The Peripheral Lymphatic System Is Impaired by the Loss of Neuronal Control Associated with Chronic Spinal Cord Injury.

Spinal cord injury (SCI) is associated with venous vascular dysfunction below the level of injury, resulting in dysregulation of tissue fluid homeostasis in afflicted skin. The purpose of this study was to determine whether loss of neuronal control in chronic SCI also affects the skin lymphatic system. Morphology of lymphatics was characterized by immunohistochemistry and lymphatic gene expression profiles determined by DNA microarray analysis. In SCI, skin lymphatic function appeared to be impaired, because the ratio of functionally dilated versus collapsed lymphatic vessels was decreased 10-fold compared with control. Consequently, the average lumen area of lymphatic vessels was almost halved, possibly due to the known impaired connective tissue integrity of SCI skin. In fact, collagenases were found to be overexpressed in SCI skin, and dermal collagen structure was impaired. Molecular profiling also suggested an SCI-specific phenotype of increased connective tissue turnover and decreased lymphatic contractility. The total number of lymphatic vessels in SCI skin, however, was doubled, pointing to enhanced lymphangiogenesis. In conclusion, these data show, for the first time, that lymphatic function and development in human skin are under neuronal control. Because peripheral venous and lymphatic vascular defects are associated with disturbed fluid homeostasis, inappropriate wound healing reactions, and impaired skin immunity, they might contribute to the predisposition of afflicted individuals to pressure ulcer formation and wound healing disorders.

Cellular and molecular changes that predispose skin in chronic spinal cord injury to pressure ulcer formation.

Patients with spinal cord injury have a predisposition to develop pressure ulcers. Specific characteristics of the patients' skin potentially involved have not yet been identified. The purpose of this investigation was to determine whether loss of neuronal control affects cellular and molecular homeostasis in the skin. Intact afflicted skin, wound edge of pressure ulcers, and control skin were analysed. Platelets, transforming growth factor-ß1, and activin A were identified by immunohistochemistry. Transforming growth factor-ß-like activity was determined by bioassay, and gene expression by DNA microarray analysis or RT-PCR. In afflicted skin, enhanced platelet extravasation was detected. Transforming growth factor-ß1 and activin A accumulated in the dermal-epidermal junction zone. Transforming growth factor-ß-like activity and activin A expression were increased in intact afflicted skin (compared to control skin) and were further enhanced in pressure ulcers. In vitro, activity was generated by fibroblast-epithelial cell interactions, which also induced activin A. Thus, loss of neuronal control in spinal cord injury appears to trigger inappropriate wound healing processes in the patients' skin. Plasma leakage and increased transforming growth factor-ß-like activity combined with shear forces potentially enhance the risk for pressure ulcer formation.

Activation of human vascular endothelium in melanoma metastases induces ICAM-1 and E-selectin expression and results in increased infiltration with effector lymphocytes.

Lymphocytic infiltration into melanoma tissue is an important prerequisite for effective antitumoral immunity. However, analysis of human metastatic melanoma has shown that leucocyte adhesion receptor expression on melanoma blood vessels is very low or absent, thereby impairing the entry of cytotoxic lymphocytes into tumor tissue. We hypothesized that adhesion molecules can be induced on melanoma vasculature allowing better infiltration of cytotoxic lymphocytes. Quantitative real-time PCR and immunofluorescence staining indicated that the adhesion molecules ICAM-1 (CD54) and E-selectin (CD62E) can be significantly induced by intralesional application of TNF alpha in tissue from human melanoma metastases either in vitro or in vivo when grafted onto immunodeficient NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice that preserved human vessels. Furthermore, activated human autologous CD3+ lymphocytes were injected intravenously into mice bearing melanoma xenografts treated with TNF-α or PBS in addition to the leucocyte chemoattractant TARC (CCL17). Significantly increased numbers of CD8+ cells were detected in TNF-α-treated melanoma metastases compared with PBS-treated controls. In addition, tumor cell apoptosis was enhanced and melanoma cell proliferation reduced as shown by TUNEL assay and KI-67 staining. We conclude that adhesion molecules can be induced on human melanoma vasculature resulting in significantly improved homing of activated autologous cytotoxic T cells to melanoma tissue and inhibition of melanoma cell proliferation. These observations should be considered when designing protocols for immunotherapy of malignant melanoma.

Increased level of circulating U2 small nuclear RNA fragments indicates metastasis in melanoma patients.

