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.

Modulation of Coenzyme Q10 content and oxidative status in human dermal fibroblasts using HMG-CoA reductase inhibitor over a broad range of concentrations. From mitohormesis to mitochondrial dysfunction and accelerated aging.

Coenzyme Q10 (CoQ10) is an endogenous lipophilic quinone, ubiquitous in biological membranes and endowed with antioxidant and bioenergetic properties, both crucial to the aging process. In fact, coenzyme Q10 synthesis is known to decrease with age in different tissues including skin. Moreover, synthesis can be inhibited by 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors such as statins, that are widely used hypocholesterolemic drugs. They target a key enzymatic step along the mevalonate pathway, involved in the synthesis of both cholesterol and isoprenylated compounds including CoQ10.In the present study, we show that pharmacological CoQ10 deprivation at concentrations of statins > 10000 nM triggers intracellular oxidative stress, mitochondrial dysfunction and generates cell death in human dermal fibroblasts (HDF). On the contrary, at lower statin concentrations, cells and mainly mitochondria, are able to partially adapt and prevent oxidative imbalance and overt mitochondrial toxicity. Importantly, our data demonstrate that CoQ10 decrease promotes mitochondrial permeability transition and bioenergetic dysfunction leading to premature aging of human dermal fibroblasts in vitro.

An optimized conditional suicide switch using doxycycline-dependent expression of human tBid.

We have examined in a HeLa model system whether tight doxycycline-dependent expression of human tBid can be used as a fast and efficient suicide switch. A stably transfected cell line expressing human tBid in a strictly dox-dependent manner showed fast and efficient killing with up to 98 % specific cell death 24 h after induction. The survival rate was only 0.6 % after eleven days of dox treatment. Very low amounts of tBid were sufficient to induce apoptosis indicating that stringent control of the dox-dependent suicide gene is essential for cell survival in the absence of inducer. Using human tBid, an endogenous effector protein with low immunogenic potential, and doxycycline, an inducer with low cytotoxicity and a long record of safe use in humans, this conditional suicide switch provides a tool for various applications, e.g., adoptive immune transfer.

Topical treatment with coenzyme Q10-containing formulas improves skin's Q10 level and provides antioxidative effects.

Ubiquinone (coenzyme Q10, Q10) represents an endogenously synthesized lipid-soluble antioxidant which is crucial for cellular energy production but is diminished with age and under the influence of external stress factors in human skin. Here, it is shown that topical Q10 treatment is beneficial with regard to effective Q10 replenishment, augmentation of cellular energy metabolism, and antioxidant effects. Application of Q10-containing formulas significantly increased the levels of this quinone on the skin surface. In the deeper layers of the epidermis the ubiquinone level was significantly augmented indicating effective supplementation. Concurrent elevation of ubiquinol levels suggested metabolic transformation of ubiquinone resulting from increased energy metabolism. Incubation of cultured human keratinocytes with Q10 concentrations equivalent to treated skin showed a significant augmentation of energy metabolism. Moreover, the results demonstrated that stressed skin benefits from the topical Q10 treatment by reduction of free radicals and an increase in antioxidant capacity.

MicroRNA profiling during human keratinocyte differentiation using a quantitative real-time PCR method.

The terminal differentiation of epidermal keratinocytes requires transcriptional and posttranscriptional regulatory mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that play key roles during differentiation processes by regulating protein expression at the posttranscriptional level. Several studies have investigated miRNA expression in murine or human skin using northern blotting, microarrays, deep sequencing, or real-time PCR (Andl et al., Curr Biol 16:1041-1049, 2006; Hildebrand et al., J Invest Dermatol 131:20-29, 2011; Sonkoly et al., PLoS One 2:e610, 2007; Yi et al., Nat Genet 38:356-362, 2006; Yi et al., Proc Natl Acad Sci U S A 106:498-502, 2009). Conventional techniques such as northern blotting and microarrays often fail to detect miRNAs of low abundance, while the per-sample cost of deep sequencing approaches is still prohibitive in many cases. In contrast, stem loop primer-based real-time PCR methods for simultaneous detection of up to 380 miRNAs allow fast, specific, and reliable miRNA profiling. These methods are suitable for in vitro material, but also for samples which are of limited availability, such as epidermal stem cells isolated from human skin biopsies. Here, we describe a general real-time PCR method for miRNA profiling using isolated epidermal stem cells, transiently amplifying cells and terminally differentiated keratinocytes of human skin.

