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Supplementary Table 1. Tumorigenic potential of cells sorted from normoxic and hypoxic spheres of the six xenograft models (take rate in percentage)

Figure S1. In the 6 models xenografted with human RCC, we identified 2 models responders to sunitinib and 4 non-responders. When treated with sunitinib, necrosis occurs earlier in the responder models

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supplementary methods

Purpose: Developing strategies to overcome resistance to sunitinib is a major challenge in human renal cell carcinoma (RCC). We hypothesized that sunitinib-induced tumor necrosis–associated hypoxia could interact with renal cancer stem cells in patients with metastatic RCC.Experimental Design: We studied tissue samples from 7 patients with primary metastatic RCC, before and after sunitinib treatment, and from six xenograft models derived from human RCC. Two xenograft models were responders to sunitinib, the four others were nonresponders. CD133/CXCR4–coexpressing cells derived from the two responder xenograft models were used for in vitro studies.Results: In the seven primary RCCs, we identified a significantly larger number of CD133/CXCR4–coexpressing cells in perinecrotic versus perivascular areas. Their numbers also significantly increased after treatment, in perinecrotic areas. We reproduced these clinical and pathologic results in all six RCC xenograft models with again a preferential perinecrotic distribution of CD133-expressing cells. Necrosis occurred at day 7 in the two responder models treated with sunitinib, whereas it occurred at day 21 in the untreated controls and in the four nonresponder models. Strikingly, when we studied the six RCC xenograft models at the time necrosis, whether spontaneous or sunitinib-induced, occurred, necrosis area correlated with stem-cell number in all 120 xenografted RCCs. When studied under experimental hypoxia, the number of CD133/CXCR4–coexpressing cells and their tumorigenic potency increased whereas their sensitivity to sunitinib decreased.Conclusions: In human RCC, sunitinib was able to generate resistance to its own therapeutic effect via induced hypoxia in perinecrotic areas where cancer stem cells were found in increased numbers. Clin Cancer Res; 21(4); 916–24. ©2014 AACR.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization, and fibroblasts isolated from these compartments have different gene expression patterns and behaviour in vitro. However, due to lack of relevant models, the contribution of skin fibroblast sub-populations to vascularization remains unknown. We thus cultured human papillary and reticular fibroblasts as cell sheets. Differential transcriptomic analysis was performed by RNA sequencing to characterize their microenvironment. Bioinformatic analysis revealed that each fibroblast population expressed specific angiogenesis and matrisome gene expression signatures resulting in specific ECM that differed both in composition and structure. The impact of secreted and ECM-bound factors was then assessed using 3D angiogenesis assays. When co-cultivated with endothelial cells, the papillary and reticular microenvironments induced the formation of distinct capillary networks mimicking the characteristics of vasculature of native dermis subcompartments (vessel diameter and density, number of branch points). Whereas conditioned media of papillary fibroblasts displayed intrinsic high angiogenic potential, reticular ones only contributed to capillary formation induced by exogenous VEGF. These results show that skin fibroblast populations regulate angiogenesis via both secreted and ECM-bound factors. Our work emphasizes the importance of papillary and reticular fibroblasts, not only for modelling dermis microenvironment but also for its vascularization.

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.

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 extracellular matrix (ECM) of blood vessels, which is composed of both the vascular basement membrane (BM) and the interstitial ECM is identified as a crucial component of the vasculature. We here focus on the unique molecular composition and scaffolding of the capillary ECM, which provides structural support to blood vessels and regulates properties of endothelial cells and pericytes. The major components of the BM are collagen IV, laminins, heparan sulfate proteoglycans and nidogen and also associated proteins such as collagen XVIII and fibronectin. Their organization and scaffolding in the BM is required for proper capillary morphogenesis and maintenance of vascular homeostasis. The BM also regulates vascular mechanosensing. A better understanding of the mechanical and structural properties of the vascular BM and interstitial ECM therefore opens new perspectives to control physiological and pathological angiogenesis and vascular homeostasis. The overall aim of this review is to explain how ECM scaffolding influences angiogenesis and capillary integrity.