UNLABELLED: Background: Melanoma is the most aggressive skin cancer and, despite recent advances in therapy, about 20% of the patients die of their disease. Early relapse detection and monitoring of therapy response are crucial for efficient treatment of advanced melanoma. Thus, there is a need for blood-based biomarkers in melanoma management. Serum-derived U2 small nuclear RNA fragments (RNU2-1f) were previously shown to be blood-based biomarkers for gastrointestinal and gynecologic malignancies. Here we examined whether RNU2-1f may also serve as diagnostic biomarker in advanced melanoma. METHODS: Circulating RNU2-1f levels were quantified by comparative reverse transcription PCR in a training cohort of patients with metastatic melanoma (n=33, thereof regionally metastasized to skin and lymph nodes, n=23, and distantly metastasized, n=10) vs. patients with benign naevi (n=16) vs. healthy controls (n=39). RESULTS were validated in an independent patient cohort with distant metastasis (n=16) vs. controls (n=18). RESULTS: Circulating RNU2-1f levels in the training cohort were significantly increased in serum of regionally and distantly metastatic patients, compared with patients with benign naevi or healthy controls (p<0.0001) and allowed accurate detection of regional (AUC 0.80) as well as distant (AUC 0.84) metastasis. In the validation cohort, increased RNU2-1f levels were confirmed and enabled highly specific detection of distant metastasis (sensitivity 81%, specificity 100%, AUC 0.94). CONCLUSIONS: This is the first report to suggest a blood-based snRNA serving as a diagnostic biomarker for melanoma metastasis. Our data provide a rationale for further defining clinical utility of circulating RNU2-1f in metastasis detection in the management of melanoma patients at risk of relapse and/or with advanced disease.

The epidermal basement membrane is a composite of separate laminin- or collagen IV-containing networks connected by aggregated perlecan, but not by nidogens.

The basement membrane between the epidermis and the dermis is indispensable for normal skin functions. It connects, and functionally separates, the epidermis and the dermis. To understand the suprastructural and functional basis of these connections, heterotypic supramolecular aggregates were isolated from the dermal-epidermal junction zone of human skin. Individual suprastructures were separated and purified by immunomagnetic beads, each recognizing a specific, molecular component of the aggregates. The molecular compositions of the suprastructures were determined by immunogold electron microscopy and immunoblotting. A composite of two networks was obtained from fibril-free suspensions by immunobeads recognizing either laminin 332 or collagen IV. After removal of perlecan-containing suprastructures or after enzyme digestion of heparan sulfate chains, a distinct network with a diffuse electron-optical appearance was isolated with magnetic beads coated with antibodies to collagen IV. The second network was more finely grained and comprised laminin 332 and laminins with α5-chains. The core protein of perlecan was an exclusive component of this network whereas its heparan sulfate chains were integrated into the collagen IV-containing network. Nidogens 1 and 2 occurred in both networks but did not form strong molecular cross-bridges. Their incorporation into one network appeared to be masked after their incorporation into the other one. We conclude that the epidermal basement membrane is a composite of two structurally independent networks that are tightly connected in a spot-welding-like manner by perlecan-containing aggregates.

Impact of thrombolysis on stroke outcome at 12 months in a population: the Bern stroke project.

BACKGROUND AND PURPOSE: Thrombolysis improves outcome of patients with acute ischemic stroke, but it is unknown whether thrombolysis has a measurable effect on long-term outcome in a defined population. METHODS: We prospectively assessed demographic data, management, and outcome of acute ischemic stroke patients admitted within 48 hours to 18 primary care hospitals of the canton of Bern (969 299 inhabitants) during 12 months. Blinded follow-up was obtained at 3 and 12 months. Predictors of mortality and favorable outcome (modified Rankin Scale score ≤2) at 3 and 12 months using logistic regression were analyzed. RESULTS: From December 2007 to December 2008, 807 patients (mean age, 72 years) were included. Median National Institutes of Health Stroke Scale score on admission was 5; 107 patients (13%) received intravenous, intra-arterial, or mechanical thrombolysis. Estimated cumulative mortality at 3 months was 20.6% and at 12 months 27.4%. Age 75 years or older, higher National Institutes of Health Stroke Scale scores, and higher Charlson comorbidity index were independent predictors of mortality at 3 and 12 months. Estimated favorable outcome at 3 months was 48.2% and at 12 months was 44.6%. Thrombolysis was the only modifiable independent predictor of favorable outcome at 3 (relative risk, 1.49; 95% CI, 1.18-1.89) and 12 months (relative risk, 1.59; 95% CI, 1.24-2.04), whereas age younger than 75 years, male gender, National Institutes of Health Stroke Scale score <4, and lower Charlson comorbidity index were nonmodifiable predictors. CONCLUSIONS: Thirteen percent of acute ischemic stroke patients admitted within 48 hours to Bernese hospitals underwent thrombolysis, which exerted a measurable effect on 3-month outcome in this population. This effect was sustained at 12 months. Age, stroke severity, Charlson comorbidity index, and male gender were independent nonmodifiable predictors of outcome.