A novel niche for skin derived precursors in non-follicular skin.

BACKGROUND: Skin derived precursors (SKP) comprise a subset of specialized dermal cells that can be distinguished from fibroblast by their capacity for spheroidal growth. Recent investigations have shown that hair follicles constitute a niche for this cell type, but their localization and their definite function in non-follicular skin remains largely unknown. OBJECTIVE: To identify the dermal niche of non-follicular SKPs and to analyze whether functional aspects correlate with this localization. METHODS: SKPs were isolated from separate anatomical regions of human abdominal skin. Fluorescence activated cell sorting then was used to obtain a pure population of non-follicular SKPs. Functional characterization of these cells was performed applying differentiation and proliferation assays. Information on specific in vivo functions was derived from histological evaluation of quantity and localization patterns. RESULTS: Sphere forming capacity and differentiation assays show that SKPs reside in the papillary part of the dermis. Further delineation revealed that the dermal capillaries represent a niche for these cells which subsequently could be isolated by FACS utilizing a perivascular marker. Whereas functional properties described for follicular SKPs could also be detected in the perivascular SKP population, histological analyses additionally point to a cross-talk with epidermal stem cells and a reduction during chronological aging. CONCLUSION: Our data show that SKPs isolated from non-follicular skin originate from a perivascular niche. Compared to their follicular counterparts, no functional differences could be observed upon cultivation, but ex vivo analyses also point to unique functions and a contribution to the phenotype of aged skin.

A comprehensive analysis of microRNA expression during human keratinocyte differentiation in vitro and in vivo.

Here, we report a comprehensive investigation of changes in microRNA (miRNA) expression profiles on human keratinocyte (HK) differentiation in vitro and in vivo. We have monitored expression patterns of 377 miRNAs during calcium-induced differentiation of primary HKs, and have compared these patterns with miRNA expression profiles of epidermal stem cells, transient amplifying cells, and terminally differentiated HKs from human skin. Apart from the previously described miR-203, we found an additional nine miRNAs (miR-23b, miR-95, miR-210, miR-224, miR-26a, miR-200a, miR-27b, miR-328, and miR-376a) that are associated with HK differentiation in vitro and in vivo. In situ hybridization experiments confirmed miR-23b as a marker of HK differentiation in vivo. Additionally, gene ontology analysis and functional validation of predicted miRNA targets using 3'-untranslated region-luciferase assays suggest that multiple miRNAs that are upregulated on HK differentiation cooperate to regulate gene expression during skin development. Our results thus provide the basis for further analysis of miRNA functions during epidermal differentiation.

Damage at the root of cell renewal--UV sensitivity of human epidermal stem cells.

BACKGROUND: The epidermis harbors adult stem cells that reside in the basal layer and ensure the continuous maintenance of tissue homeostasis. Various studies imply that stem cells generally possess specific defense mechanisms against several forms of exogenous stress factors. As sun exposition is the most prevalent impact on human skin, this feature would be of particular importance in terms of sensitivity to UV-induced DNA damage. OBJECTIVE: To investigate whether human epidermal stem cells are susceptible to UV-induced DNA damage and subsequent functional impairment. METHODS: A method to isolate human epidermal stem cells from suction blister epidermis was established and validated. Volunteers were treated with solar-simulated irradiation on test areas of the forearm and stem cells were isolated from suction blister material of this region. DNA damage was analyzed by staining for cyclobutane thymidine dimers. The functional consequences of UV-induced damages were assessed by colony forming efficiency assays and gene expression analyses. RESULTS: Compared to an unirradiated control, stem cells isolated from areas that were exposed to solar-simulated radiation showed significantly more DNA lesions. Although the number of stem cells was not reduced by this treatment, a functional impairment of stem cells could be shown by reduced colony forming efficiency and altered gene expression of stem cell markers. CONCLUSIONS: Despite their essential role in skin maintenance, epidermal stem cells are sensitive to physiological doses of UV irradiation in vivo.

