Publications by authors named "Romaric Lacroix"

48 Publications

Granulocyte microvesicles with a high plasmin generation capacity promote clot lysis and improve outcome in septic shock.

Blood 2022 Jan 13. Epub 2022 Jan 13.

AP-HM, France.

Microvesicles (MVs) have previously been shown to exert profibrinolytic capacity, which is increased in patients with septic shock (SS) with a favorable outcome. We therefore hypothesized that the plasmin generation capacity (PGC) could confer to MVs a protective effect supported by their capacity to lyse a thrombus, and we investigated the mechanisms involved. Using a MV-PGC kinetic assay, ELISA and flow cytometry, we found that granulocyte MVs (Gran-MVs) from SS patients display a heterogeneous PGC profile driven by the uPA (urokinase)/uPAR system. In vitro, these MVs lyse a thrombus according to their MV-PGC levels in a uPA/uPAR-dependent manner, as shown in a fluorescent clot lysis test and a lysis front retraction assay. Fibrinolytic activators conveyed by MVs contribute to approximately 30% of the plasma plasminogenolytic capacity of SS patients. In a murine model of SS, the injection of high PGC Gran-MVs significantly improved mouse survival and reduced the number of thrombi in vital organs. This was associated with a modification of the mouse coagulation and fibrinolysis properties toward a more fibrinolytic profile. Interestingly, mouse survival was not improved when soluble uPA was injected. Finally, using a multiplex array on plasma from SS patients, we found that neutrophil elastase correlates with the effect of high-PGC-capacity plasma and modulates the Gran-MV plasmin generation capacity by cleaving uPA-PAI-1 complexes. In conclusion, we show that high PGC level displayed by Gran-MVs reduce thrombus formation and improve survival conferring to Gran-MVs a protective role in a murine model of sepsis.
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http://dx.doi.org/10.1182/blood.2021013328DOI Listing
January 2022

[Extracellular vesicles-associated biomarkers: Opportunities and challenges in cardiovascular diseases and cancer].

Med Sci (Paris) 2021 Dec 20;37(12):1158-1165. Epub 2021 Dec 20.

Aix-Marseille Université, C2VN (Centre de recherche en cardiovasculaire et nutrition), Inserm 1263, INRAe 1260, 13000 Marseille, France - Service d'hématologie et de biologie vasculaire, CHU La Conception, AP-HM, 13005 Marseille, France.

Cardiovascular diseases and cancer are the leading causes of mortality and morbidity in the world. The search for pertinent biomarkers for risk stratification and treatment monitoring is a challenge. Rapid advances in the identification of the molecular and functional content of extracellular vesicles (EV) and ongoing progress in developing highly sensitive methodologies, identify EV as promising biomarkers easily accessible in liquid biopsies. Thanks to robust and sensitive methodologies, the measurability of biological targets on EV allows to define vesicular biomarkers pertinent for disease management. Adaptation of the pre-analytical and analytical steps to each EV-associated biomarker, technological improvement and standardization efforts driven by scientific societies are essential prerequisites to accelerate the transfer of these EV-associated biomarkers to the clinics and to support the development of personalized medicine.
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http://dx.doi.org/10.1051/medsci/2021208DOI Listing
December 2021

Dissemination of extreme levels of extracellular vesicles: tissue factor activity in patients with severe COVID-19.

Blood Adv 2021 02;5(3):628-634

Aix Marseille University, INSERM 1263, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Centre de Recherche en CardioVasculaire et Nutrition (C2VN), Marseille, France.

Coronavirus disease 2019 (COVID-19) has become one of the biggest public health challenges of this century. Severe forms of the disease are associated with a thrombo-inflammatory state that can turn into thrombosis. Because tissue factor (TF) conveyed by extracellular vesicles (EVs) has been implicated in thrombosis, we quantified the EV-TF activity in a cohort of hospitalized patients with COVID-19 (n = 111) and evaluated its link with inflammation, disease severity, and thrombotic events. Patients with severe disease were compared with those who had moderate disease and with patients who had septic shock not related to COVID-19 (n = 218). The EV-TF activity was notably increased in patients with severe COVID-19 compared with that observed in patients with moderate COVID-19 (median, 231 [25th to 75th percentile, 39-761] vs median, 25 [25th to 75th percentile, 12-59] fM; P < .0001); EV-TF was correlated with leukocytes, D-dimer, and inflammation parameters. High EV-TF values were associated with an increased thrombotic risk in multivariable models. Compared with patients who had septic shock, those with COVID-19 were characterized by a distinct coagulopathy profile with significantly higher EV-TF and EV-fibrinolytic activities that were not counterbalanced by an increase in plasminogen activator inhibitor-1 (PAI-1). Thus, this article is the first to describe the dissemination of extreme levels of EV-TF in patients with severe COVID-19, which supports the international recommendations of systematic preventive anticoagulation in hospitalized patients and potential intensification of anticoagulation in patients with severe disease.
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http://dx.doi.org/10.1182/bloodadvances.2020003308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846479PMC
February 2021

A rare coding mutation in the MAST2 gene causes venous thrombosis in a French family with unexplained thrombophilia: The Breizh MAST2 Arg89Gln variant.

