Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells.

Integrity of human skin is endangered by exposure to UV irradiation and chemical stressors, which can provoke a toxic production of reactive oxygen species (ROS) and oxidative damage. Since oxidation of proteins and metabolites occurs virtually instantaneously, immediate cellular countermeasures are pivotal to mitigate the negative implications of acute oxidative stress. We investigated the short-term metabolic response in human skin fibroblasts and keratinocytes to H2O2 and UV exposure. In time-resolved metabolomics experiments, we observed that within seconds after stress induction, glucose catabolism is routed to the oxidative pentose phosphate pathway (PPP) and nucleotide synthesis independent of previously postulated blocks in glycolysis (i.e., of GAPDH or PKM2). Through ultra-short (13)C labeling experiments, we provide evidence for multiple cycling of carbon backbones in the oxidative PPP, potentially maximizing NADPH reduction. The identified metabolic rerouting in oxidative and non-oxidative PPP has important physiological roles in stabilization of the redox balance and ROS clearance.

An integrative metabolomics and transcriptomics study to identify metabolic alterations in aged skin of humans in vivo.

BACKGROUND: Aging human skin undergoes significant morphological and functional changes such as wrinkle formation, reduced wound healing capacity, and altered epidermal barrier function. Besides known age-related alterations like DNA-methylation changes, metabolic adaptations have been recently linked to impaired skin function in elder humans. Understanding of these metabolic adaptations in aged skin is of special interest to devise topical treatments that potentially reverse or alleviate age-dependent skin deterioration and the occurrence of skin disorders. RESULTS: We investigated the global metabolic adaptions in human skin during aging with a combined transcriptomic and metabolomic approach applied to epidermal tissue samples of young and old human volunteers. Our analysis confirmed known age-dependent metabolic alterations, e.g. reduction of coenzyme Q10 levels, and also revealed novel age effects that are seemingly important for skin maintenance. Integration of donor-matched transcriptome and metabolome data highlighted transcriptionally-driven alterations of metabolism during aging such as altered activity in upper glycolysis and glycerolipid biosynthesis or decreased protein and polyamine biosynthesis. Together, we identified several age-dependent metabolic alterations that might affect cellular signaling, epidermal barrier function, and skin structure and morphology. CONCLUSIONS: Our study provides a global resource on the metabolic adaptations and its transcriptional regulation during aging of human skin. Thus, it represents a first step towards an understanding of the impact of metabolism on impaired skin function in aged humans and therefore will potentially lead to improved treatments of age related skin disorders.

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.

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.

UV radiation induces CXCL5 expression in human skin.

CXCL5 has recently been identified as a mediator of UVB-induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose-dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non-immune skin cells also contribute to UV-induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte-keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV-radiated human skin and the essential role of fibroblast-keratinocyte interaction in the regulation of epidermal CXCL5.

Krüppel-like factor 9 is a circadian transcription factor in human epidermis that controls proliferation of keratinocytes.

Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüppel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain- and loss-of-function experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.

Glutathione-conjugated sulfanylalkanols are substrates for ABCC11 and γ-glutamyl transferase 1: a potential new pathway for the formation of odorant precursors in the apocrine sweat gland.

We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.

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.

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.

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.

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.

Impact of collagen crosslinking on the second harmonic generation signal and the fluorescence lifetime of collagen autofluorescence.

BACKGROUND/PURPOSE: Collagen is the major structural protein of the skin and its crosslinks are essential for its mechanical stability. In photodamaged skin, a decrease of the mature collagen crosslink histidinohydroxylysino-norleucine was reported. In this study, we investigated the consequences and measurability of the reduced crosslinking. METHODS: In order to determine the consequences of reduced collagen crosslinking, in vitro models of reduced collagen crosslinking were established. The collagen synthesis and structure was analyzed using the signals second harmonic generation (SHG) and the fluorescence lifetime of the collagen autofluorescence by a multiphoton laser scanning microscope. RESULTS: Reduced collagen crosslinking results in a posttranscriptionally diminished collagen synthesis, a modified structure of the collagen fibers and fibrils and a higher intensity of the SHG signal. The SHG signal might be influenced by the interspaces of the collagen molecules within one collagen fibril. Because of these findings, it can be speculated that reduced collagen crosslinking changes the interspace of single collagen molecules within the collagen fibril, resulting in an enhanced SHG signal. Alternative explanations are discussed. Furthermore, the fluorescence lifetime was reduced in the in vitro models of reduced collagen crosslinking. In the crosslink sites of the collagen molecules, the main ratio of fluorescence is found. As the fluorescence lifetime is determined not only by the fluorescent molecule itself but also by its microenvironment, the change in the fluorescence lifetime might be explained by reduced crosslinking at the crosslink site. CONCLUSION: A reduction of collagen crosslinking (as seen in photodamaged skin) results in an increase of the SHG signal and a decrease of the fluorescence lifetime in vitro. In vivo measurements of the two parameters might reveal the status of collagen crosslinking and therefore help to identify the status of dermal photodamage or pathogenesis using collagen crosslinking determination.

Macrophages Are Polarized toward an Inflammatory Phenotype by their Aged Microenvironment in the Human Skin.

