Inhibition of Human Tyrosinase Requires Molecular Motifs Distinctively Different from Mushroom Tyrosinase.

Tyrosinase is the rate-limiting enzyme of melanin production and, accordingly, is the most prominent target for inhibiting hyperpigmentation. Numerous tyrosinase inhibitors have been identified, but most of those lack clinical efficacy because they were identified using mushroom tyrosinase as the target. Therefore, we used recombinant human tyrosinase to screen a library of 50,000 compounds and compared the active screening hits with well-known whitening ingredients. Hydroquinone and its derivative arbutin only weakly inhibited human tyrosinase with a half-maximal inhibitory concentration (IC50) in the millimolar range, and kojic acid showed a weak efficacy (IC50 > 500 µmol/L). The most potent inhibitors of human tyrosinase identified in this screen were resorcinyl-thiazole derivatives, especially the newly identified Thiamidol (Beiersdorf AG, Hamburg, Germany) (isobutylamido thiazolyl resorcinol), which had an IC50 of 1.1 µmol/L. In contrast, Thiamidol only weakly inhibited mushroom tyrosinase (IC50 = 108 µmol/L). In melanocyte cultures, Thiamidol strongly but reversibly inhibited melanin production (IC50 = 0.9 µmol/L), whereas hydroquinone irreversibly inhibited melanogenesis (IC50 = 16.3 µmol/L). Clinically, Thiamidol visibly reduced the appearance of age spots within 4 weeks, and after 12 weeks some age spots were indistinguishable from the normal adjacent skin. The full potential of Thiamidol to reduce hyperpigmentation of human skin needs to be explored in future studies.

Impaired glyoxalase activity is associated with reduced expression of neurotrophic factors and pro-inflammatory processes in diabetic skin cells.

Patients suffering from type II diabetes develop several skin manifestations including cutaneous infections, diabetic dermopathy, diabetic bullae and acanthosis nigricans. Diabetic micro- and macroangiopathy as well as diabetic neuropathy are believed to play a crucial role in the development of diabetic skin disorders. A reduced cutaneous nerve fibre density was reported in diabetic subjects, which subsequently leads to impaired sensory nerve functions. Using an innervated skin model, we investigated the impact of human diabetic dermal fibroblasts and keratinocytes on porcine sensory neurons. Diabetic skin cells showed a reduced capacity to induce neurite outgrowth due to a decreased support with neurotrophic factors, such as NGF. Furthermore, diabetic keratinocytes displayed insulin resistance and increased expression of pro-inflammatory cytokines demonstrating the persistent effect of diabetes mellitus on human skin cells. Dysregulations were related to a significantly reduced glyoxalase enzyme activity in diabetic keratinocytes as experimentally reduced glyoxalase activity mimicked the increase in pro-inflammatory cytokine expression and reduction in NGF. Our results demonstrate an impaired crosstalk of diabetic skin cells and sensory neurons favouring hypo-innervation. We suggest that reduced methylglyoxal detoxification contributes to an impaired neurocutaneous interaction in diabetic skin.

Genome-wide expression analysis of wounded skin reveals novel genes involved in angiogenesis.

Wound healing is a multistage process involving collaborative efforts of different cell types and distinct cellular functions. Among others, the high metabolic activity at the wound site requires the formation and sprouting of new blood vessels (angiogenesis) to ensure an adequate supply of oxygen and nutrients for a successful healing process. Thus, a cutaneous wound healing model was established to identify new factors that are involved in vascular formation and remodeling in human skin after embryonic development. By analyzing global gene expression of skin biopsies obtained from wounded and unwounded skin, we identified a small set of genes that were highly significant differentially regulated in the course of wound healing. To initially investigate whether these genes might be involved in angiogenesis, we performed siRNA experiments and analyzed the knockdown phenotypes using a scratch wound assay which mimics cell migration and proliferation in vitro. The results revealed that a subset of these genes influence cell migration and proliferation in primary human endothelial cells (EC). Furthermore, histological analyses of skin biopsies showed that two of these genes, ALBIM2 and TMEM121, are colocalized with CD31, a well known EC marker. Taken together, we identified new genes involved in endothelial cell biology, which might be relevant to develop therapeutics not only for impaired wound healing but also for chronic inflammatory disorders and/or cardiovascular diseases.