PLoS Genet 2021 01 19;17(1):e1009284. Epub 2021 Jan 19.

EA3878-GETBO, Univ Brest, Department of internal medicine and chest diseases, FCRIN_INNOVTE, CHU Brest, Brest, France.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.
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http://dx.doi.org/10.1371/journal.pgen.1009284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846112PMC
January 2021

Multifaceted role of extracellular vesicles in atherosclerosis.

Atherosclerosis 2021 02 11;319:121-131. Epub 2020 Nov 11.

Aix Marseille University, INSERM, INRAE, C2VN, Marseille, France; Department of Hematology and Vascular Biology, CHU La Conception, APHM, Marseille, France.

Extracellular vesicles (EVs) are small vesicles released by the majority of cells in response to cell activation or death stimuli. They are grouped as small EVs or exosomes, large EVs such as microvesicles (MVs) and apoptotic bodies, resulting from distinct mechanisms of generation. EVs are released into the extracellular space, in most human biological fluids and tissues, including atherosclerotic plaques. They transport complex cargo of bioactive molecules, including proteins, lipids and genetic material and are therefore involved in pathophysiological pathways of cell-cell communication. Indeed, EVs are involved in several processes such as inflammation, coagulation, vascular dysfunction, angiogenesis and senescence, contributing to the initiation and progression of atherothrombotic diseases. Consequently, they behave as a determinant of atherosclerotic plaque vulnerability leading to major cardiovascular disorders. Over the last decade, the field of EVs research has grown, highlighting their involvement in atherosclerosis. However, limitations in both detection methodologies and standardisation have hindered implementation of EVs in the clinical settings. This review summarizes the effect of EVs in atherosclerosis development, progression and severity, with specific attention devoted to their ambivalent roles in senescence and hemostasis. This review will also highlight the role of MVs as multifaceted messengers, able to promote or to attenuate atherosclerosis progression. Finally, we will discuss the main technical challenges and prerequisites of standardization for driving EVs to the clinics and delineate their relevance as emergent biomarkers and innovative therapeutic approaches in atherosclerosis.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.11.006DOI Listing
February 2021

A new hybrid immunocapture bioassay with improved reproducibility to measure tissue factor-dependent procoagulant activity of microvesicles from body fluids.

Thromb Res 2020 12 21;196:414-424. Epub 2020 Sep 21.

BioCytex, Research and Technology Department, Marseille, France. Electronic address:

Background: The procoagulant activity of tissue factor-bearing microvesicles (MV-TF) has been associated with the risk of developing venous thrombosis in cancer patients. However, MV-TF assays are limited either by i) a lack of specificity, ii) a low sensitivity, or iii) a lack of repeatability when high-speed centrifugation (HS-C) is used to isolate MV. Therefore, our objective was to develop a new hybrid "capture-bioassay" with improved reproducibility combining MV immunocapture from biofluids and measurement of their TF activity.

Materials And Methods: Factor Xa generation and flow cytometry assays were used to evaluate IMS beads performance, and to select the most effective capture antibodies. The analytical performance between IMS-based and HS-C-based assays was evaluated with various models of plasma samples (from LPS-activated blood, spiked with tumoral MV, or with saliva MV) and different biofluids (buffer, plasma, saliva, and pleural fluid).

Results: Combining both CD29 and CD59 antibodies on IMS beads was as efficient as HS-C to isolate plasmatic PS+ MV. The IMS-based strategy gave significantly higher levels of MV-TF activity than HS-C in tumor MV spiked buffer, and both pleural fluids and saliva samples. Surprisingly, lower TF values were measured in plasma due to TFPI (TF pathway inhibitor) non-specifically adsorbed onto beads. This was overcome by adding a TFPI-blocking antibody. After optimization, the new IMS-based assay significantly improved reproducibility of MV-TF bioassay versus the HS-C-based assay without losing specificity and sensitivity. In addition, this approach could identify the cellular origin of MV-TF in various biological fluids.