Defective granulation tissue formation in mice with specific ablation of integrin-linked kinase in fibroblasts - role of TGFß1 levels and RhoA activity.

Wound healing crucially relies on the mechanical activity of fibroblasts responding to TGFß1 and to forces transmitted across focal adhesions. Integrin-linked kinase (ILK) is a central adapter recruited to integrin ß1 tails in focal adhesions mediating the communication between cells and extracellular matrix. Here, we show that fibroblast-restricted inactivation of ILK in mice leads to impaired healing due to a severe reduction in the number of myofibroblasts, whereas inflammatory infiltrate and vascularization of the granulation tissue are unaffected. Primary ILK-deficient fibroblasts exhibit severely reduced levels of extracellular TGFß1, α-smooth muscle actin (αSMA) production and myofibroblast conversion, which are rescued by exogenous TGFß1. They are further characterized by elevated RhoA and low Rac1 activities, resulting in abnormal shape and reduced directional migration. Interference with RhoA-ROCK signaling largely restores morphology, migration and TGFß1 levels. We conclude that, in fibroblasts, ILK is crucial for limiting RhoA activity, thus promoting TGFß1 production, which is essential for dermal repair following injury.

Exome sequencing-based copy-number variation and loss of heterozygosity detection: ExomeCNV.

MOTIVATION: The ability to detect copy-number variation (CNV) and loss of heterozygosity (LOH) from exome sequencing data extends the utility of this powerful approach that has mainly been used for point or small insertion/deletion detection. RESULTS: We present ExomeCNV, a statistical method to detect CNV and LOH using depth-of-coverage and B-allele frequencies, from mapped short sequence reads, and we assess both the method's power and the effects of confounding variables. We apply our method to a cancer exome resequencing dataset. As expected, accuracy and resolution are dependent on depth-of-coverage and capture probe design. AVAILABILITY: CRAN package 'ExomeCNV'. CONTACT: fsathira@fas.harvard.edu; snelson@ucla.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cyclic mechanical stress downregulates endothelin-1 and its responsive genes independently of TGFß1 in dermal fibroblasts.

Mechanical forces are highly variable ranging from the ubiquitous gravity force to compression, fluid shear, torsion, tension and other forms. Mechanical forces act on cells and modulate their biological responses by regulating gene transcription, enzyme and growth factor activity. In soft connective tissues, formation of myofibroblasts strictly requires a mechanically loaded environment in addition to local transforming growth factor (TGF)-ß activity, which itself can be modulated by the mechanical status of the environment. The aim of this study was to monitor the adaptive responses of primary dermal fibroblasts towards cyclic mechanical stress under conditions of high force to better understand the regulation of gene expression in normal skin and mechanisms of gene regulation in mechanically altered fibrotic skin. Primary murine dermal fibroblasts were exposed to equi-biaxial tensile strain. Cyclic mechanical tension was applied at a frequency of 0.1 Hz (6× /min) for 24 h with a maximal increase in surface area of 15%. This treatment resulted in downregulation of alpha smooth muscle actin (αSMA) and connective tissue growth factor (CTGF) but not of TGFß1 expression. Cyclic strain also strongly reduced endothelin-1 (ET-1) expression and supplementing strained cultures with exogenous ET-1 rescued αSMA and CTGF levels. Of note, no biologically significant levels of TGFß1 activity were detected in strained cultures. We provide evidence for a novel, TGFß1-independent mechanism regulating ET-1 expression in dermal fibroblasts by biomechanical forces. Modulation of ET-1-dependent activities regulates downstream fibrotic marker genes; this pathway might therefore provide an approach to attenuate myofibroblast differentiation.

Gab2-mediated signaling promotes melanoma metastasis.

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

A Prognostic Gene Signature Expressed in Primary Cutaneous Melanoma: Synergism With Conventional Staging.