International audience; Blood perfusion of grafted tissue constructs is a hindrance to the success of stem cell-based therapies by limiting cell survival and tissue regeneration. Implantation of a pre-vascularized network engineered in vitro has thus emerged as a promising strategy for promoting blood supply deep into the construct, relying on inosculation with the host vasculature. We aimed to fabricate in vitro tissue constructs with mature microvascular networks, displaying perivascular recruitment and basement membrane, taking advantage of the angiogenic properties of dental pulp stem cells and self-assembly of endothelial cells into capillaries. Using digital scanned light-sheet microscopy, we characterized the generation of dense microvascular networks in collagen hydrogels and established parameters for quantification of perivascular recruitment. We also performed original time-lapse analysis of stem cell recruitment. These experiments demonstrated that perivascular recruitment of dental pulp stem cells is driven by PDGF-BB. Recruited stem cells participated in deposition of vascular basement membrane and vessel maturation. Mature microvascular networks thus generated were then compared to those lacking perivascular coverage generated using stem cell conditioned medium. Implantation in athymic nude mice demonstrated that in vitro maturation of microvascular networks improved blood perfusion and cell survival within the construct. Taken together, these data demonstrate the strong potential of in vitro production of mature microvasculature for improving cell-based therapies.

In vitro 3D culture systems provide promising tools for screening novel therapies and understanding drug resistance mechanisms in cancer because they are adapted for high throughput analysis. One of the main current challenges is to reproducibly culture patient samples containing cancer and stromal cells to faithfully recapitulate tumor microenvironment and move toward efficient personalized medicine. Tumors are composed of heterogeneous cell populations and characterized by chaotic vascularization in a remodeled microenvironment. Indeed, tumor angiogenesis occurs in a complex stroma containing immune cells and cancer-associated fibroblasts that secrete important amounts of cytokines, growth factors, extracellular vesicles, and extracellular matrix (ECM). This process leads to the formation of inflated, tortuous, and permeable capillaries that display deficient basement membrane (BM) and perivascular coverage. These abnormal capillaries affect responses to anti-cancer therapies such as anti-angiogenic, radio-, and immunotherapies. Current pre-clinical models are limited for investigating interactions between tumor cells and vascularization during tumor progression as well as mechanisms that lead to drug resistance. In vitro approaches developed for vascularization are either the result of engineered cell lining or based on physiological processes including vasculogenesis and sprouting angiogenesis. They allow investigation of paracrine and direct interactions between endothelial and tumor and/or stromal cells, as well as impact of biochemical and biophysical cues of the microenvironment, using either natural matrix components or functionalized synthetic hydrogels. In addition, microfluidic devices provide access to modeling the impact of shear stress and interstitial flow and growth factor gradients. In this review, we will describe the state of the art co-culture models of vascularized micro-tumors in order to study tumor progression and metastatic dissemination including intravasation and/or extravasation processes.

BackgroundEarly in the COVID-19 pandemic type 2 diabetes (T2D) was marked as a risk factor of severe disease and mortality. Inflammation is central to the aetiology of both conditions where variations in immune responses have the potential to mitigate or aggravate disease course. Identifying at risk groups based on immuno-inflammatory signatures is valuable in directing personalised care and developing potential targets for precision therapy.MethodsThis study characterised immunophenotypic variation associated with COVID-19 severity in type 2 diabetes. Broad-spectrum immunophenotyping quantified 15 leukocyte populations in peripheral circulation from a cohort of 45 hospitalised COVID-19 patients with and without type 2 diabetes.ResultsMorphological anomalies in the monocyte pool, monocytopenia specific to quiescent monocytes, and a decreased frequency of cytotoxic lymphocytes were associated with severe COVID-19 in patients with type 2 diabetes requiring intensive care. An aggravated inflammatory gene expression profile, reminiscent of the type-1 interferon pathway, underlaid the immunophenotype associated with severe disease in T2D.ConclusionShifts in T-cell and monocyte dynamics underpin a maladaptive response to SARSCoV-2. These alterations may impact type-1 interferon signalling which is the likely source of the hyperinflammation that increases voracity of COVID-19. These findings allow the identification of type 2 diabetic patients at risk of severe disease as well as providing evidence that the type-1 interferon pathway may be an actionable therapeutic target for future studies.Trial registrationNCT02671864FundingFrench National Agency of Research (ANR); European Foundation for the study of diabetes (EFSD); European Research Council (ERC); Francophone Society for Diabetes (SFD)Brief summaryMaladapted monocyte responses including class switch, morphological anomalies and systemic hyperinflammation put patients with type 2 diabetes at higher risk of severe COVID-19GRAPHICAL ABSTRACT