Conclusion: Compared to HS-C, the IMS-based measurement of MV-TF activity in body fluids improves reproducibility and makes the assay compatible with clinical practice. It can facilitate future automation.
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http://dx.doi.org/10.1016/j.thromres.2020.09.020DOI Listing
December 2020

Circulating Endothelial Cells as a Marker of Endothelial Injury in Severe COVID -19.

J Infect Dis 2020 Nov;222(11):1789-1793

Aix-Marseille University, INSERM 1263, INRAE, C2VN, Marseille, France.

Beside the commonly described pulmonary expression of the coronavirus disease 2019 (COVID-19), major vascular events have been reported. The objective of this study was to investigate whether increased levels of circulating endothelial cells (CECs) might be associated with severe forms of COVID-19. Ninety-nine patients with COVID-19 were enrolled in this retrospective study. Patients in the intensive care units (ICU) had significantly higher CEC counts than non-ICU patients and the extent of endothelial injury was correlated with putative markers of disease severity and inflammatory cytokines. Together, these data provide in vivo evidence that endothelial injury is a key feature of COVID-19.
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http://dx.doi.org/10.1093/infdis/jiaa528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7454721PMC
November 2020

MIFlowCyt-EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments.

J Extracell Vesicles 2020 3;9(1):1713526. Epub 2020 Feb 3.

Translational Nanobiology Section, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.
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http://dx.doi.org/10.1080/20013078.2020.1713526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7034442PMC
February 2020

Therapeutic targeting of soluble CD146/MCAM with the M2J-1 monoclonal antibody prevents metastasis development and procoagulant activity in CD146-positive invasive tumors.

Int J Cancer 2020 09 22;147(6):1666-1679. Epub 2020 Feb 22.

INSERM, INRAE, C2VN, UFR Pharmacie, Aix-Marseille University, Marseille, France.

Initially discovered in human melanoma, CD146/MCAM is expressed on many tumors and is correlated with cancer progression and metastasis. However, targeting CD146 remains challenging since it is also expressed on other cell types, as vessel cells, where it displays important physiological functions. We previously demonstrated that CD146 is shed as a soluble form (sCD146) that vectorizes the effects of membrane CD146 on tumor angiogenesis, growth and survival. We thus generated a novel monoclonal antibody, the M2J-1 mAb, which specifically targets sCD146, but not membrane CD146, and counteracts these effects. In our study, we analyzed the effects of sCD146 on the dissemination and the associated procoagulant phenotype in two highly invasive human CD146-positive cancer cell lines (ovarian and melanoma). Results show that sCD146 induced epithelial to mesenchymal transition, favored the generation of cancer stem cells and increased the membrane expression of tissue factor. Treatment of cancer cells with sCD146 in two experimental models (subcutaneous xenografting and intracardiac injection of cancer cells in nude mice) led to increased tumor dissemination and procoagulant activity. The M2J-1 mAb drastically reduced metastasis but also procoagulant activity, in particular by decreasing the number of circulating tumor microparticles, and blocked the relevant signaling pathways as demonstrated by RNA expression profiling experiments. Thus, our findings demonstrate that sCD146 mediates important pro-metastatic and procoagulant effects in two CD146-positive tumors. Targeting sCD146 with the newly generated M2J-1 mAb could constitute an innovative strategy for preventing dissemination and thromboembolism in many CD146-positive tumors.
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http://dx.doi.org/10.1002/ijc.32909DOI Listing
September 2020

How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients?