25-hydroxyvitamin d and advanced glycation endproducts in healthy and hypertensive subjects: are there interactions?

Advanced glycation endproducts (AGEs) accumulate during aging. Skin is the single organ of vitamin D synthesis, induced by ultraviolet B light. Accumulation of AGEs in the skin could interfere with synthesis of the vitamin, whereas the microinflammation and oxidative stress (associated with hypovitaminosis D) could amplify both the toxic effects of AGEs and their production. Clinical data on potential interactions between vitamin D3 deficiency and AGE accumulation are sparse. Here we investigated potential associations between levels of circulating vitamin D3 and those of AGEs in blood and skin with regard to markers of inflammation and oxidative stress in nondiabetic subjects. In a cross-sectional study, 146 subjects (119 healthy persons and 27 hypertensive patients; 73 male and 73 female; mean age, 57.0 ± 15.5 years) were included. Skin autofluorescence (SAF) and plasma levels of vitamin D3, AGE-associated fluorescence, high-sensitivity C-reactive protein level, and advanced oxidation protein products as well as renal function (estimated glomerular filtration rate) were determined. In a subgroup of 61 patients, N(ε)-carboxymethyllysine, soluble receptor of AGEs, and soluble vascular adhesion protein-1 were additionally analyzed. Vitamin D3 level averaged 22.5 ± 8.9 ng/mL. Prevalence of vitamin D insufficiency (20-29 ng/mL) was 43%, and that of deficiency (<20 ng/mL) 37%. The age-dependent rise in SAF was steeper in smokers and in subjects presenting arterial hypertension. No association between SAF and hypovitaminosis D was revealed. Among smokers, an inverse relationship manifested between vitamin D3 and plasma AGE-associated fluorescence as well as soluble vascular adhesion protein-1. Our data suggest that in nondiabetic adults, hypovitaminosis D does not enhance toxicity and accumulation of AGEs. Only in smokers interactions are conceivable.

Licochalcone A activates Nrf2 in vitro and contributes to licorice extract-induced lowered cutaneous oxidative stress in vivo.

The retrochalcone licochalcone A (LicA) has previously been shown to possess antimicrobial and anti-inflammatory properties. In this study, we focused on pathways responsible for the antioxidative properties of LicA. In vitro, LicA protected from oxidative stress mediated by reactive oxygen species (ROS) by activating the expression of cytoprotective phase II enzymes. LicA induced nuclear translocation of NF-E2-related factor 2 (Nrf2) in primary human fibroblasts and elevated the expression of the cytoprotective and anti-inflammatory enzymes heme oxygenase 1 and glutamate-cysteine ligase modifier subunit. LicA-treated cells displayed a higher ratio of reduced to oxidized glutathione and decreased concentrations of ROS in UVA-irradiated human dermal fibroblasts, as well as in activated neutrophils. In vivo, ultraweak photon emission analysis of skin treated with LicA-rich licorice extract revealed a significantly lowered UVA-induced luminescence, indicative for a decrease in oxidative processes. We conclude from these data that topical application of licorice extract is a promising approach to induce Nrf2-dependent cytoprotection in human skin.

Oxytocin modulates proliferation and stress responses of human skin cells: implications for atopic dermatitis.

The neuropeptide hormone oxytocin (OXT) mediates a wide spectrum of tissue-specific actions, ranging from cell growth, cell differentiation, sodium excretion to stress responses, reproduction and complex social behaviour. Recently, OXT expression was detected in keratinocytes, but expression of its receptor and function are still unexplored in human skin. Here, we showed that both OXT and its receptor are expressed in primary human dermal fibroblasts and keratinocytes. OXT-induced dose-dependent calcium fluxes in both cell types demonstrating that the OXT receptor (OXTR) is functionally expressed. We also showed that OXT decreases proliferation of dermal fibroblasts and keratinocytes in a dose-dependent manner. In order to further investigate OXT-mediated functions in skin cells, we performed OXTR knockdown experiments. OXTR knockdown in dermal fibroblasts and keratinocytes led to elevated levels of reactive oxygen species and reduced levels of glutathione (GSH). Moreover, OXTR-depleted keratinocytes exhibited an increased release of the pro-inflammatory cytokines IL6, CCL5 and CXCL10. Our data indicate that the OXT system modulates key processes which are dysregulated in atopic dermatitis (AD) such as proliferation, inflammation and oxidative stress responses. Furthermore, we detected a downregulation of the OXT system in peri-lesional and lesional atopic skin. Taken together, these data suggest that the OXT system is a novel neuroendocrine mediator in human skin homoeostasis and clinically relevant to stressed skin conditions like AD.

