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Endothelial cell interactions with their extracellular matrix are essential for vascular homeostasis and expansion. Large-scale proteomic analyses aimed at identifying components of integrin adhesion complexes have revealed the presence of several RNA Binding Proteins (RBPs) of which the functions at these sites remain poorly understood. Here, we explored the role of the RBP SAM68 (Src associated in mitosis, of 68 kDa) in endothelial cells. We found that SAM68 is transiently localized at the edge of spreading cells where it participates in membrane protrusive activity and the conversion of nascent adhesions to mechanically-loaded focal adhesions by modulation of integrin signaling and local delivery of β-actin mRNA. Furthermore, SAM68 depletion impacts cell-matrix interactions and motility through induction of key matrix genes involved in vascular matrix assembly. In a 3D environment SAM68-dependent functions in both tip and stalk cells contribute to the process of sprouting angiogenesis. Altogether, our results identify the RBP SAM68 as a novel actor in the dynamic regulation of blood vessel networks.

Cellular fibronectin (FN; also known as FN1) variants harboring one or two alternatively spliced so-called extra domains (EDB and EDA) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the extra domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both extra domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, extra domain-containing variants differentially regulate pH homeostasis, survival and TGF-β signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes. This article has an associated First Person interview with the first author of the paper.

International audience; High resolution imaging of molecules at the cell-substrate interface is required for understanding key biological processes. Here we propose a complete pipeline for multi-angle total internal reflection fluorescence microscopy (MA-TIRF) going from instrument design and calibration procedures to numerical reconstruction. Our custom setup is endowed with a homogeneous field illumination and precise excitation beam angle. Given a set of MA-TIRF acquisitions, we deploy an efficient joint deconvolution/reconstruction algorithm based on a variational formulation of the inverse problem. This algorithm offers the possibility of using various regularizations and can run on graphics processing unit (GPU) for rapid reconstruction. Moreover, it can be easily used with other MA-TIRF devices and we provide it as an open-source software. This ensemble has enabled us to visualize and measure with unprecedented nanometric resolution, the depth of molecular components of the fibronectin assembly machinery at the basal surface of endothelial cells.

International audience; The role of the extracellular matrix (ECM) in the evolution of certain diseases (e.g. fibrosis, cancer) is generally accepted but yet to be completely understood. A numerical model that captures the physical properties of the ECM, could convey certain connections between the topology of its constituents and their associated biological features. This study addresses the analysis and modeling of fibrillar networks containing Fibronectin (FN) networks, a major ECM molecule, from 2D confocal microscopy images. We leveraged the advantages of the fast discrete curvelet transform (FDCT), in order to obtain a multiscale and multidirectional representation of the FN fibrillar networks. This step was validated by performing a classification among the different variants of FN upregulated in disease states with a multi-class classification algorithm, DAG-SVM. Subsequently, we designed a method to ensure the invariance to rotation of the curvelet features. Our results indicate that the curvelets offer an appropriate discriminative model for the FN networks, that is able to characterize the local fiber geometry.

Functional interplay between tumour cells and their neoplastic extracellular matrix plays a decisive role in malignant progression of carcinomas. Here we provide a comprehensive data set of the human HNSCC-associated fibroblast matrisome. Although much attention has been paid to the deposit of collagen, we identify oncofetal fibronectin (FN) as a major and obligate component of the matrix assembled by stromal fibroblasts from head and neck squamous cell carcinomas (HNSCC). FN overexpression in tumours from 435 patients corresponds to an independent unfavourable prognostic indicator. We show that migration of carcinoma collectives on fibrillar FN-rich matrices is achieved through αvβ6 and α9β1 engagement, rather than α5β1. Moreover, αvβ6-driven migration occurs independently of latent TGF-β activation and Smad-dependent signalling in tumour epithelial cells. These results provide insights into the adhesion-dependent events at the tumour–stroma interface that govern the collective mode of migration adopted by carcinoma cells to invade surrounding stroma in HNSCC., Tumour microenvironment influences the migration of cancer cells. Here the authors analyse the proteomic constitution of the extracellular matrix and identify a role for fibronectin in regulating the collective migration of squamous cell carcinoma cells.

International audience; This communication presents a pipeline for Multi-Angle TIRF calibration from the measurement of the incident angle to the model validation. This problem is of major importance when dealing with 3D reconstruction methods from a set of MA-TIRF acquisitions since the reconstruction accuracy highly depends on the agreement between the theoretical model and the physical system. One main issue is then to build phantom samples with known geometry, or known properties, in order to adjust and/or validate the model. This paper describes such a calibration procedure using a lens as phantom sample and proposes a new model validation experiment based on a dual-color co-localization.