BACKGROUND: Current clinico-pathological American Joint Committee on Cancer (AJCC) staging of primary cutaneous melanoma is limited in its ability to determine clinical outcome, and complementary biomarkers are not available for routine prognostic assessment. We therefore adapted a gene signature, previously identified in fresh-frozen (FF) melanomas and adjacent stroma, to formalin-fixed paraffin-embedded (FFPE) melanomas. The aim was to develop a gene expression profiling (GEP) score to define patient survival probability at the time of first diagnosis. METHODS: Expression of 11 FF melanoma signature genes was quantified by reverse transcription polymerase chain reaction in an FFPE melanoma training cohort (n = 125), corresponding to the combined FF melanoma training and validation cohorts. The resulting GEP score was validated technically and clinically in an independent FFPE melanoma cohort (n = 211). All statistical tests were two-sided. RESULTS: We identified a prognostic eight-gene signature in the tumor area (tumor and adjacent tissue) of AJCC stage I-III melanomas. A signature-based GEP score correlated with melanoma-specific survival (MSS; Kaplan-Meier analysis: P < .0001) was independent of tumor stage (multivariable regression analysis: P = .0032) and stroma content (<5%-90%) and complemented conventional AJCC staging (receiver operating characteristic curve analysis: area under the curve = 0.91). In the clinical validation cohort, the GEP score remained statistically significant (P = .0131) in a multivariable analysis accounting for conventional staging. The GEP score was technically robust (reproducibility: 93%; n = 84) and clinically useful, as a binary as well as a continuous score, in predicting stage-specific patient MSS. CONCLUSIONS: The GEP score is a clinically significant prognostic tool, contributes additional information regarding the MSS of melanoma patients, and complements conventional staging.

CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells.

We investigated the functional role of CEACAM1 in a spontaneous metastasis xenograft model of human melanoma in scid mice using BRAF wildtype MeWo cells with and without RNAi mediated knockdown of CEACAM1. Tumors from the xenograft model were subjected to whole genome expression analysis and metastasis was quantified histologically. Results and identified markers were verified using tissue samples of over 100 melanoma patients. Knockdown of CEACAM1 prolonged the animals' survival by significantly reducing subcutaneous growth of MeWo tumors and spontaneous lung metastasis. Microarray analysis revealed a strong influence of CEACAM1 knockdown on the network of EMT associated genes in the xenograft tumors (e.g. downregulation of BRAF, FOSL1, NRAS and TWIST). IGFBP7 and Latexin (highest up- and downregulated expression in microarray analysis) were found to be associated with longer and shorter survival, respectively, of melanoma patients. High FOSL1 and altered TWIST1 expression were found to be correlated with shortened survival in the cohort of melanoma patients. After a stepwise selection procedure combining above markers, multivariate analysis revealed IGFBP7, Latexin and altered TWIST to be prognostic markers for death. CEACAM1 could be a target for melanoma therapy as an alternative to (or in combination with) immune checkpoint and BRAF inhibitors.

Glycoconjugate profiling of primary melanoma and its sentinel node and distant metastases: implications for diagnosis and pathophysiology of metastases.

Aiming at more precise detection of melanoma cells in sentinel lymph nodes and better understanding of the mechanisms underlying metastatic spread, expression of L1, CEACAM1, and binding of the lectins HPA, ML-I and PNA, was assessed in benign nevi (n=12), primary melanomas (PTs: n=67), their corresponding sentinel lymph nodes (SLNs: n=40), and distant metastases (DMs: n=35). Sensitivity and specificity of CEACAM1 (95-97%; 66%) and L1 (90-93%; 100%) exceeded that of the standard markers MelanA, S100, and HMB45 in single marker use. Lectin binding was found in PTs and DMs (HPA: 69% and 77%; ML-I: 82% and 77%, respectively), but rarely in SLNMs (HPA: 20%, ML-I: 20%, PNA: 5%, respectively). The highly specific and sensitive L1-11A against L1 and 4D1/C2 against CEACAM1 antibodies are a worthy completion to standard antibody panels for diagnosis of melanoma cells. Both CAMs seem to be functionally involved in lymphatic and haematogenous spread, and are thus promising target molecules for immunotoxins.

Genomic Characterization of Dysplastic Nevi Unveils Implications for Diagnosis of Melanoma.