Role of fibroblasts in the pathogenesis of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a common dermatosis that highly impairs a patient's quality of life. The recent discovery that epidermal barrier defects caused by an aberrant differentiation process of keratinocytes are comparably important to the well-characterized changes in immune response patterns attributed a crucial role to the keratinocytes. Fibroblasts are able to alter proliferation and differentiation of keratinocytes, but their role in AD is not yet fully understood. OBJECTIVE: We sought to determine the role of fibroblasts in skin proliferation and differentiation in patients with AD. METHODS: We used human 3-dimensional organotypic skin cultures consisting of atopic fibroblasts and healthy keratinocytes, as well as healthy fibroblasts and atopic keratinocytes, and compared them with their controls. The expression of differentiation markers in these organotypic cultures were analyzed by using immunohistology and quantitative RT-PCR. Furthermore, the fundamental role of fibroblast-secreted leukemia inhibitory factor was assessed by using small interfering RNA-mediated knockdown cultures. RESULTS: We observed that atopic fibroblasts influence the proliferation of keratinocytes and the terminal differentiation process, resulting in an in vivo-like morphology of AD. Subsequently, healthy fibroblasts were able to restore the structural deficits of the epidermis consisting of atopic keratinocytes. Partially, these effects were due to a reduced expression of the differentiation-associated cytokine leukemia inhibitory factor by atopic fibroblasts. CONCLUSION: These data demonstrate that fibroblasts and the modulation of fibroblast-derived factors might be new therapeutic targets for the alleviation of AD.

Epidermal nerve fibers modulate keratinocyte growth via neuropeptide signaling in an innervated skin model.

Atopic eczema is a chronic inflammatory skin disease characterized by cutaneous nerve fiber sprouting and epidermal hyperplasia, pointing to an involvement of the peripheral nervous system in cutaneous homeostasis. However, the interaction of sensory neurons and skin cells is poorly understood. Using an innervated skin model, we investigated the influence of sensory neurons on epidermal morphogenesis. Neurons induced the proliferation of keratinocytes, resulting in an increase in the epidermal thickness. Inhibition of calcitonin gene-related peptide (CGRP), but not substance P (SP) signaling, reversed this effect. Human CGRP enhanced keratinocyte proliferation and epidermal thickness in skin models, demonstrating a key role of CGRP in modulating epidermal morphogenesis, whereas SP had only a moderate effect. Innervated skin models composed of atopic skin cells showed increased neurite outgrowth, accompanied by elevated CGRP release. As atopic keratinocytes were sensitized to CGRP owing to higher expression levels of the CGRP receptor components, receptor activity-modifying protein 1 (RAMP1) and receptor component protein (RCP), atopic innervated skin models displayed a thicker epidermis than did healthy controls. We conclude that neural CGRP controls local keratinocyte growth. Our results show that the crosstalk of the cutaneous peripheral nervous system and skin cells significantly influences epidermal morphogenesis and homeostasis in healthy and atopic skin.

Atopic keratinocytes induce increased neurite outgrowth in a coculture model of porcine dorsal root ganglia neurons and human skin cells.