Tissue engineering technologies are new and promising techniques in fat tissue reconstruction. However, to assess their efficacy before any clinical application, in vivo experiments are mandatory. This study assesses whether microcomputed tomography (CT) scan imaging is suitable to analyze in vivo the behavior of injected engineered polymer and changes in fat tissue. The volume of mice inguinal fat pads and the resorption rate of different polymers were analyzed by CT scan for up to 3 months. Different biomaterials were used, including our innovative microspheres loaded with oleic acid. We were able to follow in vivo the polymer and the fat volume of the same animals during a long-term follow-up of 90 days. Semiautomatic three-dimensional quantification allowed to determine the fat volume enhancement after injection, as well as the resorption rate of our product compared to other biomaterials (i.e., polylactic and hyaluronic acid) until 90 days. Our results demonstrate the encouraging proof-of-principle evidence for the application of micro-CT scan technology to follow in vivo biodegradable polymers in a fat tissue engineering approach. This noninvasive technique offers the advantages of the long-term follow-up of fat tissue and synthetic materials in the same animals, which allows both a scientific evaluation of the measurements and the reduction of the number of animals used in in vivo protocols in accordance with the 3 "R" principles governing the use of animals in science.

Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance of in vitro applications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing (a) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, (b) three perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, (c) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9-18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced. This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

Purpose Stem cell-based attempts at regenerating chronically failing heart have not met the clinical outcomes expected by experimental studies. The efficacy of cell transplant is largely dependent on the engraftment rate, which requires cells to receive an adequate blood supply. To address this issue, we propose to develop a composite tissue engineering construct made of biocompatible pre-vascularized fibrin hydrogel. The objectives of this study are to determine co-culture conditions and to optimize fabrication of fibrin hydrogels supporting generation of capillaries and cardiomyocyte activity at the same time. Materials and methods Rat primary cardiomyocytes were co-seeded with human umbilical vein endothelial cells (HUVECs) in fibrin hydrogels. Cultures were performed in 96 well plates. Cells were cultured in a mixture of medium conditioned with human dermal fibroblasts and of cardiomyocyte medium (1:1) for 8 days. To improve co-culture of the two cell types, insulin (1 μg/mL) and/or VEGF (5 ng/mL) were added 24 hours after the initial day of culture and maintained for 24 hours. The cells treated with VEGF were also treated 5 days after initial seeding with insulin and VEGF (insulin at 1 μg/mL and VEGF at 1 or 5 ng/mL). After culture, cardiomyocytes were immuno-stained for Troponin T or Connexin-43, endothelial cells for CD31, basement membrane for collagen IV. From spinning disk images, cardiomyocyte volume, contraction and total length/ branches of capillaries were measured. Results : We first determine the best conditions allowing the generation of active cardiomyocytes and the formation of a network of mature capillaries into fibrin hydrogels. We showed that cardiomyocytes volume and contraction were not influenced by the addition of VEGF or insulin. For cells treated with VEGF for 24 hours, total length and total branches of capillaries were significantly increased over other conditions without supplements (control), or with insulin alone or with VEGF. We also observed lumen formation and basement membrane deposition. Conclusions These results may provide a new model of vascularized human heart tissue for in vitro studies, displaying cardiomyocyte contractility together with lumenized capillary formation. Furthermore, our data may help overcome current obstacles toward the success of cardiac cell therapy for damaged myocardium. In such approach, endothelial capillaries will serve as structural and trophic support through inosculation with the host cardiac vasculature after epicardial graft. As a perspective of this work, and in order to improve the cardiomyocyte alignment and the development of capillaries, we will design specific PDMS mold to allow the formation of a micro-tissue. Application of human cardiac cells and implantation in in vivo model is expected. Acknowledgement/Funding ESC research grant, Fondation de l'avenir, Societe Francaise de Cardiologie, Institut Servier

