Involvement of platelet-derived VWF in metastatic growth of melanoma in the brain.

BACKGROUND: The prognosis of patients with brain metastases (BM) is poor despite advances in our understanding of the underlying pathophysiology. The high incidence of thrombotic complications defines tumor progression and the high mortality rate. We, therefore, postulated that von Willebrand factor (VWF) promotes BM via its ability to induce platelet aggregation and thrombosis. METHODS: We measured the abundance of VWF in the blood and intravascular platelet aggregates of patients with BM, and determined the specific contribution of endothelial and platelet-derived VWF using in vitro models and microfluidics. The relevance for the brain metastatic cascade in vivo was demonstrated in ret transgenic mice, which spontaneously develop BM, and by the intracardiac injection of melanoma cells. RESULTS: Higher levels of plasma VWF in patients with BM were associated with enhanced intraluminal VWF fiber formation and platelet aggregation in the metastatic tissue and peritumoral regions. Platelet activation triggered the formation of VWF multimers, promoting platelet aggregation and activation, in turn enhancing tumor invasiveness. The absence of VWF in platelets, or the blocking of platelet activation, abolished platelet aggregation, and reduced tumor cell transmigration. Anticoagulation and platelet inhibition consistently reduced the number of BM in preclinical animal models. CONCLUSIONS: Our data indicate that platelet-derived VWF is involved in cerebral clot formation and in metastatic growth of melanoma in the brain. Targeting platelet activation with low-molecular-weight heparins represents a promising therapeutic approach to prevent melanoma BM.

Urothelial Carcinoma of the Bladder Induces Endothelial Cell Activation and Hypercoagulation.

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.

Claudin-1 decrease impacts epidermal barrier function in atopic dermatitis lesions dose-dependently.

The transmembrane protein claudin-1 is a major component of epidermal tight junctions (TJs), which create a dynamic paracellular barrier in the epidermis. Claudin-1 downregulation has been linked to atopic dermatitis (AD) pathogenesis but variable levels of claudin-1 have also been observed in healthy skin. To elucidate the impact of different levels of claudin-1 in healthy and diseased skin we determined claudin-1 levels in AD patients and controls and correlated them to TJ and skin barrier function. We observed a strikingly broad range of claudin-1 levels with stable TJ and overall skin barrier function in healthy and non-lesional skin. However, a significant decrease in TJ barrier function was detected in lesional AD skin where claudin-1 levels were further reduced. Investigations on reconstructed human epidermis expressing different levels of claudin-1 revealed that claudin-1 levels correlated with inside-out and outside-in barrier function, with a higher coherence for smaller molecular tracers. Claudin-1 decrease induced keratinocyte-autonomous IL-1ß expression and fostered inflammatory epidermal responses to non-pathogenic Staphylococci. In conclusion, claudin-1 decrease beyond a threshold level results in TJ and epidermal barrier function impairment and induces inflammation in human epidermis. Increasing claudin-1 levels might improve barrier function and decrease inflammation and therefore be a target for AD treatment.

Tight Junction barriers in human hair follicles - role of claudin-1.

Barrier function of hair follicles (HFs) is of great interest because they might be an entry port for allergens/pathogens, but could on the other hand be used for drug delivery or vaccination. Therefore we investigated tight junction (TJ) barrier function in human HFs. We show that there is a TJ barrier in the outermost living layer bordering to the environment from the infundibulum to the lower central part and between Henle's and Huxles layer of anagen HFs. In club hair typical for catagen and telogen HFs a TJ barrier is found surrounding the club. This demonstrates that there is a continuous TJ barrier along interfollicular epidermis and HFs in different phases of HF cycle. However, interestingly, in cell culture experiments we can show that barrier is less tight in HF keratinocytes compared to interfollicular keratinocytes. Knock-down of the TJ protein claudin-1, which we demonstrate here to be less expressed in HFs of lesional atopic dermatitis skin, results in impaired barrier function, decreased proliferation and increased apoptosis of hair keratinocytes. This is in line with a hair growth phenotype in claudin-1 deficient patients (NISCH syndrome) and corresponding knock-out mice and indicates an important role of claudin-1 in HF barrier function and growth.

Biphasic influence of Staphylococcus aureus on human epidermal tight junctions.

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.

Diverse regulation of claudin-1 and claudin-4 in atopic dermatitis.

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.

A custom tailored model to investigate skin penetration in porcine skin and its comparison with human skin.

Reliable models for the determination of skin penetration and permeation are important for the development of new drugs and formulations. The intention of our study was to develop a skin penetration model which (1) is viable and well supplied with nutrients during the period of the experiment (2) is mimicking human skin as far as possible, but still is independent from the problems of supply and heterogeneity, (3) can give information about the penetration into different compartments of the skin and (4) considers specific inter-individual differences in skin thickness. In addition, it should be quick and inexpensive (5) and without ethical implications (6). Using a chemically divers set of four topically approved active pharmaceutical ingredients (APIs), namely diclofenac, metronidazole, tazarotene, and terbinafine, we demonstrated that the model allows reliable determination of drug concentrations in different layers of the viable epidermis and dermis. For APIs susceptible for skin metabolism, the extent of metabolic transformation in epidermis and dermis can be monitored. Furthermore, a high degree of accordance in the ability for discrimination of skin concentrations of the substances in different layers was found in models derived from porcine and human skin. Viability, proliferation, differentiation and markers for skin barrier function were surveyed in the model. This model, which we call 'Hamburg model of skin penetration' is particularly suited to support a rational ranking and selection of dermatological formulations within drug development projects.