Skin of patients suffering from atopic eczema displays a higher epidermal nerve fiber density, associated with neurogenic inflammation and pruritus. Using an in vitro coculture system, allowing a spatially compartmented culture of somata from porcine dorsal root ganglion neurons and human primary skin cells, we investigated the influence of dermal fibroblasts and keratinocytes on neurite outgrowth. In comparison with dermal fibroblasts, keratinocytes induced more branched and less calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. By adding neutralizing antibodies, we showed that nerve growth factor (NGF) and glial cell-line-derived neurotrophic factor (GDNF) are pivotal neurotrophic factors of skin cell-induced neurite outgrowth. Keratinocytes and dermal fibroblasts secreted different ratios of neurotrophic factors, influencing morphology and CGRP immunoreactivity of neurites. To investigate changes of the peripheral nervous system in the pathogenesis of atopic eczema in vitro, we analyzed neurite outgrowth mediated by atopic skin cells. Atopic keratinocytes produced elevated levels of NGF and mediated an increased outgrowth of CGRP-positive sensory fibers. Our results demonstrate the impact of dermal fibroblasts and keratinocytes on skin innervation and emphasize the role of keratinocytes as key players of hyperinnervation in atopic eczema.

DNA methylation regulates lineage-specifying genes in primary lymphatic and blood endothelial cells.

During embryonic development, the lymphatic system emerges by transdifferentiation from the cardinal vein. Although lymphatic and blood vasculature share a close molecular and developmental relationship, they display distinct features and functions. However, even after terminal differentiation, transitions between blood endothelial cells (BEC) and lymphatic endothelial cells (LEC) have been reported. Since phenotypic plasticity and cellular differentiation processes frequently involve epigenetic mechanisms, we hypothesized that DNA methylation might play a role in regulating cell type-specific expression in endothelial cells. By analyzing global gene expression and methylation patterns of primary human dermal LEC and BEC, we identified a highly significant set of genes, which were differentially methylated and expressed. Pathway analyses of the differentially methylated and upregulated genes in LEC revealed involvement in developmental and transdifferentiation processes. We further identified a set of novel genes, which might be implicated in regulating BEC-LEC plasticity and could serve as therapeutic targets and/or biomarkers in vascular diseases associated with alterations in the endothelial phenotype.

Identification of new genes involved in human adipogenesis and fat storage.

Since the worldwide increase in obesity represents a growing challenge for health care systems, new approaches are needed to effectively treat obesity and its associated diseases. One prerequisite for advances in this field is the identification of genes involved in adipogenesis and/or lipid storage. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. The primary screen showed that 459 genes affected adipogenesis and/or lipid accumulation after knock-down. Out of these hits, 333 could be validated in a secondary screen using independent siRNAs and 110 genes were further regulated on the gene expression level during adipogenesis. Assuming that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we performed InCell-Western analysis for the most striking hits to distinguish between the two phenotypes. Beside well known regulators of adipogenesis and neutral lipid storage (i.e. PPARγ, RXR, Perilipin A) the screening revealed a large number of genes which have not been previously described in the context of fatty tissue biology such as axonemal dyneins. Five out of ten axonemal dyneins were identified in our screen and quantitative RT-PCR-analysis revealed that these genes are expressed in preadipocytes and/or maturing adipocytes. Finally, to show that the genes identified in our screen are per se druggable we performed a proof of principle experiment using an antagonist for HTR2B. The results showed a very similar phenotype compared to knock-down experiments proofing the "druggability". Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point to identify anti-obesity compounds targeting the adipose tissue.

UV radiation induces the release of angiopoietin-2 from dermal microvascular endothelial cells.

In human skin, ultraviolet radiation (UVR)-induced erythema is characterized by the inflammatory and angiogenic activation of dermal endothelial cells. Recently, it has been shown that the release of angiopoietin-2 (Ang-2) from cytoplasmic storages of activated endothelial cells is crucial for the induction of inflammation and angiogenesis. Therefore, we hypothesized that UVR exposure induces the release of Ang-2 from endothelial cells controlling the early steps of erythema formation. In an in vivo study, suction blister fluids generated from UV-irradiated skin showed significantly increased concentrations of Ang-2, vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNFα). Likewise, in vitro UVR exposure of human dermal microvascular endothelial cells (HDMECs) triggered the release of Ang-2 that enhanced the pro-inflammatory response of these cells and facilitated their detachment from smooth muscle cells as evidenced by employing a three-dimensional co-culture spheroid model. These effects were inhibited by angiopoietin-1 (Ang-1), which competes with Ang-2 for binding the endothelial cell Tie2 receptor. Collectively, these observations suggest that UVR triggers the release of endothelial Ang-2 which may promote the destabilization and pro-inflammatory phenotype of the microvascular endothelium. As Ang-1 counteracts UVR-induced effects, stimulating the Ang-1 activity may represent a strategy to stabilize the dermal microcirculatory system, thus protecting against UVR-induced skin damages.