Blood Adv 2019 12;3(24):4238-4251

Laboratoire d'Hématologie, CHU Estaing 1, Clermont-Ferrand, France.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive leukemia for which we developed a nationwide network to collect data from new cases diagnosed in France. In a retrospective, observational study of 86 patients (2000-2013), we described clinical and biological data focusing on morphologies and immunophenotype. We found expression of markers associated with plasmacytoid dendritic cell origin (HLA-DRhigh, CD303+, CD304+, and cTCL1+) plus CD4 and CD56 and frequent expression of isolated markers from the myeloid, B-, and T-lymphoid lineages, whereas specific markers (myeloperoxidase, CD14, cCD3, CD19, and cCD22) were not expressed. Fifty-one percent of cytogenetic abnormalities impact chromosomes 13, 12, 9, and 15. Myelemia was associated with an adverse prognosis. We categorized chemotherapeutic regimens into 5 groups: acute myeloid leukemia (AML)-like, acute lymphoid leukemia (ALL)-like, lymphoma (cyclophosphamide, doxorubicin, vincristine, and prednisone [CHOP])-like, high-dose methotrexate with asparaginase (Aspa-MTX) chemotherapies, and not otherwise specified (NOS) treatments. Thirty patients received allogeneic hematopoietic cell transplantation (allo-HCT), and 4 patients received autologous hematopoietic cell transplantation. There was no difference in survival between patients receiving AML-like, ALL-like, or Aspa-MTX regimens; survival was longer in patients who received AML-like, ALL-like, or Aspa-MTX regimens than in those who received CHOP-like regimens or NOS. Eleven patients are in persistent complete remission after allo-HCT with a median survival of 49 months vs 8 for other patients. Our series confirms a high response rate with a lower toxicity profile with the Aspa-MTX regimen, offering the best chance of access to hematopoietic cell transplantation and a possible cure.
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http://dx.doi.org/10.1182/bloodadvances.2019000647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929390PMC
December 2019

Ticagrelor attenuates the increase of extracellular vesicle concentrations in plasma after acute myocardial infarction compared to clopidogrel.

J Thromb Haemost 2020 03 9;18(3):609-623. Epub 2020 Jan 9.

First Chair and Department of Cardiology, Medical University of Warsaw, Poland.

Background: Platelet P2Y12 antagonist ticagrelor reduces mortality after acute myocardial infarction (AMI) compared to clopidogrel, but the underlying mechanism is unknown. Because activated platelets, leukocytes, and endothelial cells release proinflammatory and prothrombotic extracellular vesicles (EVs), we hypothesized that the release of EVs is more efficiently inhibited by ticagrelor compared to clopidogrel.

Objectives: We compared EV concentrations and EV procoagulant activity in plasma of patients after AMI treated with ticagrelor or clopidogrel.

Methods: After percutaneous coronary intervention, 60 patients with first AMI were randomized to ticagrelor or clopidogrel. Flow cytometry was used to determine concentrations of EVs from activated platelets (CD61 , CD62p ), fibrinogen , phosphatidylserine (PS ), leukocytes (CD45 ), endothelial cells (CD31 , 146 ), and erythrocytes (CD235a ) in plasma at randomization, after 72 hours and 6 months of treatment. A fibrin generation test was used to determine EV procoagulant activity.

Results: Concentrations of platelet, fibrinogen , PS , leukocyte, and erythrocyte EVs increased 6 months after AMI compared to the acute phase of AMI (P ≤ .03). Concentrations of platelet EVs were lower on ticagrelor compared to clopidogrel after 6 months (P = .03). Concentrations of fibrinogen , PS , and leukocyte EVs were lower on ticagrelor compared to clopidogrel both after 72 hours and 6 months (P ≤ .03). Concentrations of endothelial EVs and EV procoagulant activity did not differ between patient groups and over time (P ≥ .17).

Conclusions: Ticagrelor attenuates the increase of EV concentrations in plasma after acute myocardial infarction compared to clopidogrel. The ongoing release of EVs despite antiplatelet therapy might explain recurrent thrombotic events after AMI and worse clinical outcomes on clopidogrel compared to ticagrelor.
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http://dx.doi.org/10.1111/jth.14689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7065161PMC
March 2020

Extracellular vesicles and coagulation in blood from healthy humans revisited.

J Extracell Vesicles 2019 8;8(1):1688936. Epub 2019 Nov 8.

Laboratory of Experimental Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.

: In 2001, we studied the presence and coagulant properties of "microparticles" in the blood of healthy humans. Since then, multiple improvements in detection, isolation and functional characterization of the now called "extracellular vesicles" (EVs) have been made, and shortcomings were identified. : To revisit the presence and function of EVs in blood from healthy humans. : Blood was collected from 20 healthy donors. EV-containing plasma was prepared according to new guidelines, and plasma was diluted to prevent swarm detection. Single EVs were measured by flow cytometry with known sensitivity of fluorescence and light scatter. The haemostatic properties of EVs were measured by thrombin-, fibrin-, and plasmin generation. Plasma concentrations of thrombin-antithrombin complexes and prothrombin fragment 1 + 2 were measured to assess the coagulation status . : Compared to 2001, the total concentrations of detected EVs increased from 190- to 264-fold. In contrast to 2001, however, EVs are non-coagulant which we show can be attributed to improvements in blood collection and plasma preparation. No relation is present between the plasma concentrations of EVs and either TAT or F1 + 2. Finally, we show that EVs support plasmin generation. : Improvements in blood collection, plasma preparation and detection of EVs reveal that results from earlier studies have to be interpreted with care. Compared to 2001, higher concentrations of EVs are detected in blood of healthy humans which promote fibrinolysis rather than coagulation.
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http://dx.doi.org/10.1080/20013078.2019.1688936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853244PMC
November 2019

Comparison of the Response to Rituximab between Myelin Oligodendrocyte Glycoprotein and Aquaporin-4 Antibody Diseases.