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

Immunohistochemical analyses point to epidermal origin of human Merkel cells.

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, ß1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. ß1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.

Alteration of tight junction proteins is an early event in psoriasis: putative involvement of proinflammatory cytokines.

Psoriasis is an inflammatory skin disease characterized by hyperproliferation of keratinocytes, impaired barrier function, and pronounced infiltration of inflammatory cells. Tight junctions (TJs) are cell-cell junctions that form paracellular barriers for solutes and inflammatory cells. Altered localization of TJ proteins in the epidermis was described in plaque-type psoriasis. Here we show that localization of TJ proteins is already altered in early-stage psoriasis. Occludin, ZO-1, and claudin-4 are found in more layers than in normal epidermis, and claudin-1 and -7 are down-regulated in the basal and in the uppermost layers. In plaque-type psoriasis, the staining patterns of occludin and ZO-1 do not change, whereas the claudins are further down-regulated. Near transmigrating granulocytes, all TJ proteins except for junctional adhesion molecule-A are down-regulated. Treatment of cultured keratinocytes with interleukin-1beta and tumor necrosis factor-alpha, which are present at elevated levels in psoriatic skin, results in an increase of transepithelial resistance at early time points and a decrease at later time points. Injection of interleukin-1beta into an ex vivo skin model leads to an up-regulation of occludin and ZO-1, resembling TJ protein alteration in early psoriasis. Our results show for the first time that alteration of TJ proteins is an early event in psoriasis and is not the consequence of the more profound changes found in plaque-type psoriasis. Our data indicate that cytokines are involved in alterations of TJ proteins observed in psoriasis.

Regulation of epidermal tight-junctions (TJ) during infection with exfoliative toxin-negative Staphylococcus strains.

Tight Junction (TJ) proteins have been shown to exert a barrier function within the skin. Here, we study the fate of TJ proteins during the challenge of the skin by bacterial colonization and infection. We investigated the influence of various exfoliative toxin-negative Staphylococcus strains on TJ, adherens junction (AJ), desmosomal proteins, and actin in a human keratinocyte infection culture and in a porcine skin infection model. We found that the pathogen Staphylococcus aureus downregulates TJ and subsequently AJ and desmosomal proteins, including atypical protein kinase C, an essential player in TJ formation, at the cell-cell borders of keratinocytes in a time and concentration dependent manner. Little changes in protein and RNA levels were seen, indicating redistribution of proteins. In cultured keratinocytes, a reduction of transepithelial resistance was observed. Staphylococcus epidermidis shows only minor effects. All strains induced enhanced expression of occludin and ZO-1 at the beginning of colonization/infection. Thus, we demonstrate that TJ are likely to be involved in skin infection of exfoliative toxin-negative S. aureus. As we did not find a change in actin, and as changes of TJ preceded alterations of AJs and desmosomes, we suggest that S. aureus targets TJ.

Differential induction of connexins 26 and 30 in skin tumors and their adjacent epidermis.

Gap junctions (GJs) have been shown to play a role in tumor progression including a variety of keratinocyte-derived and non-keratinocyte-derived skin tumors. Here we show that the synthesis of the GJ proteins connexin 26 and connexin 30 (Cx26 and Cx30) is induced in keratinocyte-derived epithelial skin tumors whereas there is either no change or a downregulation of Cx43. Cx26, Cx30, and Cx43 are absent in non-epithelial skin tumors. Further, Cx26 and Cx30 are induced in the epidermis adjacent to malignant melanoma but absent in the epidermis adjacent to benign non-epithelial skin lesions (melanocytic nevi and angioma). The keratinocyte-derived skin tumors are very heterogeneous regarding the Cx26/Cx30 pattern in the epidermis at the periphery of the tumors. We did not observe any difference in the localization of the very similar proteins Cx26 and Cx30 but a variation in intensity of immunoreactivity. As the staining patterns of Cx26 and Cx30 antibodies are not identical to those of CK6, a marker for hyperproliferation, and CK17, a marker for trauma, we discuss that the induction of these gap junctional proteins exceeds a reflection of reactive hyperproliferative or traumatized epidermis. We further discuss the putative roles of these gap junctional proteins in tumor progression.

Expression and localization of tight junction-associated proteins in human hair follicles.

Tight junctions (TJ) are barrier-forming intercellular junctions selectively sealing cells and controlling the paracellular pathway. They have been well-characterized in simple epithelia and endothelia but have only recently been described in stratified epithelia such as epidermis, oesophagus and oral mucosa. Various epithelial layers which are partly in morphogenic continuity with the epidermis and develop therefrom during early fetal life, build the human hair follicle. The barrier function of these epithelial layers seems to be important for the universal continuity of the barrier represented by the skin. We show the presence of the TJ proteins ZO-1, occludin, and various claudins in the hair follicle and demonstrate their impressive heterogeneous distribution pattern within a given stratum as well as within its different epithelial layers. Coexpression of the various TJ proteins, arguing for typical TJ structures, can be observed especially in cell layers facing the hair shaft and the stratum corneum, and in addition at the border between the outer and inner root sheaths. Usually they are found in close proximity to desmosomal and adherens junction proteins. The morphological and biological importance of these findings and the possible roles of TJ in hair follicles, e.g. in follicular penetration, are discussed.