Dermal penetration of creatine from a face-care formulation containing creatine, guarana and glycerol is linked to effective antiwrinkle and antisagging efficacy in male subjects.

BACKGROUND: The dermal extracellular matrix provides stability and structure to the skin. With increasing age, however, its major component collagen is subject to degeneration, resulting in a gradual decline in skin elasticity and progression of wrinkle formation. Previous studies suggest that the reduction in cellular energy contributes to the diminished synthesis of cutaneous collagen during aging. AIMS: To investigate the potential of topically applied creatine to improve the clinical signs of skin aging by stimulating dermal collagen synthesis in vitro and in vivo. PATIENTS/METHODS: Penetration experiments were performed with a pig skin ex vivo model. Effects of creatine on dermal collagen gene expression and procollagen synthesis were studied in vitro using cultured fibroblast-populated collagen gels. In a single-center, controlled study, 43 male Caucasians applied a face-care formulation containing creatine, guarana extract, and glycerol to determine its influence on facial topometric features. RESULTS: Cultured human dermal fibroblasts supplemented with creatine displayed a stimulation of collagen synthesis relative to untreated control cells both on the gene expression and at the protein level. In skin penetration experiments, topically applied creatine rapidly reached the dermis. In addition, topical in vivo application of a creatine-containing formulation for 6 weeks significantly reduced the sagging cheek intensity in the jowl area as compared to baseline. This result was confirmed by clinical live scoring, which also demonstrated a significant reduction in crow's feet wrinkles and wrinkles under the eyes. CONCLUSIONS: In summary, creatine represents a beneficial active ingredient for topical use in the prevention and treatment of human skin aging.

TRP-channel-specific cutaneous eicosanoid release patterns.

Analyzing mechanisms and key players in peripheral nociception nonneuronal skin cells are getting more and more into focus. Herein we investigated the functional expression of TRPV1 and TRPA1 in human keratinocytes and fibroblasts and assessed proinflammatory lipid mediator release upon their stimulation as well as sensory effects after topical application, combining in vitro and in vivo approaches. In vitro, the expression of functional TRPV1 and TRPA1 channels on fibroblasts and keratinocytes was confirmed via immunofluorescence, qualitative real time (RT) polymerase chain reaction, and cellular Ca(2+) influx measurements. Additionally, the agonists allyl isothiocyanate (TRPA1) and capsaicin (TRPV1) induce a differential secretion pattern of the eicosanoids PGE(2) and LTB(4) in human dermal fibroblasts and keratinocytes, which was also detectable invivo, analyzing suction blister fluid at various times after short-term topical application. Capsaicin provoked the release of LTB(4) at 2 and 24 hours. In contrast, PGE(2) levels were reduced upon stimulation. Allyl isothiocyanate, however, increased PGE(2) levels only at 24 hours, but did not alter LTB(4) levels. In parallel, heat pain thresholds were reduced by both agents after short-term topical application, but only AITC provoked a long-lasting local erythema. In conclusion, the agonist-induced activation of nociceptors by TRPA1 and TRPV1 elicits painful sensations, whereas nonneuronal tissue cells respond with differential release of inflammatory mediators, thus influencing local vasodilatation and neuronal sensitization. These results have implications for the application of transient receptor potential antagonists to improve inflammatory skin conditions and pain management.

Lotus leaf extract and L-carnitine influence different processes during the adipocyte life cycle.