Ann Neurol 2020 02 27;87(2):256-266. Epub 2019 Nov 27.

Public Assistance Hospitals of Marseille, Department of Neurology, Timone Hospital, Marseille.

Objective: To compare response to rituximab (RTX) between adult patients positive for myelin oligodendrocyte glycoprotein (MOG) and aquaporin-4 (AQP4) antibodies.

Methods: We prospectively studied adult patients with MOG or AQP4 antibodies who received RTX under an individualized dosing schedule adapted to the biological effect of RTX monitored by memory B-cell measurement. Memory B cells were counted monthly and when relapse occurred. The biological effect of RTX was considered significant with <0.05% memory B cells in peripheral blood lymphocytes.

Results: In 16 patients with MOG antibodies and 29 with AQP4 antibodies, mean follow-up was 19 (range = 9-38) and 38 (13-79) months. Under RTX, 10 relapses occurred in 6 of 16 (37.5%) patients with MOG antibodies, and 13 occurred in 7 of 29 (24%) with AQP4 antibodies. The median time of relapse after the most recent infusion was 2.6 (0.6-5.8) and 7 (0.8-13) months, respectively (p < 0.001). Memory B cells had reemerged in 2 of 10 (20%) relapses in patients with MOG antibodies and 12 of 13 (92.5%) with AQP4 antibodies (p < 0.001).

Interpretation: In AQP4 antibody-associated disorder, relapse mostly occurs when the biological effect of RTX decreases, which argues for treatment efficacy. In MOG antibody-associated disorder, the efficacy of RTX is not constant, because one-third of patients showed relapse despite an effective biological effect of RTX. In this subpopulation, memory B-cell depletion was unable to prevent relapse, which was probably caused by different immunological mechanisms. These findings should be used to improve treatment strategies for MOG antibody-associated disorder. ANN NEUROL 2020;87:256-266.
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http://dx.doi.org/10.1002/ana.25648DOI Listing
February 2020

Increasing the sensitivity of the human microvesicle tissue factor activity assay.

Thromb Res 2019 Oct 19;182:64-74. Epub 2019 Jul 19.

Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, France; Department of Hematology and Vascular Biology, CHU La Conception, APHM, Marseille, France.

Introduction: The TF-FVIIa complex is the primary activator of coagulation. Elevated levels of microvesicle (MV) bearing tissue factor (TF)-dependent procoagulant activity are detectable in patients with an increased risk of thrombosis. Several methods have been described to measure MV TF activity but they are hampered by limited sensitivity and specificity. The aim of this work was to increase the sensitivity of the MV TF activity assay (called Chapel Hill assay).

Material And Methods: Improvements of the MV TF activity assay included i/ speed and time of centrifugation, ii/ use of a more potent inhibitory anti-TF antibody iii/ use of FVII and a fluorogenic substrate to increase specificity.

Results: The specificity of the MV TF activity assay was demonstrated by the absence of activity on MV derived from a knock-out-TF cell line using an anti-human TF monoclonal antibody called SBTF-1, which shows a higher TF inhibitory effect than the anti-human TF monoclonal antibody called HTF-1. Experiments using blood from healthy individuals, stimulated or not by LPS, or plasma spiked with 3 different levels of MV, demonstrated that the new assay was more sensitive and this allowed detection of MV TF activity in platelet free plasma (PFP) samples from healthy individuals. However, the assay was limited by an inter-assay variability, mainly due to the centrifugation step.

Conclusions: We have improved the sensitivity of the MV TF activity assay without losing specificity. This new assay could be used to evaluate levels of TF-positive MV as a potential biomarker of thrombotic risk in patients.
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http://dx.doi.org/10.1016/j.thromres.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825876PMC
October 2019

Microvesicles and Cancer Associated Thrombosis.

Semin Thromb Hemost 2019 Sep 20;45(6):593-603. Epub 2019 Aug 20.

Aix-Marseille University, INSERM, INRA, C2VN UMR_S1263, UFR de Pharmacie, Marseille, France.