High expression of the extracellular matrix component tenascin-C in the tumor microenvironment correlates with decreased patient survival. Tenascin-C promotes cancer progression and a disrupted tumor vasculature through an unclear mechanism. Here, we examine the angiomodulatory role of tenascin-C. We find that direct contact of endothelial cells with tenascin-C disrupts actin polymerization, resulting in cytoplasmic retention of the transcriptional coactivator YAP. Tenascin-C also downregulates YAP pro-angiogenic target genes, thus reducing endothelial cell survival, proliferation, and tubulogenesis. Glioblastoma cells exposed to tenascin-C secrete pro-angiogenic factors that promote endothelial cell survival and tubulogenesis. Proteomic analysis of their secretome reveals a signature, including ephrin-B2, that predicts decreased survival of glioma patients. We find that ephrin-B2 is an important pro-angiogenic tenascin-C effector. Thus, we demonstrate dual activities for tenascin-C in glioblastoma angiogenesis and uncover potential targeting and prediction opportunities.

Cell migration is a multistep process initiated by extracellular matrix components that leads to cytoskeletal changes and formation of different protrusive structures at the cell periphery. Lumican, a small extracellular matrix leucine-rich proteoglycan, has been shown to inhibit human melanoma cell migration by binding to α2β1 integrin and affecting actin cytoskeleton organization. The aim of this study was to determine the effect of lumican overexpression on the migration ability of human colon adenocarcinoma LS180 cells. The cells stably transfected with plasmid containing lumican cDNA were characterized by the increased chemotactic migration measured on Transwell filters. Lumican-overexpressing cells presented the elevated filamentous to monomeric actin ratio and gelsolin up-regulation. This was accompanied by a distinct cytoskeletal actin rearrangement and gelsolin subcellular relocation, as observed under laser scaning confocal microscope. Moreover, LS180 cells overexpressing lumican tend to form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin at the submembrane region of the cells. In conclusion, the elevated level of lumican secretion to extracellular space leads to actin cytoskeletal remodeling followed by an increase in migration capacity of human colon LS180 cells. These data suggest that lumican expression and its presence in ECM has an impact on colon cancer cells motility and may modulate invasiveness of colon cancer.

International audience; In recent years, special attention has been paid to finding new pro-angiogenic factors which could be used in gene therapy of vascular diseases such as critical limb ischaemia (CLI). Angiogenesis, the formation of new blood vessels, is a complex process dependent on different cytokines, matrix proteins, growth factors and other pro- or anti-angiogenic stimuli. Numerous lines of evidence suggest that key mediators of angiogenesis, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) together with fibroblast growth factor2 (FGF2) are involved in regulation of the normal and pathological process of angiogenesis. However, less information is available on the complex interactions between these and other angiogenic factors. The aim of this study was to characterise the effect of fibroblast growth factor2 on biological properties of human endothelial progenitor cells with respect to the expression level of other regulatory cytokines. Ectopic expression of FGF2 in EP cells stimulates their pro-angiogenic behaviour, leading to increased proliferation, migration and tube formation abilities. Moreover, we show that the expression profile of VEGF and other pro-angiogenic cytokines, such as HGF, MCP2, and interleukins, is affected differently by FGF2 in EPC. In conclusion, we provide evidence that FGF2 directly affects not only the biological properties of EP cells but also the expression pattern and secretion of numerous chemocytokines. Our results suggest that FGF2 could be applied in therapeutic approaches for CLI and other ischaemic diseases of the vascular system in vivo.

Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) with anti-tumor activity. We recently demonstrated that lumican inhibits the migration of melanoma cells and identified beta1 integrin as mediator of this effect [M.F. D'Onofrio, S. Brézillon, T. Baranek, C. Perreau, P.J. Roughley, F.X. Maquart, Y. Wegrowski, Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican, Biochem. Biophys. Res. Commun. 365 (2008) 266-272]. The aim of the present work was to study beta1 integrin, focal adhesion complexes, actin distribution and expression in the presence of lumican substratum in comparison to type I collagen or fibronectin substrata in A375 human melanoma cells. The protein distribution was investigated by immunocytochemistry and confocal microscopy. In parallel, their expression was evaluated by Western immunoblotting and Real-time Reverse Transcription-PCR analyses. The interaction of melanoma cells with the lumican substratum resulted in heterogeneous distribution of beta1 integrin on cell membrane after 24h of seeding. Concomitantly, a reorganization of actin stress fibers and a significant decrease in vinculin immunostaining at focal adhesion complexes were observed. No alteration of the expression was detected at protein and mRNA levels. However, a cytosolic accumulation of vinculin focal adhesion protein was observed on lumican substratum by confocal microscopy. Moreover, vinculin expression was significantly increased in cytosolic fractions in comparison to cells seeded on type I collagen or fibronectin substrata. Our results suggest that lumican induces an alteration of the link between actin filaments and beta1 integrin, characterized by a cytosolic accumulation of vinculin focal adhesion protein, which could lead to a destabilization of focal adhesion complexes. In addition, focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) was significantly decreased. Therefore, the cytoskeleton remodeling and the decreased pFAK phosphorylation induced by lumican in melanoma cells might explain, at least in part, the anti-invasive effect of this SLRP.

