Tumour-derived exosomes (TEX) are a subset of extracellular vesicles (EVs) present in body fluids of patients with cancer. The role of this exosome subset in melanoma progression has been of interest ever since ex vivo studies of exosomes produced by melanoma cell lines were shown to suppress anti-melanoma immune responses. To study the impact of melanoma-derived exosomes (MTEX) present in patients' plasma on melanoma progression, isolation of MTEX from total plasma exosomes is necessary. We have developed an immunoaffinity-based method for MTEX capture from plasma of melanoma patients. Using mAb 763.74 specific for the CSPG4 epitope uniquely expressed on melanoma cells, we separated MTEX from non-tumour cell-derived exosomes and evaluated the protein cargo of both fractions by quantitative flow cytometry. Melanoma-associated antigens were carried by MTEX but were not detectable in exosomes produced by normal cells. Separation of plasma-derived MTEX from non-MTEX provides an opportunity for future evaluation of MTEX as potential biomarkers of melanoma progression and as surrogates of melanoma in tumour liquid biopsy studies.

To evaluate the feasibility of an innovative peer intervention promoting healthy eating and physical activity, which purposefully selected peer facilitators according to socioeconomic status to target less-advantaged overweight receivers.

In vitro expansion of late endothelial progenitor cells (EPCs) might yield a cell therapy product useful for myocardial and leg ischaemia, but the influence of EPC expansion on the angiogenic properties of these cells is unknown. In the present study, we investigated the effect of in vitro EPC expansion on vascular endothelial growth factor (VEGF) receptor expression. EPCs were obtained from CD34(+) cord blood cells and expanded for up to 5 weeks. Real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that VEGFR2 expression, contrary to VEGFR1 and VEGFR3 expression, was significantly higher on expanded EPCs than on freshly isolated CD34(+) cells or on human umbilical vein endothelial cells (HUVECs). Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs. The impact of VEGFR2 increase was studied on the three theoretical steps of angiogenesis, i.e., EPC proliferation, migration and differentiation. VEGFR2 up-regulation had no effect on VEGF-induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin alpha(6) subunit overexpression. In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC-based angiogenic therapy.

Front-of-Package nutrition labels (FoPLs) are intended to help reduce the incidence of nutrition-related non-communicable diseases through an improvement in diet quality. FoPLs have been shown to improve the nutritional quality of purchases and have been associated with improved diet quality, which is in turn associated with reduced risk of non-communicable diseases. However, the potential impact of FoPLs on reducing mortality from chronic diseases has never been estimated.

Fluid challenge (FC) is one of the most common practices in Intensive Care Unit (ICU). The present study aimed to evaluate whether echocardiographic assessment of the response to FC at the end of the infusion or 20 min later could affect the results of the FC.

Zenker carcinoma is still being treated empirically because of the lack of evidence- based guidelines. We report for the first time about the genetic examination of this rare entity. The revealed mutations show genetic similarities with HPV(-)HNSCC which suggests that well-known therapeutic strategies may be applicable for this disease.

Exosomes are nanovesicles involved in intercellular communications. They are released by a variety of cell types; however, their presence in the inner ear has not been described in the literature. The aims of this study were to determine if exosomes are present in the inner ear and, if present, characterize the changes in their protein content in response to ototoxic stress. In this laboratory investigation, inner ear explants of 5-day-old Wistar rats were cultured and treated with either cisplatin or gentamicin. Hair cell damage was assessed by confocal microscopy. Exosomes were isolated using ExoQuick, serial centrifugation, and mini-column methods. Confirmation and characterization of exosomes was carried out using transmission electron microscopy (TEM), ZetaView, BCA protein analysis, and proteomics. Vesicles with a typical size distribution for exosomes were observed using TEM and ZetaView. Proteomic analysis detected typical exosome markers and markers for the organ of Corti. There was a statistically significant reduction in the exosome protein level and number of particles per cubic centimeter when the samples were exposed to ototoxic stress. Proteomic analysis also detected clear differences in protein expression when ototoxic medications were introduced. Significant changes in the proteomes of the exosomes were previously described in the context of hearing loss and ototoxic treatment. This is the first report describing exosomes derived from the inner ear. These findings may present an opportunity to conduct further studies with the hope of using exosomes as a biomarker to monitor inner ear function in the future.