Background Microvascular obstruction (MVO) is associated with poor outcome after ST-segment elevation myocardial infarction (STEMI). Vascular endothelial growth factor-A (VEGF-A) is a vascular permeability inducer playing a key role in MVO pathogenesis. We aimed to assess whether VEGF-A levels are associated with MVO, when evaluated by magnetic resonance imaging (MRI) in STEMI patients. Methods The multicenter prospective PREGICA study included a CMR substudy with all consecutive patients with a first STEMI who had undergone cardiac MRI at baseline and at 6-month follow-up. Patients with initial TIMI flow >1 were excluded. VEGF-A levels were measured in blood samples drawn at inclusion. Results Between 2010 and 2017, 147 patients (mean age 57 ± 10 years; 84% males) were included. MVO was present in 65 (44%) patients. After multivariate analysis, higher troponin peak (OR 1.005; 95% CI 1.001–1.008; p = 0.007) and VEGF-A levels (OR 1.003; 95% CI 1.001–1.005; p = 0.015) were independently associated with MVO. When considering only patients with successful percutaneous coronary intervention (final TIMI flow 3, n = 130), higher troponin peak (p = 0.004) and VEGF-A levels (p = 0.03) remained independently predictive of MVO. Moreover, MVO was associated with adverse left ventricular (LV) remodeling and VEGF-A levels were significantly and inversely correlated with LV ejection fraction (EF) at 6-month follow-up. Conclusion Our results show that VEGF-A levels were independently associated with MVO during STEMI and correlated with mid-term LVEF alteration. VEGF-A could therefore be considered as a biomarker of MVO in STEMI patients and be used to stratify patient prognosis.

International audience

International audience

International audience; Angiogenesis is a complex process leading to the growth of new blood vessels from existing vasculature, triggered by local proangiogenic factors such as VEGF. An excess of angiogenesis is a recurrent feature of various pathologic conditions such as tumor growth. Phostines are a family of synthetic glycomimetic compounds that exhibit anticancer properties, and the lead compound 3-hydroxy-4,5-bis-benzyloxy-6-benzyloxymethyl-2-phenyl2-oxo-2λ5-[1,2]oxaphosphinane (PST 3.1a) shows antiglioblastoma properties both in vitro and in vivo. In the present study, we assessed the effect of PST 3.1a on angiogenesis and endothelial metabolism. In vitro, PST 3.1a (10 µM) inhibited all steps that regulate angiogenesis, including migration, proliferation, adhesion, and tube formation. In vivo, PST 3.1a reduced intersegmental vessel formation and vascularization of the subintestinal plexus in zebrafish embryos and also altered pathologic angiogenesis and glioblastoma progression in vivo. Mechanistically, PST 3.1a altered interaction of VEGF receptor 2 and glycosylation-regulating protein galectin-1, a key component regulating angiogenesis associated with tumor resistance. Thus, these data show that use of PST 3.1a is an innovative approach to target angiogenesis.-Bousseau, S., Marchand, M., Soleti, R., Vergori, L., Hilairet, G., Recoquillon, S., Le Mao, M., Gueguen, N., Khiati, S., Clarion, L., Bakalara, N., Martinez, M. C., Germain, S., Lenaers, G., Andriantsitohaina, R. Phostine 3.1a as a pharmacological compound with antiangiogenic properties against diseases with excess vascularization.

