Background Despite recent progress, the prognosis of patients with transthyretin (TTR) cardiac amyloidosis remains poor; this is primarily due to late diagnosis, when irreversible damage has already occurred. Today’s diagnostic work-up still relies on peripheral tissue or a cardiac biopsy, while circulating levels of TTR or other plasma markers have little diagnostic value. Although extracellular vesicles (EVs, as key mediators of intercellular communication) may reflect disease-specific molecular changes, their protein cargo has not yet been explored in the context of TTR amyloidosis (ATTR) cardiomyopathy. Objectives To characterize the plasma EV proteome in ATTR cardiomyopathy and identify potential biomarkers for pathophysiological pathways, diagnosis, or prognosis. Methods We performed mass-spectrometry-based, label-free, proteomic profiling of plasma EVs from 65 patients with hypertrophic cardiomyopathy due to TTR amyloidosis (the ATTR+ group, n=41) or non-amyloid cardiac disease (the ATTR- group, n=24). The groups were matched by age and sex. Results A distinct protein signature comprising 117 deregulated proteins was identified in EVs from ATTR+ patients. The ATTR+ EVs were enriched in proteins associated with vascular homeostasis, coagulation, and inflammation. At least 18 of these proteins formed an interconnected network centered on plasmin/plasminogen. Notably, EV levels of TTR and plasminogen levels were elevated, while the level of alpha2-antiplasmin (plasmin’s primary inhibitor) was low. This imbalance is particularly relevant because plasmin is known to promote amyloidogenesis via TTR cleavage. Conclusions Our findings provide new insights into the molecular mechanisms underlying ATTR cardiomyopathy and suggest that plasma EV proteins are potential diagnostic or prognostic biomarkers and/or therapeutic targets. CONDENSED ABSTRACT TTR amyloidosis (ATTR) causes severe cardiac damage, which is often diagnosed late. Through a comparative proteomic analysis of plasma extracellular vesicles (EVs) in patients with ATTR cardiomyopathy vs. patients with other cardiomyopathies, we identified several proteins of relevance to the pathophysiology of ATTR. Our analysis is the first to have highlighted an enrichment of plasmin/plasminogen (known to initiate the amyloidogenic process) and TTR in circulating EVs. Our results might foster the development of (i) diagnostic and prognostic markers for ATTR cardiomyopathy that do not require invasive procedures, and (ii) new therapeutic strategies. ![Figure][1] ETHICAL APPROVAL The present analysis was based on blood samples collected as part of a research project entitled "Study of the myocardial microenvironment and toxicity of amyloid proteins in patients with cardiac amyloidosis", which was approved by an institutional review board (CPP Sud- Méditerranée II, Marseille, France; approval references: 2021T2-12/2021-A00950-41 and 2022-A02416-37). ### Competing Interest Statement The authors have declared no competing interest. [1]: pending:yes

The fibrotic stroma characterizing pancreatic ductal adenocarcinoma (PDAC) derives from a progressive tissue rigidification, which induces epithelial mesenchymal transition and metastatic dissemination. The aim of this study was to investigate the influence of matrix stiffness on PDAC progression by analyzing the proteome of PDAC-derived extracellular vesicles (EVs). PDAC cell lines (mPDAC and KPC) were grown on synthetic supports with a stiffness close to non-tumor (NT) or tumor tissue (T), and the protein expression levels in cell-derived EVs were analyzed by a quantitative MS E label-free mass spectrometry approach. Our analysis figured out 15 differentially expressed proteins (DEPs) in mPDAC-EVs and 20 DEPs in KPC-EVs in response to matrix rigidification. Up-regulated proteins participate to the processes of metabolism, matrix remodeling, and immune response, altogether hallmarks of PDAC progression. A multimodal network analysis revealed that the majority of DEPs are strongly related to pancreatic cancer. Interestingly, among DEPs, 11 related genes ( ACTB/ANXA7/C3/IGSF8/LAMC1/LGALS3/PCD6IP/SFN/TPM3/VARS/YWHAZ ) for mPDAC-EVs and 9 ( ACTB/ALDH2/GAPDH/HNRNPA2B/ITGA2/NEXN/PKM/RPN1/S100A6 ) for KPC-EVs were significantly overexpressed in tumor tissues according to gene expression profiling interaction analysis (GEPIA). Concerning the potential clinical relevance of these data, the cluster of ACTB, ITGA2, GAPDH and PKM genes displayed an adverse effect ( p  < 0.05) on the overall survival of PDAC patients.