In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling.

Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI.

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a-d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), beta-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and beta-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and beta-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B(2) receptor, which transiently interacts with beta-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/beta-arrestin complex, freeing internalized receptors from beta-arrestins and promoting recycling and resensitization.

Exosomes are nanovesicles part of a recently described intercellular communication system. Their properties seem promising as a biomarker in cancer research, where more sensitive monitoring and therapeutic applications are desperately needed. In the case of head and neck squamous cell carcinoma (HNSCC), overall survival often remains poor, although huge technological advancements in the treatment of this disease have been made. In the following review, diagnostic and therapeutic properties are highlighted and summarised. Impressive first results have been obtained but more research is needed to implement these innovative techniques into daily clinical routines.

Severe SARS-CoV-2 infections are characterized by lymphopenia, but the mechanisms involved are still elusive. Based on our knowledge of HIV pathophysiology, we hypothesized that SARS-CoV-2 infection-mediated lymphopenia could also be related to T cell apoptosis. By comparing intensive care unit (ICU) and non-ICU COVID-19 patients with age-matched healthy donors, we found a strong positive correlation between plasma levels of soluble FasL (sFasL) and T cell surface expression of Fas/CD95 with the propensity of T cells to die and CD4 T cell counts. Plasma levels of sFasL and T cell death are correlated with CXCL10 which is part of the signature of 4 biomarkers of disease severity (ROC, 0.98). We also found that members of the Bcl-2 family had modulated in the T cells of COVID-19 patients. More importantly, we demonstrated that the pan-caspase inhibitor, Q-VD, prevents T cell death by apoptosis and enhances Th1 transcripts. Altogether, our results are compatible with a model in which T-cell apoptosis accounts for T lymphopenia in individuals with severe COVID-19. Therefore, a strategy aimed at blocking caspase activation could be beneficial for preventing immunodeficiency in COVID-19 patients.

Angiopoietin-like 4 (ANGPTL4) is a target of hypoxia that accumulates in the endothelial extracellular matrix. While ANGPTL4 is known to regulate angiogenesis and vascular permeability, its context-dependent role related to vascular endothelial growth factor (VEGF) has been suggested in capillary morphogenesis. We here thus develop in vitro 3D models coupled to imaging and morphometric analysis of capillaries to decipher ANGPTL4 functions either alone or in the presence of VEGF. ANGPTL4 induces the formation of barely branched and thin endothelial capillaries that display linear adherens junctions. However, ANGPTL4 counteracts VEGF-induced formation of abundant ramified capillaries presenting cell-cell junctions characterized by VE-cadherin containing reticular plaques and serrated structures. We further deciphered the early angiogenesis steps regulated by ANGPTL4. During the initial activation of endothelial cells, ANGPTL4 alone induces cell shape changes but limits the VEGF-induced cell elongation and unjamming. In the growing sprout, ANGPTL4 maintains cohesive VE-cadherin pattern and sustains moderate 3D cell migration but restricts VEGF-induced endothelium remodeling and cell migration. This effect is mediated by differential short- and long-term regulation of P-Y1175-VEGFR2 and ERK1-2 signaling by ANGPTL4. Our in vitro 3D models thus provide the first evidence that ANGPTL4 induces a specific capillary morphogenesis but also overcomes VEGF effect.

The members of the lysyl oxidase (LOX) family are amine oxidases, which initiate the covalent cross-linking of the extracellular matrix (ECM), regulate ECM stiffness, and contribute to cancer progression. The aim of this study was to build the first draft of the interactome of the five members of the LOX family in order to determine its molecular functions, the biological and signaling pathways mediating these functions, the biological processes it is involved in, and if and how it is rewired in cancer. In vitro binding assays, based on surface plasmon resonance and bio-layer interferometry, combined with queries of interaction databases and interaction datasets, were used to retrieve interaction data. The interactome was then analyzed using computational tools. We identified 31 new interactions and 14 new partners of LOXL2, including the α5β1 integrin, and built an interactome comprising 320 proteins, 5 glycosaminoglycans, and 399 interactions. This network participates in ECM organization, degradation and cross-linking, cell-ECM interactions mediated by non-integrin and integrin receptors, protein folding and chaperone activity, organ and blood vessel development, cellular response to stress, and signal transduction. We showed that this network is rewired in colorectal carcinoma, leading to a switch from ECM organization to protein folding and chaperone activity.

