 receptor (MC1R) whose activation is controlled by two secreted ligands, α‐melanocyte stimulating hormone (αMSH) and agouti signal protein (ASP). As melanin is extremely stable, lasting years in biological tissues, the mechanism underlying the relatively rapid decrease in visible pigmentation elicited by ASP is of obvious interest. In this study, the effects of ASP and αMSH on the regulation of melanin synthesis and on visible pigmentation were assessed in normal murine melanocytes and were compared with the quick depigmenting effect of the tyrosinase inhibitor, phenylthiourea (PTU). αMSH increased pheomelanin levels prior to increasing eumelanin content over 4 days of treatment. Conversely, ASP switched off the pigment synthesis pathway, reducing eu‐ and pheo‐melanin synthesis within 1 day of treatment that was proportional to the decrease in tyrosinase protein level and activity. These results demonstrate that the visible depigmentation of melanocytes induced by ASP does not require the degradation of existing melanin but rather is due to the dilution of existing melanin by melanocyte turnover, which emphasizes the importance of pigment distribution to visible color.

Contribution of UVB and UVA to UV-dependent stimulation of cyclooxygenase-2 expression in artificial epidermis

Both UVB (280-320 nm) and UVA (320-400 nm) radiation lead to an enhanced expression of cyclooxygenase-2 (COX-2) in epidermal cells in various in-vitro and in-vivo models. It is demonstrated here that the expression of COX-2 is induced in artificial human epidermis exposed to simulated solar light (>290 nm). Employing filters eliminating specified regions from the simulated solar spectrum, the UVB and UVA-2 (320-350 nm) regions are shown to fully account for induction of COX-2 mRNA and protein as well as the enhanced production of prostaglandin E(2) after irradiation. At the protein level, approximately 70% of the total induction by solar light is due to light in the UVA-2 region. UVA-1 (350-400 nm), visible light and IR radiation are practically ineffective. COX-2 induction by simulated solar light is attenuated in the presence of inhibitors of p38(MAPK) or of c-Jun-N-terminal kinases (JNK), whereas COX-2 induction by UVA is blocked only by inhibition of JNK. UV-induced COX-2 expression is not affected by inhibition of the MEK 1,2/ERK 1,2 pathways.

Reconstituted 3-dimensional human skin of various ethnic origins as an in vitro model for studies of pigmentation

Reconstituted 3-dimensional human skin equivalents containing melanocytes and keratinocytes on an artificial dermal substitute are gaining popularity for studies of skin metabolism because they exhibit morphological and growth characteristics similar to human epidermis. In this study, we show that such a pigmented epidermis model can be used to assess the regulation of pigmentation by known melanogenic compounds. In monolayers or in melanocyte-keratinocyte co-cultures, melanocyte-keratinocyte interactions are missing or are spatially limited. The commercial skin equivalents used in this study were derived from epidermal cells obtained from donors of three different ethnic origins (African- American, Asian, and Caucasian), and they reflect those distinct skin phenotypes. We used these pigmented human epidermis models to test compounds for potential effects on pigmentation in a more physiologically relevant context, which allows further characterization and validation of interesting melanogenic factors. We used known melanogenic stimulators (alpha-melanocyte-stimulating hormone and 3,4-dihydroxyphenylalanine) and inhibitors (hydroquinone, arbutin, kojic acid, and niacinamide) and examined their effects on the production of melanin and its distribution in upper layers of the skin. Our studies indicate that commercial skin equivalents provide a convenient and cost-effective alternative to animal testing for evaluating the regulation of mammalian pigmentation by melanogenic factors and for elucidating their mechanisms of action.

[41] Topically applied antioxidants in skin protection

Publisher Summary Reactive oxygen species (ROS) and free radicals can be generated in skin by ultraviolet (UV) irradiation, ultrasonication, toxic or allergic chemical noxes, or even during normal metabolic processes of cells. More than 10 10 ROS are generated per day and per cell under normal physiological conditions, which have to be dealt with by the endogenous antioxidant system. Oxidative stress arises when the balance between antioxidant and prooxidant processes drifts in favor of a prooxidant status. Some of the general events in the early phase of oxidative stress response in skin are depletion of endogenous intra- and intercellular antioxidants, the enhancement of the intracellular hydroperoxide level, and the induction of specific signal transduction pathways. This chapter presents an example of a screening strategy for the successful selection of in vivo functional topical antioxidants using special in vitro, ex vivo, and in vivo methods. The screening protocol used is subdivided into three steps. In the primary step, the efficacy of antioxidants is evaluated in vitro in cultures of primary skin cells, and the biocompatibility of the selected efficient antioxidants is proved in vitro and in vivo. In the second step, the antioxidant efficacy is tested ex vivo in a clinical pilot study using biopsies from in vivo -pretreated skin sites. In the third step, the in vivo efficacy of preselected antioxidants has been confirmed in clinical studies on panels of human volunteers.