Analysis of tissue-specific gene expression using laser capture microdissection.

Epithelial tissues exhibit optimal conditions for studying cellular differentiation since the differentiation status of a single cell can be determined by its distance to the basal membrane. For that reason Laser Capture Microdissection (LCM) may serve as a perfect tool to compare the characteristics of cells that have been collected from different strata of the epithelium. However, as cell boundaries are not visible in untreated tissue sections, samples have to be stained to allow for sufficient structural orientation. This usually results in a considerable reduction of RNA content in the dissected specimen. To circumvent this problem, we have established a modified hematoxylin/eosin staining protocol that concurrently allows visualization of important structures and the subsequent isolation of sufficient RNA amounts to be used for linear amplification and quantitative analyses.

In situ localization of epidermal stem cells using a novel multi epitope ligand cartography approach.

Precise knowledge of the frequency and localization of epidermal stem cells within skin tissue would further our understanding of their role in maintaining skin homeostasis. As a novel approach we used the recently developed method of multi epitope ligand cartography, applying a set of described putative epidermal stem cell markers. Bioinformatic evaluation of the data led to the identification of several discrete basal keratinocyte populations, but none of them displayed the complete stem cell marker set. The distribution of the keratinocyte populations within the tissue was remarkably heterogeneous, but determination of distance relationships revealed a population of quiescent cells highly expressing p63 and the integrins alpha(6)/beta(1) that represent origins of a gradual differentiation lineage. This population comprises about 6% of all basal cells, shows a scattered distribution pattern and could also be found in keratinocyte holoclone colonies. The data suggest that this population identifies interfollicular epidermal stem cells.

Deregulation of versican and elastin binding protein in solar elastosis.

Several changes in skin appearance including loss of elasticity and wrinkle formation are associated with alterations in the composition of the dermal extracellular matrix. They are induced by intrinsic aging or by environmental factors such as UV light referred to as photoaging. A general characteristic in the histology of photoaged skin is the accumulation of elastotic material suggesting impaired formation and/or massive breakdown of elastic fibres. In order to shed light on some of the underlying mechanisms we tracked two of the major players in elastic fibre formation in different skin conditions: EBP (elastin binding protein), a regulator of elastic fibre assembly and VER (versican), a component of functional elastic fibres as well as non-functional elastotic material. Using quantitative RT-PCR on skin biopsies we found that the expression levels of VER and EBP were unaltered during intrinsic skin aging. Upon acute UV stress however, VER and EBP showed different regulation patterns: VER mRNA increased after 6 h and was further up-regulated until 24 h. The EBP mRNA by contrast was reduced after 6 h but showed massive induction at 24 h after acute UV stress. In chronically sun-exposed skin, VER protein was accumulated similar to elastotic material in the extracellular space, whereas its mRNA level was consistently reduced compared to sun-protected skin. The EBP mRNA by contrast showed slightly increased expression levels in the sun-exposed area compared to its sun-protected counterpart. Based on these data we propose a model which may help to explain parts of the mechanisms leading to the formation of elastotic masses. We further hypothesize that the presence of elastotic material triggers some yet unknown feedback mechanism(s) resulting in altered expression patterns of VER and EBP in chronically sun-exposed skin.

White Tea extract induces lipolytic activity and inhibits adipogenesis in human subcutaneous (pre)-adipocytes.

