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.

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.

An irreversible loss of subcutaneous adipose tissue in patients after tumor removal or deep dermal burns makes soft tissue engineering one of the most important challenges in biomedical research. The ideal scaffold for adipose tissue engineering has yet not been identified though biodegradable polymers gained an increasing interest during the last years. In the present study we synthesized two novel biodegradable polymers, poly(ε-caprolactone-co-urethane-co-urea) (PEUU) and poly[(L-lactide-co-ε-caprolactone)-co-(L-lysine ethyl ester diisocyanate)-block-oligo(ethylene glycol)-urethane] (PEU), containing different types of hydrolytically cleavable bondings. Solutions of the polymers at appropriate concentrations were used to fabricate fleeces by electrospinning. Ultrastructure, tensile properties, and degradation of the produced fleeces were evaluated. Adipose-derived stem cells (ASCs) were seeded on fleeces and morphology, viability, proliferation and differentiation were assessed. The biomaterials show fine micro- and nanostructures composed of fibers with diameters of about 0.5 to 1.3 µm. PEUU fleeces were more elastic, which might be favourable in soft tissue engineering, and degraded significantly slower compared to PEU. ASCs were able to adhere, proliferate and differentiate on both scaffolds. Morphology of the cells was slightly better on PEUU than on PEU showing a more physiological appearance. ASCs differentiated into the adipogenic lineage. Gene analysis of differentiated ASCs showed typical expression of adipogenetic markers such as PPARgamma and FABP4. Based on these results, PEUU and PEU meshes show a promising potential as scaffold materials in adipose tissue engineering.

Movie S2 This is similar to movie 1, but with a more direct interaction. This is to indicate the heterogeneous nature of the nerve interactions with keratinocytes Movie S3 A 3-D resolution of nerve fiber innervation of keratinocytes showing synaptic bouton-like structure penetrating and exiting keratinocytes; corresponds to figure 2dMovie S4 A bottom to top Z projection of the sample from figure 2d and Movie 3, indicating that the nerve fiber could be within one or | QUESTIONS ADDRESSEDA deregulation of miR expression in skin-associated diseases such as psoriasis and atopic dermatitis has been reported previously. 4,5However, to our knowledge, no study has been published yet, showing that specific secretory miRs become deregulated in the course of skin ageing and are involved in human skin cell crosstalk. Thus, we analysed, if secreted miRs might play a role in age-related dysfunctions. | EXPERIMENTAL DESIGNWe generated miR profiles of interstitial fluid collected from younger and older volunteers after suction blistering and identified agedependent differences in miR levels (eg miR-126). In vitro, we analysed whether miR-126 is age-dependently secreted by dermal endothelial cells and incorporated by other skin cells. | RESULTS | Secretory miR levels during skin ageingTo investigate the levels of secreted miRs during skin ageing, human interstitial fluid of the skin was analysed. 6 Quantitative PCR-based microRNA profiling was performed, and 175 miRs were detected with a C t -value <40 which was sufficient to be considered in the following analysis ( Figure S1 and Table S1). Setting a threshold of 1.5-fold regulation (up/down), we identified 61 miRs that were regulated in an age-related manner in suction blister fluids collected from young and old volunteers. The results could be confirmed in an independent experiment for three selected miRs (miR-100, miR-126 and miR-223) | Age-related miR-100 , miR-126 and miR-223 levels in cultured endothelial cellsIn vitro analysis was performed to delineate whether the age-related miR expression changes are triggered during cellular ageing. We cultivated dermal endothelial cells isolated from volunteers aged between 22 and 73 years and determined miR-100, miR-126 and miR-223 abundance in the supernatant 48 hours after cell seeding. All three miRs were significantly decreased in the supernatant of endothelial cell populations isolated from aged volunteers compared to the younger cell population ( Figure S2A-C). In contrast, intra-cellular miR levels detected for miR-100 and miR-126 (miR-223 was not analysed) in the intra-cellular compartment remain stable throughout age ( Figure S3A-B).*contributed equally to this work F I G U R E 1 Age-related miR expression changes in human suction blister fluids of the skin. A-C: Different miRs were selected from miRNA profiling for validation from qRT-PCR profiling. The suction blister fluid of 20 healthy volunteers of two different age groups (18-28 years and 66-75 years) was collected, and the suction blister fluids of 2 volunteers ...