A well-defined risk factor and precursor for cutaneous melanoma is the dysplastic nevus. These benign tumors represent clonal hyperproliferation of melanocytes that are in a senescent-like state, but with occasional malignant transformation events. To portray the mutational repertoire of dysplastic nevi in patients with the dysplastic nevus syndrome and to determine the discriminatory profiles of melanocytic nevi (including dysplastic nevi) from melanoma, we sequenced exomes of melanocytic nevi including dysplastic nevi (n = 19), followed by a targeted gene panel (785 genes) characterization of melanocytic nevi (n = 46) and primary melanomas (n = 42). Exome sequencing revealed that dysplastic nevi harbored a substantially lower mutational load than melanomas (21 protein-changing mutations versus >100). Known "driver" mutations in genes for melanoma, including CDKN2A, TP53, NF1, RAC1, and PTEN, were not found among any melanocytic nevi sequenced. Additionally, melanocytic nevi including dysplastic nevi showed a significantly lower frequency and a different UV-associated mutational signature. These results show that although melanocytic nevi and dysplastic nevi harbor stable genomes with relatively few alterations, progression into melanomas requires additional mutational processes affecting key tumor suppressors. This study identifies molecular parameters that could be useful for diagnostic platforms.

Transcriptional dissection of melanoma identifies a high-risk subtype underlying TP53 family genes and epigenome deregulation.

BACKGROUND: Melanoma is a heterogeneous malignancy. We set out to identify the molecular underpinnings of high-risk melanomas, those that are likely to progress rapidly, metastasize, and result in poor outcomes. METHODS: We examined transcriptome changes from benign states to early-, intermediate-, and late-stage tumors using a set of 78 treatment-naive melanocytic tumors consisting of primary melanomas of the skin and benign melanocytic lesions. We utilized a next-generation sequencing platform that enabled a comprehensive analysis of protein-coding and -noncoding RNA transcripts. RESULTS: Gene expression changes unequivocally discriminated between benign and malignant states, and a dual epigenetic and immune signature emerged defining this transition. To our knowledge, we discovered previously unrecognized melanoma subtypes. A high-risk primary melanoma subset was distinguished by a 122-epigenetic gene signature ("epigenetic" cluster) and TP53 family gene deregulation (TP53, TP63, and TP73). This subtype associated with poor overall survival and showed enrichment of cell cycle genes. Noncoding repetitive element transcripts (LINEs, SINEs, and ERVs) that can result in immunostimulatory signals recapitulating a state of "viral mimicry" were significantly repressed. The high-risk subtype and its poor predictive characteristics were validated in several independent cohorts. Additionally, primary melanomas distinguished by specific immune signatures ("immune" clusters) were identified. CONCLUSION: The TP53 family of genes and genes regulating the epigenetic machinery demonstrate strong prognostic and biological relevance during progression of early disease. Gene expression profiling of protein-coding and -noncoding RNA transcripts may be a better predictor for disease course in melanoma. This study outlines the transcriptional interplay of the cancer cell's epigenome with the immune milieu with potential for future therapeutic targeting. FUNDING: National Institutes of Health (CA154683, CA158557, CA177940, CA087497-13), Tisch Cancer Institute, Melanoma Research Foundation, the Dow Family Charitable Foundation, and the Icahn School of Medicine at Mount Sinai.

Inactivation of active and latent transforming growth factor beta by free thiols: potential redox regulation of biological action.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine with important roles in inflammation, wound repair, and cancer. Cells secrete TGF-beta as a latent protein complex, consisting of disulfide-bonded homodimers of growth factor and latency-associated propeptide. Latency regulates extracellular TGF-beta action by controlling the levels of active growth factor available. We report here that active and latent TGF-beta were inactivated in vitro by reduction of the growth factor dimer under physiological conditions. We also demonstrate that the latency-associated propeptide has chaperone-like activity and partially protects TGF-beta from inactivation. TGF-beta inactivation occurred upon incubation with the physiological redox agents, cysteine, homocysteine, and reduced glutathione. Inactivation was temperature- and dose-dependent. While inactivation by physiological concentrations of redox agents was partial at 37 degrees C, active and latent TGF-beta were completely inactivated by raising the temperature in the presence of the redox agents. The mechanism of TGF-beta inactivation involved the generation of biologically inactive growth factor monomer and required the presence of free thiol groups, since thiol blockers protected TGF-beta from reduction. We conclude that non-enzymatic redox reactions may be involved in the regulation of extracellular TGF-beta activity. This might be of particular relevance in wound repair (e.g. in burns), as a mechanism protecting from excess TGF-beta activity, as well as in conditions involving redox dysregulation, such as reperfusion injury of the heart, Alzheimer's disease, and cancer.