While aminoglycosides (AG) have been used for decades, debate remains on their optimal dosing strategy. We investigated the international practices of AG usage specifically regarding dosing and therapeutic drug monitoring (TDM) in critically ill patients. We conducted a prospective, multicentre, observational, cohort study in 59 intensive-care units (ICUs) in 5 countries enrolling all ICU patients receiving AG therapy for septic shock.

Although most physicians in medical settings have to deliver bad news, the skills of delivering bad news to patients have been given insufficient attention. Delivering bad news is a complex communication task that includes verbal and nonverbal skills, the ability to recognize and respond to patients' emotions and the importance of considering the patient's environment such as culture and social status. How bad news is delivered can have consequences that may affect patients, sometimes over the long term.

Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND) plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab). This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100-1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK) cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future.

Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives. The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair.

Lysyl oxidases are major actors of microenvironment and extracellular matrix (ECM) remodeling. These cross-linking enzymes are thus involved in many aspects of physiopathology, including tumor progression, fibrosis and cardiovascular diseases. We have already shown that Lysyl Oxidase-Like 2 (LOXL2) regulates collagen IV deposition by endothelial cells and angiogenesis. We here provide evidence that LOXL2 also affects deposition of other ECM components, including fibronectin, thus altering structural and mechanical properties of the matrix generated by endothelial cells. LOXL2 interacts intracellularly and directly with collagen IV and fibronectin before incorporation into ECM fibrillar structures upon exocytosis, as demonstrated by TIRF time-lapse microscopy. Furthermore, surface plasmon resonance experiments using recombinant scavenger receptor cysteine-rich (SRCR) domains truncated for the catalytic domain demonstrated their direct binding to collagen IV. We thus used directed mutagenesis to investigate the role of LOXL2 catalytic domain. Neither enzyme activity nor catalytic domain were necessary for collagen IV deposition and angiogenesis, whereas the SRCR domains were effective for these processes. Finally, surface coating with recombinant SRCR domains restored deposition of collagen IV by LOXL2-depleted cells. We thus propose that LOXL2 SRCR domains orchestrate scaffolding of the vascular basement membrane and angiogenesis through interactions with collagen IV and fibronectin, independently of the enzymatic cross-linking activity.

The understanding of endothelium-extracellular matrix interactions during the initiation of new blood vessels is of great medical importance; however, the mechanobiological principles governing endothelial protrusive behaviours in 3D microtopographies remain imperfectly understood. In blood capillaries submitted to angiogenic factors (such as vascular endothelial growth factor, VEGF), endothelial cells can transiently transdifferentiate in filopodia-rich cells, named tip cells, from which angiogenesis processes are locally initiated. This protrusive state based on filopodia dynamics contrasts with the lamellipodia-based endothelial cell migration on 2D substrates. Using two-photon polymerization, we generated 3D microstructures triggering endothelial phenotypes evocative of tip cell behaviour. Hexagonal lattices on pillars ("open"), but not "closed" hexagonal lattices, induced engagement from the endothelial monolayer with the generation of numerous filopodia. The development of image analysis tools for filopodia tracking allowed to probe the influence of the microtopography (pore size, regular vs. elongated structures, role of the pillars) on orientations, engagement and filopodia dynamics, and to identify MLCK (myosin light-chain kinase) as a key player for filopodia-based protrusive mode. Importantly, these events occurred independently of VEGF treatment, suggesting that the observed phenotype was induced through microtopography. These microstructures are proposed as a model research tool for understanding endothelial cell behaviour in 3D fibrillary networks.