BACKGROUND: The cellular and molecular mechanisms of adipose tissue biology have been studied extensively over the last two decades. Adipose tissue growth involves both an increase in fat cell size and the formation of mature adipocytes from precursor cells. To investigate how natural substances influence these two processes, we examined the effects of lotus leaf extract (Nelumbo nucifera-extract solution obtained from Silab, France) and L-carnitine on human preadipocytes and adipocytes. METHODS: For our in vitro studies, we used a lotus leaf extract solution alone or in combination with L-carnitine. Utilizing cultured human preadipocytes, we investigated lotus leaf extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. Studies on human adipocytes were performed aiming to elucidate the efficacy of lotus leaf extract solution to stimulate lipolytic activity. To further characterize lotus leaf extract solution-mediated effects, we determined the expression of the transcription factor adipocyte determination and differentiation factor 1 (ADD1/SREBP-1c) on the RNA- and protein level utilizing qRT-PCR and immunofluorescence analysis. Additionally, the effect of L-carnitine on beta-oxidation was analyzed using human preadipocytes and mature adipocytes. Finally, we investigated additive effects of a combination of lotus leaf extract solution and L-carnitine on triglyceride accumulation during preadipocyte/adipocyte differentiation. RESULTS: Our data showed that incubation of preadipocytes with lotus leaf extract solution significantly decreased triglyceride accumulation during adipogenesis without affecting cell viability. Compared to controls, adipocytes incubated with lotus leaf extract solution exhibited a significant increase in lipolysis-activity. Moreover, cell populations cultivated in the presence of lotus leaf extract solution showed a decrease in adipocyte differentiation capacity as indicated by a decrease in the ADD1/SREBP-1c signal. Importantly, our results demonstrated that a combination of lotus leaf extract solution and L-carnitine reduced triglyceride accumulation to a greater extent compared to incubation with either substance alone. CONCLUSIONS: Overall, our data demonstrate that a combination of lotus leaf extract and L-carnitine reduced triglyceride accumulation in human (pre)adipocytes by affecting different processes during the adipocyte life cycle. For this reason, this combination might represent a treatment option for obesity-related diseases.

Aging and chronic sun exposure cause distinct epigenetic changes in human skin.

Epigenetic changes are widely considered to play an important role in aging, but experimental evidence to support this hypothesis has been scarce. We have used array-based analysis to determine genome-scale DNA methylation patterns from human skin samples and to investigate the effects of aging, chronic sun exposure, and tissue variation. Our results reveal a high degree of tissue specificity in the methylation patterns and also showed very little interindividual variation within tissues. Data stratification by age revealed that DNA from older individuals was characterized by a specific hypermethylation pattern affecting less than 1% of the markers analyzed. Interestingly, stratification by sun exposure produced a fundamentally different pattern with a significant trend towards hypomethylation. Our results thus identify defined age-related DNA methylation changes and suggest that these alterations might contribute to the phenotypic changes associated with skin aging.

A new protocol for functional analysis of adipogenesis using reverse transfection technology and time-lapse video microscopy.

Since the worldwide increase in obesity represents a growing challenge for healthcare systems, research focusing on fat cell metabolism has become a focal point of interest. Here, we describe a small interfering RNA (siRNA)-technology-based screening method to study fat cell differentiation in human primary preadipocytes that could be further developed towards an automated middle-throughput screening procedure. First, we established optimal conditions for the reverse transfection of human primary preadipocytes demonstrating that an efficient reverse transfection of preadipocytes is technically feasible. Aligning the processes of reverse transfection and fat cell differentiation utilizing peroxisome proliferator-activated receptor gamma (PPAR gamma)-siRNA, we showed that preadipocyte differentiation was suppressed by knock-down of PPAR gamma, the key regulator of fat cell differentiation. The use of fluorescently labelled fatty acids in combination with fluorescence time-lapse microscopy over a longer period of time enabled us to quantify the PPAR gamma phenotype. Additionally, our data demonstrate that reverse transfection of human cultured preadipocytes with TIP60 (HIV-1 Tat-interacting protein 60)-siRNA lead to a TIP60 knock-down and subsequently inhibits fat cell differentiation, suggesting a role of this protein in human adipogenesis. In conclusion, we established a protocol that allows for an efficient functional and time-dependent analysis by quantitative time-lapse microscopy to identify novel adipogenesis-associated genes.