Microvesicles (MVs) are small membrane enclosed structures released into the extracellular space by virtually all cell types. Their composition varies according to the cell origin and the stimulus which caused their formation. They harbor functional molecules and participate in intercellular communication. Endothelium, inflammatory cells, and cancer cells produce procoagulant MVs which contribute to cancer-associated thrombosis (CAT) in animal models. The tissue factor (TF) conveyed by these MVs was shown to play a key role in different animal models of experimental CAT. Alternatively, other molecular mechanisms involving polyphosphates or phosphatidylethanolamine could also be involved. In clinical practice, an association between an increase in the number of TF-positive or the procoagulant activity of these MVs and the occurrence of CAT has indeed been demonstrated in pancreatic-biliary cancers, suggesting that they could behave as a biomarker predictive for CAT. However, to date, this association was not confirmed in other types of cancer. Potential causes explaining this limited associated between MVs and CAT are (1) the diversity of mechanisms associating MVs and different types of cancer; (2) a more complex role of MVs in hemostasis integrating their anticoagulant and fibrinolytic activity; and (3) the lack of sensitivity, reproducibility, and standardization of current methodologies permitting measurement of MVs. Each of these hypotheses constitutes an interesting exploration path for a future reassessment of the clinical interest of the MVs in CAT.
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http://dx.doi.org/10.1055/s-0039-1693476DOI Listing
September 2019

Involvement of Platelets in Cancers.

Semin Thromb Hemost 2019 Sep 5;45(6):569-575. Epub 2019 Aug 5.

Aix Marseille Univ, INSERM, INRA, C2VN, Marseille, France.

Cancer-related venous thromboembolism (VTE) is frequent and constitutes the second leading cause of death in patients with cancer. High platelet count is one of independent predictive factors of cancer-associated VTE. Besides the implication of platelets in cancer-associated VTE, recent clinical and experimental evidences support that platelets play several roles in the progression of malignancies and inversely, cancer can also influence platelet count and activity. The objective of this report is to review the current literature regarding the role of platelets in cancer through experimental results and population-based studies. Platelets are implicated in cancer progression and metastasis through proangiogenic factors (growth factors and signaling pathways), antiangiogenic factors (angiostatin, endostatin, thrombospondin-1), and matrix metalloproteinases. In addition, platelets are involved in cancer-associated thrombosis and thus tumor cell-induced platelet activation, through anionic phospholipids on their surface, released soluble factors, such as P-selectin, CD40 ligand, platelet factor 4, thrombospondin-1 or beta-thromboglobulin, tumor cell procoagulant proteins (tissue factor, urokinase-type plasminogen activator, plasminogen activator inhibitor type 1), and microparticles. Due to these different mechanisms, platelets may represent a potential therapeutic target. The main current treatments against platelets are: (1) acetylsalicylic acid (aspirin) and nonsteroidal anti-inflammatory drugs, nonselective cyclo-oxygenase (COX)-1 and COX-2 inhibitors, which are associated with decreased cancer incidence and better overall survival and (2) irreversible inhibitor of P2Y12 subtype which decreases cancer incidence. Platelets are key players in tumor growth, metastasis, and cancer-associated thrombosis. This multifaceted role identifies them as a relevant therapeutic target for prevention of cancer occurrence and treatment of cancer.
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http://dx.doi.org/10.1055/s-0039-1693475DOI Listing
September 2019

Extracellular vesicles from T cells overexpress miR-146b-5p in HIV-1 infection and repress endothelial activation.

Sci Rep 2019 07 16;9(1):10299. Epub 2019 Jul 16.

Aix Marseille Univ, INSERM, C2VN, Marseille, France.

Human immunodeficiency virus type 1 (HIV-1) infection promotes a generalized activation of host responses that involves not only CD4 T cells, but also cells of the microenvironment, which are not directly infected, such as endothelial cells. The mechanisms triggering HIV-1-associated vascular alterations remain poorly understood. Extracellular vesicles (EVs), implicated in cell-to-cell communication, have been recently described as carriers of microRNAs (miRNAs). Here, we show that miR-146b-5p is upregulated in both CD4 T cells, CD4 T cell-derived EVs and circulating EVs obtained from antiretroviral therapy-naive HIV-1-infected patients. We further demonstrate that EVs from T cell line overexpressing miR-146b-5p mimics (miR-146b-EVs): 1) protect their miRNA cargo from RNase degradation, 2) transfer miR-146b-5p mimics into endothelial cells and 3) reduce endothelial inflammatory responses in vitro and in vivo in the lungs of mice through the downregulation of nuclear factor-κB-responsive molecules. These data advance our understanding on chronic inflammatory responses affecting endothelial homeostasis, in infectious and non-infectious diseases and pave the way for potential new anti-inflammatory strategies.
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http://dx.doi.org/10.1038/s41598-019-44743-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635508PMC
July 2019

Randomized controlled trial protocol to investigate the antiplatelet therapy effect on extracellular vesicles (AFFECT EV) in acute myocardial infarction.