Conventional chemotherapy used against cancer is mostly limited due to their non-targeted nature, affecting normal tissue and causing undesirable toxic effects to the affected tissue. With the aim of improving these treatments both therapeutically and in terms of their safety, numerous studies are currently being carried out using nanoparticles (NPs) as a vector combining tumor targeting and carrying therapeutic tools. In this context, it appears that nucleolin, a molecule over-expressed on the surface of tumor cells, is an interesting therapeutic target. Several ligands, antagonists of nucleolin of various origins, such as AS1411, the F3 peptide and the multivalent pseudopeptide N6L have been developed and studied as therapeutic tools against cancer. Over the last ten years or so, numerous studies have been published demonstrating that these antagonists can be used as tumor targeting agents with NPs from various origins. Focusing on nucleolin ligands, the aim of this article is to review the literature recently published or under experimentation in our research team to evaluate the efficacy and future development of these tools as anti-tumor agents.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with β-catenin. We found that the Wnt/β-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing β-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/β-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased β-catenin stabilization. In conclusion, in this study, we identified β-catenin as a new nucleolin interactor and suggest that the Wnt/β-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.

Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.

In this study, a novel anticancer reagent based on polyplexes nanoparticles was developed. These nanoparticles are obtained by mixing negatively charged polyelectrolytes with the antitumour cationically-charged pseudopeptide N6L. Using two in vivo experimental tumor pancreatic models based upon PANC-1 and mPDAC cells, we found that the antitumour activity of N6L is significantly raised via its incorporation in polyplexed nanoparticles. Study of the mechanism of action using affinity isolation and si-RNA experiments indicated that N6L-polyplexes are internalized through their interaction with nucleolin. In addition, using a very aggressive model of pancreatic cancer in which gemcitabine, a standard of care for this type of cancer, has a weak effect on tumour growth, we observed that N6L-polyplexes administration has a stronger efficacy than gemcitabine. Biodistribution studies carried out in tumour-bearing mice indicated that N6L-polyplexes localises in tumour tissue, in agreement with its antitumour effect. These results support the idea that N6L nanoparticles could develop into a promising strategy for the treatment of cancer, especially hard-to-treat pancreatic cancers.

ABSTRACT Claudin-16 protein (CLDN16) is a component of tight junctions (TJ) with a restrictive distribution so far demonstrated mainly in the kidney. Here, we demonstrate the expression of CLDN16 also in the tooth germ and show that claudin-16 gene (CLDN16) mutations result in amelogenesis imperfecta (AI) in the 5 studied patients with familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). To investigate the role of CLDN16 in tooth formation, we studied a murine model of FHHNC and showed that CLDN16 deficiency led to altered secretory ameloblast TJ structure, lowering of extracellular pH in the forming enamel matrix, and abnormal enamel matrix protein processing, resulting in an enamel phenotype closely resembling human AI. This study unravels an association of FHHNC owing to CLDN16 mutations with AI, which is directly related to the loss of function of CLDN16 during amelogenesis. Overall, this study indicates for the first time the importance of a TJ protein in tooth formation and underlines the need to establish a specific dental follow-up for these patients. © 2015 American Society for Bone and Mineral Research.

Background: Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.Methods: EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.Results: The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.Conclusion and general significance: The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties. (C) 2014 Elsevier B.V. All rights reserved.

Tooth development is regulated by a series of reciprocal inductive signaling between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissue formation. We report a delay in enamel deposition and formation that is clearly distinguishable in the growing incisor and associated with a significant reduction of MMP-3 and MMP-20 expression in tooth germs of KO mice. Insufficient basement membrane degradation is evidenced by a persistent laminin immunostaining, resulting in a delay of both odontoblast and ameloblast differentiation. Consequently, enamel volume and thickness are decreased in adult mutant teeth but enamel maturation and tooth morphology are normal, as shown by micro-computed tomographic (micro-CT), nanoindentation, and scanning electron microscope analyses. In addition, the dentino-enamel junction appears as a rough calcified layer of approximately 10 +/- 5 mu m thick (mean +/- SD) in both molars and growing incisors of KO adult mice. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal crosstalk during tooth development by regulating the expression of MMPs. The mild tooth phenotype observed in EMMPRIN KO mice suggests that the direct effect of EMMPRIN may be limited to a short time window, comprised between basement membrane degradation allowing direct cell contact and calcified matrix deposition. (C) 2014 Elsevier Inc. All rights reserved.

Dry eye is a common disease that develops as a result of alteration of tear fluid, leading to osmotic stress and a perturbed epithelial barrier. Matrix metalloproteinase-9 (MMP-9) may be important in dry eye disease, as its genetic knockout conferred resistance to the epithelial disruption. We show that extracellular matrix metalloproteinase inducer (EMMPRIN; also termed CD147), an inducer of MMP expression, participates in the pathogenesis of dry eye through MMP-mediated cleavage of occludin, an important component of tight junctions. EMMPRIN expression was increased on the ocular surface of dry eye patients and correlated with those of MMP-9. High osmolarity in cell culture, mimicking dry eye conditions, increased both EMMPRIN and MMP-9 and resulted in the disruption of epithelial junctions through the cleavage of occludin. Exogenously added recombinant EMMPRIN had similar effects that were abrogated in the presence of the MMP inhibitor marimastat. Membrane occludin immunostaining was markedly increased in the apical corneal epithelium of both EMMPRIN and MMP-9 knock-out mice. Furthermore, an inverse correlation between EMMPRIN and occludin membrane staining was consistently observed both in vitro and in vivo as a function of corneal epithelial cells differentiation. These data suggest a possible role of EMMPRIN in regulating the amount of occludin at the cell surface in homeostasis beyond pathological situations such as dry eye disease, and EMMPRIN may be essential for the formation and maintenance of organized epithelial structure. (Ant J Pathol 2011, 179:1278-1286. DOI: 10.1016/j.ajpath.2011.05.036)

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr(-/-) lung when compared to wild-type (Cftr(+/+)) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr(-/-) lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr(-/-) mice compared to Cftr(+/+). Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr(+/+) lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr(-/-) lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr(-/-) lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.