CEACAM1 expression in cutaneous malignant melanoma predicts the development of metastatic disease.

PURPOSE: The cell adhesion molecule CEACAM1 is involved in intercellular adhesion and subsequent signal transduction events in a number of epithelia. CEACAM1 downregulation has been demonstrated in colorectal and prostate carcinomas. This study sought to analyze whether its expression in malignant melanoma is associated with metastasis. PATIENTS AND METHODS: CEACAM1 expression was immunohistochemically evaluated in 100 primary cutaneous malignant melanomas and correlated with metastasis in a 10-year follow-up. Furthermore, CEACAM1 expression was analyzed in metastatic lesions (11 distant metastases and six sentinel lymph node metastases). Univariate Kaplan-Meier analysis and multivariate Cox proportional hazard regression analysis adjusted for standard prognostic indicators were performed to assess the prognostic relevance of CEACAM1 expression. RESULTS: A total of 28 of 40 patients with CEACAM1-positive primary melanomas developed metastatic disease, compared with only six of 60 patients with CEACAM1-negative melanomas. Often, the strongest CEACAM1 expression was observed at the invading front. In addition, CEACAM1 expression was preserved in the metastatic lesions. Kaplan-Meier analysis revealed a highly significant association between CEACAM1 expression and metastasis (P <.0001); multivariate Cox regression analysis, including CEACAM1 expression status adjusted for tumor thickness, presence of ulceration, and mitotic rate, confirmed that CEACAM1 is an independent factor for the risk of metastasis and demonstrated that the predictive value of CEACAM1 expression is superior to that of tumor thickness. CONCLUSION: Expression of the cell adhesion molecule CEACAM1 in the primary tumors in melanoma patients is associated with the subsequent development of metastatic disease. This raises the possibility of a functional role for this cell adhesion molecule in the metastatic spread it indicates.

Cyclic mechanical strain induces TGFß1-signalling in dermal fibroblasts embedded in a 3D collagen lattice.

Many tissues are constantly exposed to mechanical stress, e.g. shear stress in vascular endothelium, compression forces in cartilage or tensile strain in the skin. Dermal fibroblasts can differentiate into contractile myofibroblasts in a process requiring the presence of TGFß1 in addition to mechanical load. We aimed at investigating the effect of cyclic mechanical strain on dermal fibroblasts grown in a three-dimensional environment. Therefore, murine dermal fibroblasts were cultured in collagen gels and subjected to cyclic tension at a frequency of 0.1 Hz (6 cycles/min) with a maximal increase in surface area of 10 % for 24 h. This treatment resulted in a significant increase in active TGFß1 levels, leaving the amount of total TGFß1 unaffected. TGFß1 activation led to pSMAD2-mediated transcriptional elevation of downstream mediators, such as CTGF, and an auto-induction of TGFß1, respectively.

Rhodocetin antagonizes stromal tumor invasion in vitro and other alpha2beta1 integrin-mediated cell functions.

The pleiotropic effects of Calloselasma rhodostoma venom is caused by various toxins, among them kistrin and ancrod, which block platelet activation triggered by RGD-dependent integrins and the blood clotting cascade, respectively. Here, we demonstrate that rhodocetin, another component of this venom, acts as alpha2beta1 integrin inhibiting disintegrin and antagonizes important cellular responses to type I collagen. Cell adhesion, migration, and collagen lattice contraction in vitro were specifically inhibited by rhodocetin, whereas expression of collagen-degrading matrix metalloproteases was differently modulated. Moreover, cell invasion of HT1080 fibrosarcoma cells into a type I collagen matrix, but not into a fibrin gel or a basement membrane-extracted matrigel was efficiently blocked by rhodocetin. Unlike its natural ligand collagen, rhodocetin failed to cluster alpha2beta1 integrin, despite similar binding affinities. Hence, in the absence of focal adhesions cells do not attach firmly to rhodocetin and do not respond with any of alpha2beta1-triggered cell reactions, except for MMP-1 production. Therefore, this disintegrin may be a valuable tool to specifically target stromal tumor invasion and to manipulate other alpha2beta1 integrin-mediated functions, such as excessive scar contraction and fibrosis. Rhodocetin might be therapeutically useful because of its lack of interference with RGD-dependent integrins, low molecular mass, high solubility, and biochemical stability.