Anja Knott

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

BackgroundThe 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.MethodsFor 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.ResultsOur 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.ConclusionWhite 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.

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.

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 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.

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.

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.

Poly(A) tail shortening correlates with mRNA repression in tropoelastin regulation

BACKGROUND It has been shown for various organisms that expression of tropoelastin (TE) is high during fetal and neonatal growth and that it is reduced in adulthood by an unknown mechanism. OBJECTIVE To highlight the process of TE mRNA repression in vivo, total RNA from human skin biopsies was analyzed and TE mRNA expression was compared in fetal and adult donors. METHODS TaqMan Real-Time PCR, Poly(A) tail length assay, immunoblot. RESULTS In this study a more than 30-fold reduction of mature TE mRNA was detected whereas the decline on pre-mRNA level was not pronounced. This finding supports the hypothesis that the repression of mature TE mRNA is for the most part due to posttranscriptional mechanisms. Since deadenylation-dependent mRNA destabilization is the major decay pathway for most mRNAs, poly(A) tail length of mature TE mRNA was analyzed in fetal and adult human skin, lung and uterus, showing a profound reduction of poly(A) tail length in the adult samples. While TE mRNA is repressed in adult tissues in vivo, TGF-ß(1) has been shown to induce expression of TE mRNA in vitro on the posttranscriptional level. To analyze the underlying mechanism, TE mRNA poly(A) tail length was analyzed in human dermal fibroblasts after treatment with TGF-ß(1)in vitro. Besides the expected increase in TE expression, TGF-ß(1) treatment resulted in a significant stabilization of TE mRNA poly(A) tail length. CONCLUSION Our findings correlate for the first time TE expression level with poly(A) tail length and suggest that maintenance of poly(A) tail and deadenylation of TE mRNA might be general mechanisms involved in the regulation of TE expression.

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

BackgroundAging 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.ResultsWe 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.ConclusionsOur 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.

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.

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.