INTRODUCTION:Snail, a family of transcriptional repressors implicated in cell movement, has been correlated with tumour invasion. The Plasminogen Activation (PA) system, including urokinase plasminogen activator (uPA), its receptor and its inhibitor, plasminogen activator inhibitor type 1(PAI-1), also plays a key role in cancer invasion and metastasis, either through proteolytic degradation or by non-proteolytic modulation of cell adhesion and migration. Thus, Snail and the PA system are both over-expressed in cancer and influence this process. In this study we aimed to determine if the activity of SNAI1 (a member of the Snail family) is correlated with expression of the PA system components and how this correlation can influence tumoural cell migration.METHODS:We compared the invasive breast cancer cell-line MDA-MB-231 expressing SNAI1 (MDA-mock) with its derived clone expressing a dominant-negative form of SNAI1 (SNAI1-DN). Expression of PA system mRNAs was analysed by cDNA microarrays and real-time quantitative RT-PCR. Wound healing assays were used to determine cell migration. PAI-1 distribution was assessed by immunostaining.RESULTS:We demonstrated by both cDNA microarrays and real-time quantitative RT-PCR that the functional blockade of SNAI1 induces a significant decrease of PAI-1 and uPA transcripts. After performing an in vitro wound-healing assay, we observed that SNAI1-DN cells migrate more slowly than MDA-mock cells and in a more collective manner. The blockade of SNAI1 activity resulted in the redistribution of PAI-1 in SNAI1-DN cells decorating large lamellipodia, which are commonly found structures in these cells.CONCLUSIONS:In the absence of functional SNAI1, the expression of PAI-1 transcripts is decreased, although the protein is redistributed at the leading edge of migrating cells in a manner comparable with that seen in normal epithelial cells.

Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents.

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein enriched on tumor cells and normal epithelia. It is mainly known for its ability to induce matrix metalloproteinase production in fibroblasts following epithelial-stromal interaction. We sought to examine whether EMMPRIN has a broader role promoting fibroblast-to-myofibroblast differentiation. Because alpha-smooth muscle actin (alphaSMA) is considered a marker of this differentiation process, we analyzed the effect of EMMPRIN on its expression in corneal and skin fibroblasts by Western blots, immunocytochemistry, and a functional assay of collagen lattice contraction. Increasing EMMPRIN expression by cDNA transfection or by treatment with exogenously added recombinant EMMPRIN resulted in an up-regulation of alphaSMA expression. EMMPRIN also increased the contractile properties of the treated fibroblasts as demonstrated by the immunohistochemical appearance of stress fibers and by the accelerated contraction of fibroblast-embedded collagen lattices. Blocking EMMPRIN expression by small interfering RNA inhibited alphaSMA and collagen gel contraction induced not only by EMMPRIN but also by transforming growth factor-beta, a major mediator of myofibroblast differentiation that also regulated EMMPRIN expression. These findings, combined with the fact that EMMPRIN and alphaSMA colocalized to the same cells in the stroma of pathological corneas, expand on the mechanism by which EMMPRIN remodels extracellular matrix during wound healing and cancer.

Cancer is a complex and dynamic process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process, we need to study each one of the levels involved, which allows the scale change, and to integrate this knowledge. A matricellular protein, PAI-1, is able to induce in vitro cell behaviour modifications, morphological changes, and to promote cell migration. PAI-1 influences the mesenchymo-amaeboid transition. This matricellular protein should be considered as a potential 'launcher' of the metastatic process acting at the molecular, cellular, tissular levels and, as a consequence, at the organism's level.

Le processus cancéreux est une dynamique complexe, dont l'intégration de tous les paramètres nécessite de mieux comprendre les événements observés aux différentes échelles et d'en modéliser les interactions. La modification d'assemblages moléculaires « matricellulaires » peut déclencher le processus métastatique. In vitro, un microenvironnement cellulaire, très enrichi en PAI-1 matriciel, entraîne une modification du comportement des cellules qui adoptent le mode de migration amiboïde et présentent toutes les caractéristiques morphologiques de l'état post-transition mésenchymateuse-amiboïde et ses conséquences. PAI-1 apparaît donc comme une protéine matricellulaire capable de déclencher le processus à l'échelle moléculaire, cellulaire, et tissulaire, susceptible d'engager un pronostic concernant l'organisme entier. Pour citer cet article : M. Malo et al., C. R. Biologies 329 (2006).