Platelets 2020 26;31(1):26-32. Epub 2018 Dec 26.

1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland.

Activated platelets contribute to thrombosis and inflammation by the release of extracellular vesicles (EVs) exposing P-selectin, phosphatidylserine (PS) and fibrinogen. P2Y12 receptor antagonists are routinely administered to inhibit platelet activation in patients after acute myocardial infarction (AMI), being a combined antithrombotic and anti-inflammatory therapy. The more potent P2Y12 antagonist ticagrelor improves cardiovascular outcome in patients after AMI compared to the less potent clopidogrel, suggesting that greater inhibition of platelet aggregation is associated with better prognosis. The effect of ticagrelor and clopidogrel on the release of EVs from platelets and other P2Y12-exposing cells is unknown. This study compares the effects of ticagrelor and clopidogrel on (1) the concentrations of EVs from activated platelets (primary end point), (2) the concentrations of EVs exposing fibrinogen, exposing PS, from leukocytes and from endothelial cells (secondary end points) and (3) the procoagulant activity of plasma EVs (tertiary end points) in 60 consecutive AMI patients. After the percutaneous coronary intervention, patients will be randomized to antiplatelet therapy with ticagrelor (study group) or clopidogrel (control group). Blood will be collected from patients at randomization, 48 hours after randomization and 6 months following the index hospitalization. In addition, 30 age- and gender-matched healthy volunteers will be enrolled in the study to investigate the physiological concentrations and procoagulant activity of EVs using recently standardized protocols and EV-dedicated flow cytometry. Concentrations of EVs will be determined by flow cytometry. Procoagulant activity of EVs will be determined by fibrin generation test. The compliance and response to antiplatelet therapy will be assessed by impedance aggregometry. We expect that plasma from patients treated with ticagrelor (1) contains lower concentrations of EVs from activated platelets, exposing fibrinogen, exposing PS, from leukocytes and from endothelial cells and (2) has lower procoagulant activity, when compared to patients treated with clopidogrel. Antiplatelet therapy effect on EVs may identify a new mechanism of action of ticagrelor, as well as create a basis for future studies to investigate whether lower EV concentrations are associated with improved clinical outcomes in patients treated with P2Y12 antagonists.
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http://dx.doi.org/10.1080/09537104.2018.1557616DOI Listing
April 2020

A new assay to evaluate microvesicle plasmin generation capacity: validation in disease with fibrinolysis imbalance.

J Extracell Vesicles 2018 16;7(1):1494482. Epub 2018 Jul 16.

Aix-Marseille Université, C2VN, UMR-1263, INSERM, INRA 1260, UFR de Pharmacie, Marseille, France.

Among extracellular vesicles, leukocyte-derived microvesicles (LMVs) have emerged as complex vesicular structures. Primarily identified as procoagulant entities, they were more recently ascribed to plasmin generation capacity (MV-PGC). The objectives of this work were (1) to develop a new hybrid bio-assay combining the specific isolation of LMVs and measurement of their PGC, and compare its performance to the original method based on centrifugation, (2) to validate MV-PGC in septic shock, combining increased levels of LMVs and fibrinolytic imbalance. Using plasma sample spiked with LMVs featuring different levels of PGC, we demonstrated that CD15-beads specifically extracted LMVs. The MV dependency of the test was demonstrated using electron microscopy, high speed centrifugation, nanofiltration and detergent-mediated solubilization and the MV-PGC specificity using plasmin-specific inhibitors, or antibodies blocking elastase or uPA. Thanks to a reaction booster (ε-ACA), we showed that the assay was more sensitive and reproducible than the original method. Moreover, it exhibited a good repeatability, inter-operator and inter-experiment reproducibility. The new immunomagnetic bio-assay was further validated in patients with septic shock. As a result, we showed that MV-PGC values were significantly lower in septic shock patients who died compared to patients who survived, both at inclusion and 24 h later (1.4 [0.8-3.0] 3.1 [1.7-18]  × 10/min,  = 0.02; 1.4 [1-1.6] 5.2 [2.2-16]  × 10/min,  = 0.004). Interestingly, combining both MV-PGC and PAI-1 in a ratio significantly improved the predictive value of PAI-1. This strategy, a hybrid capture bioassay to specifically measure LMV-PGC using for the first time, opens new perspectives for measuring subcellular fibrinolytic potential in clinical settings with fibrinolytic imbalance.
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http://dx.doi.org/10.1080/20013078.2018.1494482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052415PMC
July 2018

CD34+ Hematopoietic Stem Cell Count Is Predictive of Vascular Event Occurrence in Children with Sickle Cell Disease.