BACKGROUND Chronic limb ischemia is a serious clinical problem. Patients who do not qualify for standard treatment may benefit from novel gene therapies. OBJECTIVES This study evaluated angiogenesis following intramuscular injections of angiogenic plasmid Ang-1 in Fisher rats. MATERIAL AND METHODS Twenty rats had plasmids injected intramuscularly in their hind limbs. The study group consisted of 10 animals which received the Ang-1 plasmid, while the control group consisted of 10 rats that received an empty plasmid. All the animals were euthanized after 12 weeks and tissue samples from the hind limb thigh muscles and internal organs were harvested for histological and immunohistochemical examinations. To assess the angiogenesis the number of vessels in the hind limb muscles visualized by the SMA and FVIII markers was counted for each animal in five separate microscopic fields. RESULTS There were no pathological lesions or any signs of neoplastic angiogenesis in any of the 20 rats. The number of vessels visualized by the FVIII marker in the study group was two times higher than in the control group (median: 12, range: 7-25 vs. median: 6, range: 2-15; p < 0.0001). The median estimated that the number of vessels visualized by the SMA marker is 63% higher in the study group compared to the control group (median: 6.5, range: 1-12 vs. median: 4, range: 0-10; p = 0.0008). CONCLUSIONS Intramuscular injections of Ang-1 plasmids induced angiogenesis in the rat hind limb muscles.

In the case of melanoma, advances in therapies are slow, which raises the need to evaluate new therapeutic strategies and natural products with potential cancer cell inhibiting effect. Pseudofactin II (PFII), a novel cyclic lipopeptide biosurfactant has been isolated from the Arctic strain of Pseudomonas fluorescens BD5. The aim of this study was to investigate the effect of PFII on A375 melanoma cells compared with the effect of PFII on Normal Human Dermis Fibroblast (NHDF) cells and elucidate the underlying mechanism of PFII cytotoxic activity. Melanoma A375 cells and NHDF cells were exposed to PFII or staurosporine and apoptotic death was assessed by monitoring caspase 3-like activity and DNA fragmentation. From time-dependent monitoring of lactate dehydrogenase (LDH) release, Ca(2+) influx, and a correlation between Critical Micelle Concentration (CMC) we concluded that cell death is the consequence of plasma membrane permeabilisation by micelles. This finding suggests that pro-apoptotic mechanism of PFII is different from previously described cyclic lipopeptides. The mechanism of PFII specificity towards malignant cells remains to be discovered. The results of this study show that PFII could be a new promising anti-melanoma agent.

Aims Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line. Main methods Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic β- and γ-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms. Key findings A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican. Significance Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.

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Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) involved in the control of melanoma growth and invasion. The aim of the present study was to analyse the role of lumican in the regulation of the development of lung metastasis.B16F1 melanoma cells stably transfected with lumican expressing plasmid (Lum-B16F1) were injected to syngenic mice. The lung metastasis was compared to mice injected with mock-transfected B16F1 cells (Mock-B16F1). The expression of lumican, cyclin D1, apoptotic markers, vascular endothelium growth factor (VEGF) and Von Willebrand Factor (vWF) within lung metastasis nodules was investigated by immunohistochemistry. In parallel, cells cultured in presence of lumican were assayed for apoptosis and motility.We observed that the number and the size of lung metastasis nodules were significantly decreased in mice injected with Lum-B16F1 cells in comparison to Mock-B16F1 cells. This was associated with an increase of tumour cell apoptosis within metastasis nodules but the cell proliferation rate remained constant in the two mice groups. In contrast, the VEGF immunostaining and the number of blood vessels within the lung metastasis nodules were decreased in the lumican-expressing tumours. In vitro, a significant decrease of apoptotic markers in wild type B16F1 cells incubated with increasing amounts of lumican core protein was observed. In addition, pseudotubes formation on Matrigel(R) and the migratory capacity of endothelial cells was inhibited by lumican. Altogether, our results indicate that lumican decreases lung metastasis development not only by inducing tumour cell apoptosis but also by inhibiting angiogenesis.

The dynamic reorganization of the actin cytoskeleton is regulated by a number of actin binding proteins (ABPs). Four human colon adenocarcinoma cell lines – parental and three selected sublines, which differ in motility and metastatic potential, were used to investigate the expression level and subcellular localization of selected ABPs. Our interest was focused on cofilin and ezrin. These proteins are essential for cell migration and adhesion. The data received for the three more motile adenocarcinoma sublines (EB3, 3LNLN, 5W) were compared with those obtained for the parental LS180 adenocarcinoma cells and fibroblastic NRK cells. Quantitative densitometric analysis and confocal fluorescence microscopy were used to examine the expression levels and subcellular distribution of the selected ABPs. Our data show distinct increase in the level of cofilin in adenocarcinoma cells accompanied by the reduction of inactive phosphorylated form of cofilin. In more motile cells, cofilin was accumulated at cellular periphery in co-localization with actin filaments. Furthemore, we indicated translocation of ezrin towards the cell periphery within more motile cells in comparison with NRK and parental adenocarcinoma cells. In summary, our data indicate the correlation between migration ability of selected human colon adenocarcinoma sublines and subcellular distribution as well as the level of cofilin and ezrin. Therefore these proteins might be essential for the higher migratory activity of invasive tumor cells.