BACKGROUND: The dramatic increase in obesity-related diseases emphasizes the need to elucidate the cellular and molecular mechanisms underlying fat metabolism. To investigate how natural substances influence lipolysis and adipogenesis, we determined the effects of White Tea extract on cultured human subcutaneous preadipocytes and adipocytes. METHODS: For our in vitro studies we used a White Tea extract solution that contained polyphenols and methylxanthines. Utilizing cultured human preadipocytes we investigated White Tea extract solution-induced inhibition of triglyceride incorporation during adipogenesis and possible effects on cell viability. In vitro studies on human adipocytes were performed aiming to elucidate the efficacy of White Tea extract solution to stimulate lipolytic activity. To characterize White Tea extract solution-mediated effects on a molecular level, we analyzed gene expression of essential adipogenesis-related transcription factors by qRT-PCR and determined the expression of the transcription factor ADD1/SREBP-1c on the protein level utilizing immunofluorescence analysis. RESULTS: Our data show that incubation of preadipocytes with White Tea extract solution significantly decreased triglyceride incorporation during adipogenesis in a dose-dependent manner (n = 10) without affecting cell viability (n = 10). These effects were, at least in part, mediated by EGCG (n = 10, 50 µM). In addition, White Tea extract solution also stimulated lipolytic activity in adipocytes (n = 7). Differentiating preadipocytes cultivated in the presence of 0.5% White Tea extract solution showed a decrease in PPARγ, ADD1/SREBP-1c, C/EBPα and C/EBPδ mRNA levels. Moreover, the expression of the transcription factor ADD1/SREBP-1c was not only decreased on the mRNA but also on the protein level. CONCLUSION: White Tea extract is a natural source that effectively inhibits adipogenesis and stimulates lipolysis-activity. Therefore, it can be utilized to modulate different levels of the adipocyte life cycle.

A novel treatment option for photoaged skin.

BACKGROUND: DNA damage as a result of ultraviolet (UV) exposure plays an important role in the progression of cutaneous aging. Both folic acid and creatine have been linked to the process of DNA protection and repair. AIMS: This study aims to investigate the effects of a commercially available folic acid- and creatine-containing formulation to fight the clinical signs of premature skin aging. PATIENTS/METHODS: Both in vitro and in vivo home-in-use studies using a folic acid- and creatine-containing formulation were performed aiming to elucidate the efficacy in terms of improvement of skin regeneration, protection from UV-induced DNA damage (Comet assay), reduction of wrinkle volume, and skin visco-elasticity. Furthermore, clinical evaluation and photography were carried out to determine the improvement of clinically graded parameters after treatment. RESULTS: Cultured full-thickness epidermal skin models supplemented with folic acid and creatine after epithelial perturbation showed an accelerated skin regeneration compared to untreated control models. Similarly, application of a folic acid- and creatine-containing formulation significantly improved epidermal turnover in vivo as evidenced by smaller corneocytes derived from the treated sites relative to the vehicle-treated sides. In addition, topical in vivo application of this formulation significantly protected from UV-induced DNA lesions, increased skin firmness, and reduced wrinkle volume compared to untreated control areas. Expert grading confirmed a significant decrease of fine and coarse wrinkles in the periocular region as well as overall wrinkles, tactile roughness, and laxity. CONCLUSIONS: Taken together, these results show that the combination of folic acid and creatine significantly accelerates epidermal skin regeneration in vitro and in vivo. Together with the finding of improved biomechanical skin properties, we conclude that the described topical formulation provides an effective treatment option for (photo)-aged skin.

Natural Arctium lappa fruit extract improves the clinical signs of aging skin.