Cydia pomonella granulovirus, in particular CpGV-M isolate, is used as a biological control against the codling moth (CM), Cydia pomonella. As a result of intensive control over the years, codling moth populations have developed resistance against this isolate. This resistance is now called type I resistance. Isolates, among them, CpGV-R5, have been found that are able to overcome type I resistance. Both CpGV-M and CpGV-R5 are used in orchards to control the codling moth. High resolution melting (HRM) has been adapted to differentiate between CpGV-M and CpGV-R5 isolates. Specific PCR primers have been designed for the CpGV p38 gene, encompassing the variable region responsible for the ability to overcome resistance. Because each amplicon has a specific melting point, it is possible to identify the CpGV-M and CpGV-R5 genotypes and to quantify their relative proportion. This method has been validated using mixtures of occlusion bodies of each isolate at various proportions. Then, the HRM has been used to estimate the proportion of each genotype in infected larvae or in occlusion bodies (OBs) extracted from dead larvae. This method allows a rapid detection of genotype replication and enables the assessment of either success or failure of the infection in field conditions.

Hypothermic and normothermic ex vivo liver perfusions promote organ recovery after donation after circulatory death (DCD). We tested whether these perfusions can reduce the risk of hepatocellular carcinoma (HCC) recurrence in a 1h-DCD syngeneic transplantation model, using Fischer F344 rats. DCD grafts were machine perfused for 2h with hypothermic perfusion (HOPE) or normothermic perfusion (NORMO), and transplanted. After reperfusion, we injected HCC cells into the vena porta. On day 28 after transplantation, we assessed tumour volumes by MRI. Control rats included transplantations with Fresh and non-perfused DCD livers. We observed apoptotic-necrotic hepatocyte foci in all DCD grafts, which were more visible than in the Fresh liver grafts. Normothermic perfusion allowed a faster post-transplant recovery, with lower day 1 levels of transaminases compared with the other DCD. Overall, survival was similar in all four groups and all animals developed HCCs. Total tumor volume was lower in the Fresh liver recipients compared to the DCD and DCD+HOPE recipients. Volumes in DCD+NORMO recipients were not significantly different from those in the Fresh group. This experiment confirms that ischemia/reperfusion injury promotes HCC cell engraftment/growth after DCD liver transplantation. Using the present extreme 1h ischemia model, both hypothermic and normothermic perfusions were not effective in reducing this risk.

The adjunction of autologous platelet-rich plasma (PRP) is emerging as a promising approach to enhance the long-term survival of fat grafting, but there are still insufficient data on its efficacy. The aim of this in vivo study was to assess the effect of the addition of non-activated PRP on fat graft outcome.Human adipose tissue mixed with 20% of non-activated PRP was injected under the scalp skin of nude Balb/cAnNRj mice and compared to grafted fat mixed with 20% of saline. The fat graft volume was analyzed by a computed tomography scan until day 90 and immunohistochemistry was then performed to assess adipocyte viability and graft revascularization.At day 90, the volume of fat graft was not enhanced by PRP compared to the saline control group. However, immunohistochemistry showed that PRP significantly increased the fat graft area occupied by intact adipocytes compared to the saline group (72.66% vs. 60.78%, respectively; p 0.05). Vascularity was also significantly higher in the PRP group compared to the control group (6695 vs. 4244 CD31The adjunction of non-activated-PRP to fat grafts significantly increased adipocyte viability and tissue vascularity. However, in contrast to other studies adding activated-PRP, non-activated-PRP did not increase residual fat graft volume until day 90. Further studies are therefore needed to understand whether PRP has a positive effect on fat graft volume. As 3D computed tomography scan is a reproducible and precise technique, it should be used to analyze fat graft volume changes over time.

Malignant pleural mesothelioma (MPM) is a thoracic aggressive cancer caused by asbestos exposure, which is difficult to diagnose and treat. Here, we characterized an in vivo orthotopic xenograft model consisting of human mesothelioma cells (designed as H2052/484) derived from a pleural NCI-H2052 tumor injected in partially immunodeficient athymic mice. We assessed tumor formation and tumor-dependent patterns of inflammation. H2052/484 cells conserved their mesothelioma phenotype and most characteristics from the parental NCI-H2052 cells. After intra-thoracic injection of H2052/484 cells, thoracic tumors developed in nearly all mice (86%) within 14 days, faster than from parental NCI-H2052 cells. When the mice were euthanized, the pleural lavage fluid was examined for immune cell profiles. The pleural immune cell population increased with tumor development. Interestingly, the proportion of myeloid-derived suppressor cell and macrophage (especially CD206+ M2 macrophages) populations increased in the pleural fluid of mice with large mesothelioma development, as previously observed in immunocompetent mice. This reliable orthotopic model recapitulates human mesothelioma and may be used for the study of new treatment strategies.

Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine implicated in acute and chronic inflammatory diseases. MIF is overexpressed in various tumors. It displays a number of functions that provide a direct link between the process of inflammation and tumor growth. Our group recently identified the MIF-receptor CD74 as an independent prognostic factor for overall survival in patients with malignant pleural mesothelioma. In the present study, we compared the levels of expression of MIF and CD74 in different human mesothelioma cell lines and investigated their physiopathological functions in vitro and in vivo. Human mesothelioma cells expressed more CD74 and secreted less MIF than non tumoral MeT5A cells, suggesting a higher sensitivity to MIF. In mesothelioma cells, high MIF levels were associated with a high multiplication rate of cells. In vitro, reduction of MIF or CD74 levels in both mesothelioma cell lines showed that the MIF/CD74 signaling pathway promoted tumor cell proliferation and protected MPM cells from apoptosis. Finally, mesothelioma cell lines expressing high CD74 levels had a low tumorigenic potential after xenogeneic implantation in athymic nude mice. All these data highlight the complexity of the MIF/CD74 signaling pathway in the development of mesothelioma.

The novel class of bis-arylaminoacenaphthylenes (Ar-BIANH2) was employed for the preparation of zirconium and yttrium complexes to be used as catalysts for cyclohydroamination of a number of primary and secondary aminoalkenes. The complex [(2,6-iPr2C6H3-BIAN)Zr(NMe2)2(Z1-NHMe2)] was isolated and completely characterized, including X-ray diffraction analysis. Despite its easy and almost quantitative isolation, it showed only moderate catalytic performance in the intramolecular hydroamination, irrespective of the cyclization precursor used. On the other hand, in situ generated YIII complexes obtained using the same class of ligands were found to be very active, leading to the hydroamination of substrates including those normally reluctant in undergoing cyclization such as those featuring an internal non-activated CQC double bond. Electron donating substituents and especially steric hindrance on the ligand improve the performance of the catalysts, allowing us to decrease the catalyst loading to 1 mol% in the latter case.

International audience; Primary percutaneous coronary intervention (PCI) is the best available reperfusion strategy for acute ST-segment elevation myocardial infarction (STEMI), with nearly 95% of occluded coronary vessels being reopened in this setting. Despite re-establishing epicardial coronary vessel patency, primary PCI may fail to restore optimal myocardial reperfusion within the myocardial tissue, a failure at the microvascular level known as no-reflow (NR). NR has been reported to occur in up to 60% of STEMI patients with optimal coronary vessel reperfusion. When it does occur, it significantly attenuates the beneficial effect of reperfusion therapy, leading to poor outcomes. The pathophysiology of NR is complex and incompletely understood. Many phenomena are known to contribute to NR, including leukocyte infiltration, vasoconstriction, activation of inflammatory pathways and cellular oedema. Vascular damage and haemorrhage may also play important roles in the establishment of NR. In this review, we describe the pathophysiological mechanisms of NR and the tools available for diagnosing it. We also describe the microvasculature and the endothelial mechanisms involved in NR, which may provide relevant therapeutic targets for reducing NR and improving the prognosis for patients.

