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The dynamic change from a globular conformation to an elongated fiber determines the ability of von Willebrand factor (VWF) to trap platelets. Fiber formation is favored by the anchorage of VWF to the endothelial cell surface, and VWF-platelet aggregates on the endothelium contribute to inflammation, infection, and tumor progression. Although P-selectin and ανβ3-integrins may bind VWF, their precise role is unclear, and additional binding partners have been proposed. In the present study, we evaluated whether the endothelial glycocalyx anchors VWF fibers to the endothelium. Using microfluidic experiments, we showed that stabilization of the endothelial glycocalyx by chitosan oligosaccharides or overexpression of syndecan-1 (SDC-1) significantly supports the binding of VWF fibers to endothelial cells. Heparinase-mediated degradation or impaired synthesis of heparan sulfate (HS), a major component of the endothelial glycocalyx, reduces VWF fiber–dependent platelet recruitment. Molecular interaction studies using flow cytometry and live-cell fluorescence microscopy provided further evidence that VWF binds to HS linked to SDC-1. In a murine melanoma model, we found that protection of the endothelial glycocalyx through the silencing of heparanase increases the number of VWF fibers attached to the wall of tumor blood vessels. In conclusion, we identified HS chains as a relevant binding factor for VWF fibers at the endothelial cell surface in vitro and in vivo.

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs.

Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal–related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance.

Cancer-related venous thromboembolisms (VTE) are associated with metastasis and reduced survival in patients with urothelial cancer of the bladder. Although previous reports suggest the contribution of tissue factor and podoplanin, the mechanistic linkage between VTE and bladder cancer cell–derived molecules is unknown. Therefore, we compared distinct procoagulant pathways in four different cell lines. In vitro findings were further confirmed by microfluidic experiments mimicking the pathophysiology of tumor blood vessels and in tissue samples of patients with bladder cancer by transcriptome analysis and immunohistology. In vitro and microfluidic experiments identified bladder cancer–derived VEGF-A as highly procoagulant because it promoted the release of von Willebrand factor (VWF) from endothelial cells and thus platelet aggregation. In tissue sections from patients with bladder cancer, we found that VWF-mediated blood vessel occlusions were associated with a poor outcome. Transcriptome data further indicate that elevated expression levels of enzymes modulating VEGF-A availability were significantly connected to a decreased survival in patients with bladder cancer. In comparison with previously postulated molecular players, we identified tumor cell–derived VEGF-A and endothelial VWF as procoagulant mediators in bladder cancer. Therapeutic strategies that prevent the VEGF-A–mediated release of VWF may reduce tumor-associated hypercoagulation and metastasis in patients with bladder cancer. Implications: We identified the VEGF-A–mediated release of VWF from endothelial cells to be associated with bladder cancer progression.

Tight junctions are important for skin barrier function. The tight junction protein claudin 1 (Cldn-1) has been reported to be down-regulated in nonlesional skin of atopic dermatitis (AD) patients. In contrast, we did not observe a significant down-regulation of Cldn-1 in nonlesional skin of the AD cohort used in this study. However, for the first time, a significant down-regulation of Cldn-1 in the upper and lower epidermal layers of lesional skin was detected. In addition, there was a significant up-regulation of Cldn-4 in nonlesional, but not lesional, AD skin. For occludin, no significant alterations were observed. In an AD-like allergic dermatitis mouse model, Cldn-1 down-regulation in eczema was significantly influenced by dermal inflammation, and significantly correlated with hallmarks of eczema (ie, increased keratinocyte proliferation, altered keratinocyte differentiation, increased epidermal thickness, and impaired barrier function). In human epidermal equivalents, the addition of IL-4, IL-13, and IL-31 resulted in a down-regulation of Cldn-1, and Cldn1 knockdown in keratinocytes resulted in abnormal differentiation. In summary, we provide the first evidence that Cldn-1 and Cldn-4 are differentially involved in AD pathogenesis. Our data suggest a role of Cldn-1 in AD eczema formation triggered by inflammation.

BACKGROUND: Birch bark has a long lasting history as a traditional medicinal remedy to accelerate wound healing. Recently, the efficacy of birch bark preparations has also been proven clinically. As active principle pentacyclic triterpenes are generally accepted. Here, we report a comprehensive study on the underlying molecular mechanisms of the wound healing properties of a well-defined birch bark preparation named as TE (triterpene extract) as well as the isolated single triterpenes in human primary keratinocytes and porcine ex-vivo wound healing models. METHODOLOGY/PRINCIPAL FINDINGS: We show positive wound healing effects of TE and betulin in scratch assay experiments with primary human keratinocytes and in a porcine ex-vivo wound healing model (WHM). Mechanistical studies elucidate that TE and betulin transiently upregulate pro-inflammatory cytokines, chemokines and cyclooxygenase-2 on gene and protein level. For COX-2 and IL-6 this increase of mRNA is due to an mRNA stabilizing effect of TE and betulin, a process in which p38 MAPK and HuR are involved. TE promotes keratinocyte migration, putatively by increasing the formation of actin filopodia, lamellipodia and stress fibers. Detailed analyses show that the TE components betulin, lupeol and erythrodiol exert this effect even in nanomolar concentrations. Targeting the actin cytoskeleton is dependent on the activation of Rho GTPases. CONCLUSION/SIGNIFICANCE: Our results provide insights to understand the molecular mechanism of the clinically proven wound healing effect of birch bark. TE and betulin address the inflammatory phase of wound healing by transient up-regulation of several pro-inflammatory mediators. Further, they enhance migration of keratinocytes, which is essential in the second phase of wound healing. Our results, together with the clinically proven efficacy, identify birch bark as the first medical plant with a high potential to improve wound healing, a field which urgently needs effective remedies.

Tight junction (TJ) proteins are involved in a number of cellular functions, including paracellular barrier formation, cell polarization, differentiation, and proliferation. Altered expression of TJ proteins was reported in various epithelial tumors. Here, we used tissue samples of human cutaneous squamous cell carcinoma (SCC), its precursor tumors, as well as sun-exposed and non-sun-exposed skin as a model system to investigate TJ protein alteration at various stages of tumorigenesis. We identified that a broader localization of zonula occludens protein (ZO)-1 and claudin-4 (Cldn-4) as well as downregulation of Cldn-1 in deeper epidermal layers is a frequent event in all the tumor entities as well as in sun-exposed skin, suggesting that these changes result from chronic UV irradiation. In contrast, SCC could be distinguished from the precursor tumors and sun-exposed skin by a frequent complete loss of occludin (Ocln). To elucidate the impact of down-regulation of Ocln, we performed Ocln siRNA experiments in human keratinocytes and uncovered that Ocln downregulation results in decreased epithelial cell-cell adhesion and reduced susceptibility to apoptosis induction by UVB or TNF-related apoptosis-inducing ligand (TRAIL), cellular characteristics for tumorigenesis. Furthermore, an influence on epidermal differentiation was observed, while there was no change of E-cadherin and vimentin, markers for epithelial-mesenchymal transition. Ocln knock-down altered Ca(2+)-homeostasis which may contribute to alterations of cell-cell adhesion and differentiation. As downregulation of Ocln is also seen in SCC derived from other tissues, as well as in other carcinomas, we suggest this as a common principle in tumor pathogenesis, which may be used as a target for therapeutic intervention.