BACKGROUND: Subclinical, chronic tissue inflammation involving the generation of cytokines (e.g., interleukin-6 and tumor necrosis factor-alpha) might contribute to the cutaneous aging process. AIMS: This study aims to screen for an active ingredient with anti-inflammatory (i.e., reduction of interleukin-6 and tumor necrosis factor-alpha) and matrix-stimulating efficacy which improves the clinical signs of skin aging in vivo. PATIENTS/METHODS: In vitro studies with pure Arctiin were performed investigating the inhibition of cytokine induction and stimulation of collagen neo-synthesis. In vivo home-in-use studies using an Arctium lappa fruit extract-containing formulation were carried out to determine procollagen and hyaluronan synthesis, hyaluronan synthase-2 gene expression, and reduction of wrinkle volume after treatment. RESULTS: In vitro studies on human dermal fibroblasts and monocyte-derived dendritic cells supplemented with pure Arctiin showed relative to untreated control cells a stimulation of collagen synthesis and a decrease in interleukin-6 and tumor necrosis factor-alpha concentration, respectively. In addition, topical in vivo application of an A. lappa fruit extract-containing formulation for 12 weeks significantly stimulated procollagen synthesis and increased hyaluronan synthase-2 expression as well as hyaluronan levels compared to vehicle-treated control areas. Similarly, after a 4-week treatment with an A. lappa fruit extract-containing formulation, wrinkle volume in the crow's feet area was significantly reduced as compared to treatment with the vehicle. CONCLUSIONS: Our data show that topical treatment with a natural A. lappa fruit extract significantly improves the metabolism of the dermal extracellular matrix and leads to a visible wrinkle reduction in vivo. In conclusion, A. lappa fruit extract represents a targeted means to regenerate dermal structures and, thus, offers an effective treatment option for mature skin.

Expression of decorin and collagens I and III in different layers of human skin in vivo: a laser capture microdissection study.

Extracellular matrix (ECM) organization is a complex process that requires the coordinated efforts of many molecules. For the regulation of collagen fiber diameter, the proteoglycan decorin appears to be of major relevance. To investigate the role of decorin in the process of (photo-)aging in more detail, full-thickness punch biopsies were isolated from human buttock skin. Single exposure with two minimal erythemal doses of solar simulated irradiation caused down-regulation of decorin mRNA in young (n = 5) and old subjects (n = 5) after 24 h. Interestingly, decorin mRNA was elevated with age. To test the hypothesis that a decreased collagen-to-decorin-ratio impairs collagen structure we also investigated collagens I and III gene expression. Both were down-regulated with increasing age and after single UV-irradiation. As determined by laser capture microdissection-quantitative real time-Polymerase chain reaction (n = 11), decorin is mostly present in the reticular dermis while being absent from the papillary dermis. Minor expression was also observed in the epidermis. However, in contrast to full-thickness skin biopsies age-dependent changes in collagens I, III, and decorin expression could not be observed with this methodology indicating technical limitations. Together with our finding that collagens I and III mRNA are similarly expressed in the reticular and papillary dermis and are down-regulated by UV, our studies support the idea of a major role of decorin in ECM organization. Altered expression of decorin mRNA in the different dermal strata and a decrease in the collagen-to-decorin ratio inflicted by both age and ultraviolet irradiation possibly affect collagen bundle diameter and subsequently the mechanical properties of human skin.

Conditional cell suicide using dox-dependent caspase-2 expression.

BACKGROUND: Adoptive immune transfer is used as an efficient treatment modality to achieve a graft-versus-leukemia effect in persisting or relapsing residual leukemic disease. Safety considerations dictate the need for equipping the transferred cells with a conditional suicide mechanism to eliminate donor T cells when graft-versus-host disease occurs. We have examined in a model system using HeLa cells whether doxycycline (dox)-dependent expression of pro-apoptotic proteins could be used as a potential new strategy for conditional cell elimination. METHODS: Four constructs encoding pro-apoptotic proteins were tested in transient transfections to identify suitable cell death inducers. Murine caspase-2 placed under Tet-control was chosen for stable transfection into cell lines carrying different dox-dependent transregulators. The efficiency of cell death induction and the expression patterns of caspase-2 were analyzed in the respective clones. RESULTS: Different levels of induced cell death were obtained depending on the properties of the transregulators used to control target gene expression. High expression levels of caspase-2 in the presence of dox were required to achieve efficient induction of cell death, while tight repression in the absence of inducer was not necessary for cell survival. Dox treatment for 48 h resulted in 94% cell death indicating a very efficient conditional suicide mechanism. CONCLUSIONS: We propose that the principle of using pro-apoptotic cellular proteins placed under appropriate dox-dependent regulation may represent an alternative conditional suicide mechanism to the frequently used herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir-system, which harbors immunological and toxicological risks.