Objective Juvenile dermatomyositis (JDM) is an inflammatory pediatric myopathy characterized by focal capillary loss in muscle, followed by progressive recovery upon adequate treatment with immunomodulating drugs, although some patients remain refractory to treatment. While the underlying mechanism of capillary depletion remains uncertain, recent studies have identified an up-regulation of type I interferon (IFN) expression specific to JDM. Given that myogenic precursor cells (MPCs) exert proangiogenic activity during normal skeletal muscle regeneration, we hypothesized that they may also modulate vascular remodeling/angiogenesis during JDM. The aim of this study was to investigate that hypothesis. Methods Human cell cocultures were used to analyze angiogenic properties in patients with JDM, patients with Duchenne's muscular dystrophy (DMD) (control patients for vascular remodeling), and healthy control subjects. Transcriptome analysis was used to examine muscle-derived MPCs. Histologic analysis of type I IFN in muscle biopsy samples was also performed. Results Using human cell cocultures, we showed highly angiogenic properties of MPCs from JDM patients in association with the expression of an angiogenic molecular signature. Transcriptome analysis of MPCs freshly isolated from muscle samples revealed type I IFN as the master regulator of the most up-regulated genes in JDM-derived MPCs. Functionally, treatment of normal MPCs with type I IFN recapitulated the molecular pattern and the proangiogenic functions of JDM-derived MPCs. In vivo histologic investigation showed that MPCs synthesized type I IFN and major proangiogenic molecules in JDM muscle. Moreover, MPCs derived from JDM muscles that were characterized by strong vasculopathy produced higher levels of type I IFN, confirming MPCs as a cellular source of type I IFN during JDM, and this correlated with the severity of the disease. Conclusion These results demonstrate a new type I IFN pathway in JDM that activates the production of angiogenic effectors by MPCs, triggering their proangiogenic function to promote vessel recovery and muscle reconstruction.

Summary 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., Graphical Abstract, Highlights • Endothelial cells (ECs) promote myogenesis • Myogenic progenitor cells (MPCs) stimulate angiogenesis as they differentiate • EC- and MPC-derived Apelin, Oncostatin M, and Periostin promote myo-angiogenesis • Restorative macrophages stimulate myo-angiogenesis via Oncostatin M secretion, In this study, Chazaud et al. demonstrate that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted to couple myogenesis and angiogenesis during skeletal muscle regeneration. EC- and MPC-derived Apelin, Oncostatin M, and Periostin controlled myogenesis/angiogenesis coupling and were required for myogenesis and vessel formation. They show that, via the production of Oncostatin M, restorative macrophages promoted myogenesis/angiogenesis coupling.

Scientific Reports 6: Article number: 20927; published online: 18 February 2016; updated: 23 September 2016 The original version of this Article contained a typographical error in the spelling of the author E. Gazquez, which was incorrectly given as E. Gazguez. This has now been corrected in the PDFand HTML versions of the Article.

Background— Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell–cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. Methods and Results— We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4 -deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4 -deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4 -deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. Conclusions— These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.

Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. LOXL2 belongs to the lysyl oxidase family of secreted enzymes involved in ECM crosslinking. Knockdown experiments in Tg(fli1:egfp)y1 zebrafish embryos resulted in lack of intersegmental vessel circulation and demonstrated LOXL2 involvement in proper capillary formation. Further investigation in vitro by loss and gain of function experiments confirmed that LOXL2 was required for tubulogenesis in 3D fibrin gels and demonstrated that this enzyme was required for collagen IV assembly in the ECM. In addition, LOXL2 depletion down-regulated cell migration and proliferation. These data suggest a major role for LOXL2 in the organization of endothelial basal lamina and in the downstream mechanotransductive signaling. Altogether, our study provides the first evidence for the role of LOXL2 in regulating angiogenesis through collagen IV scaffolding.

Dans le modele parametrique de la theorie des reponses aux items (TRI), l’estimation du niveau d’aptitude ( ) du repondant a fait l’objet de plusieurs procedures differentes, certaines basees uniquement sur le protocole de reponses observe et d’autres, appuyees sur un modele de population contraignant. Peu d’etudes cependant ont entrepris d’etablir les merites comparatifs de ces procedures. Au moyen d’une large experimentation Monte Carlo, nous etudions le biais, la precision et l’efficacite de capture de quatre estimateurs de capacite : MV, BME, EAP, WARM, auxquels nous ajoutons un estimateur MV winsorise (WINS) et un autre extrapole (EXT), qui permettent tous deux l’utilisation de l’estimateur MV pour les scores parfaits (tous items reussis) et nuls (tous items echoues).