Aging of the skin is accompanied by cellular as well as tissue environmental changes, ultimately reducing the ability of the tissue to regenerate and adequately respond to external stressors. Macrophages are important gatekeepers of tissue homeostasis, and it has been reported that their number and phenotype change during aging in a site-specific manner. How aging affects human skin macrophages and what implications this has for the aging process in the tissue are still not fully understood. Using single-cell RNA-sequencing analysis, we show that there is at least a 50% increase of macrophages in human aged skin, which appear to have developed from monocytes and exhibit more proinflammatory M1-like characteristics. In contrast, the cell-intrinsic ability of aged monocytes to differentiate into M1 macrophages was reduced. Using coculture experiments with aged dermal fibroblasts, we show that it is the aged microenvironment that drives a more proinflammatory phenotype of macrophages in the skin. This proinflammatory M1-like phenotype in turn negatively influenced the expression of extracellular matrix proteins by fibroblasts, emphasizing the impact of the aged macrophages on the skin phenotype.

Interference and co-existence of staphylococci and Cutibacterium acnes within the healthy human skin microbiome.

Human skin is populated by trillions of microbes collectively called the skin microbiome. Staphylococcus epidermidis and Cutibacterium acnes are among the most abundant members of this ecosystem, with described roles in skin health and disease. However, knowledge regarding the health beneficial effects of these ubiquitous skin residents is still limited. Here, we profiled the staphylococcal and C. acnes landscape across four different skin sites of 30 individuals (120 skin samples) using amplicon-based next-generation sequencing. Relative abundance profiles obtained indicated the existence of phylotype-specific co-existence and exclusion scenarios. Co-culture experiments with 557 staphylococcal strains identified 30 strains exhibiting anti-C. acnes activities. Notably, staphylococcal strains were found to selectively exclude acne-associated C. acnes and co-exist with healthy skin-associated phylotypes, through regulation of the antimicrobial activity. Overall, these findings highlight the importance of skin-resident staphylococci and suggest that selective microbial interference is a contributor to healthy skin homeostasis.

Qualification of a new and precise automatic tool for the assessment of hair diameters in phototrichograms.

BACKGROUND/PURPOSE: To automatically assess hair growth during cosmetic trials, incorporating parameters such as anagen-to-telogen rate, growth rate, and especially hair diameter. METHODS: We designed and qualified a new and automatic phototrichogram system based on a high-resolution DSLR camera system (theoretical resolution of 2.557 µm/pixel) and modular macrolens system with fixed focus, combined with a trainable pattern recognition software for automated analysis. RESULTS: We improved the standard routine for dermatological phototrichogram technique to overcome inaccuracy in thickness measurements due to hair swelling by using an alternative immersion fluid, and increased the effective resolution for hair size and thickness measurement to <4 µm. After having qualified manual measurements as gold standard for the determination of hair diameters, we established a new trainable automatic picture analysis software able to locate and measure individual hairs in length and thickness even in picture series taken from the same skin area at different time points. Comparisons between manual and automatic measurements of the same hairs showed a >90% correlation, and by comparing the automatic results with manual measurements of the same images without individual hair annotation, we could find a correlation of at least 80%. CONCLUSION: According to the results and findings generated in this qualification study, we have a reliable tool now that enables us to test cosmetic products for hair treatment in a highly automated way with a sufficient degree of precision and accuracy to detect even small changes in hair diameter during cosmetic trials.

Qualification of an automated device to objectively assess the effect of hair care products on hair shine.

The authors developed and qualified an automated routine screening tool to quantify hair shine. This tool is able to separately record individual properties of hair shine such as specular reflection and multiple reflection, as well as additional features such as sparkle, parallelism of hair fibers, and hair color, which strongly affect the subjective ranking by individual readers. A side-by-side comparison of different hair care and styling products with regard to hair shine using the automated screening tool in parallel with standard panel assessment showed that the automated system provides an almost identical ranking and the same statistical significances as the panel assessment. Provided stringent stratification of hair fibers for color and parallelism, the automated tool competes favorably with panel assessments of hair shine. In this case, data generated with the opsira Shine-Box are clearly superior over data generated by panel assessment in terms of reliability and repeatability, workload and time consumption, and sensitivity and specificity to detect differences after shampoo, conditioner, and leave-in treatment. The automated tool is therefore well suited to replace standard panel assessments in claim support, at least as a screening tool. A further advantage of the automated system over panel assessments is the fact that absolute numeric values are generated for a given hair care product, whereas panel assessments can only give rankings of a series of hair care products included in the same study. Thus, the absolute numeric data generated with the automated system allow comparison of hair care products between studies or at different time points after treatment.

A method to analyze the influence of mechanical strain on dermal collagen morphologies.

Collagen fibers and their orientation play a major role in the mechanical behavior of soft biological tissue such as skin. Here, we present a proof-of-principle study correlating mechanical properties with collagen fiber network morphologies. A dedicated multiphoton stretching device allows for mechanical deformations in combination with a simultaneous analysis of its collagen fiber network by second harmonic generation imaging (SHG). The recently introduced Fiber Image Network Evaluation (FINE) algorithm is used to obtain detailed information about the morphology with regard to fiber families in collagen network images. To demonstrate the potential of our method, we investigate an isotropic and an anisotropic ex-vivo dorsal pig skin sample under quasi-static cyclic stretching and relaxation sequences. Families of collagen fibers are found to form a partially aligned collagen network under strain. We find that the relative force uptake is accomplished in two steps. Firstly, fibers align within their fiber families and, secondly, fiber families orient in the direction of force. The maximum alignment of the collagen fiber network is found to be determined by the largest strain. Isotropic and anisotropic samples reveal a different micro structural behavior under repeated deformation leading to a similar force uptake after two stretching cycles. Our method correlates mechanical properties with morphologies in collagen fiber networks.