Stem Cell Rev Rep 2018 Oct;14(5):694-701

Inserm UMR-S1140, Faculté de Pharmacie, Paris, France.

Background/objectives: Sickle cell disease (SCD) complications mostly result from vascular dysfunction, concerning systemic microvasculature and cerebral large vessels. The aim of this cohort study was to identify potential circulating biomarkers predictive for further vascular event occurrence in pediatric SCD.

Methods: We consecutively enrolled 108 children with SCD at steady state, aged 3-18 years old (median 9.8 years). Hematology, coagulation, hemolysis, endothelial, platelet and vascular activation parameters were recorded at inclusion. Neurovascular and systemic vascular events were prospectively recorded during a mean follow-up period of 27 months.

Results: Patients at steady state displayed significantly higher hemolysis and platelet activation markers, higher leukocyte, CD34+ hematopoietic stem cell and microvesicle counts, and a pro-coagulant profile compared to controls matched for age and ethnicity. Circulating endothelial cell or nucleosome level did not differ. During the follow-up period, 36 patients had at least one neurovascular (n = 12) or systemic vascular event (n = 25). In a multivariate model, high CD34+ cell count was the best predictor for the occurrence of a vascular event (OR 1.2 for 1000 cell/mL increase, 95% CI [1.049-1.4], p = 0.013, sensitivity 53%, specificity 84% for a threshold of 8675 cells/mL).

Conclusion: CD34+ cell count at steady state is a promising biomarker of further vascular event in children with SCD.
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http://dx.doi.org/10.1007/s12015-018-9835-8DOI Listing
October 2018

Biomarkers for the risk of thrombosis in pancreatic adenocarcinoma are related to cancer process.

Oncotarget 2018 May 29;9(41):26453-26465. Epub 2018 May 29.

Centre de Recherche sur l'Inflammation (CRI), INSERM UMR 1149, University of Paris Diderot, Sorbonne Paris Cité, Paris, France.

Background: Venous thrombo-embolic events (VTE) frequently occur in patients with pancreatic ductal adenocarcinoma (PDAC) and contribute to high morbidity and mortality.

Objectives: To determine whether VTE biomarkers are related to cancer, inflammation or precancerous states and to assess their relevance to predict VTE in PDAC.

Patients And Methods: We compared VTE biomarkers in patients with PDAC ( = 42), intraductal papillary mucinous neoplasm of the pancreas (IPMN, = 48) or chronic pancreatitis ( = 50). PDAC patients were followed-up for 6 months.

Results: Factor VIII, D-dimers, von Willebrand factor, free tissue factor pathway inhibitor and microvesicle-tissue factor (MV-TF) activity were higher in PDAC patients compared to patients with IPMN or chronic pancreatitis. PDAC patients with metastasis presented higher D-dimers and MV-TF activity compared to patients with localized lesions, but elevation of D-dimers was dependent on tumor size. In multivariate analysis, elevated D-dimers (≥2.16 µg/mL) or MV-TF activity (≥2.37 pg/mL) were significant risk factors for VTE in PDAC patients, after adjustment for age and sex (HR 4.9 [1.0-23.1] or HR 10.5 [1.5-72.4], mean [interquartile range], respectively). Cumulative probability of VTE at 6 months was higher in patients with elevated D-dimers (56.3% vs 15.6%, 0.009) and in patients with high MV-TF activity (74.3% vs 21.7%, 0.01).

Conclusions: VTE biomarkers including D-dimers and MV-TF activity are not related to inflammation but rather to cancer process and dissemination. D-dimers and MV-TF activity are associated to future VTE in PDAC patients and could help identify patients who could benefit from thromboprophylaxis.
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http://dx.doi.org/10.18632/oncotarget.25458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995170PMC
May 2018

A novel anti-CD146 antibody specifically targets cancer cells by internalizing the molecule.

Oncotarget 2017 Dec 28;8(68):112283-112296. Epub 2017 Nov 28.

INSERM UMR-S 1076, Aix-Marseille University, UFR Pharmacy, Marseille, France.

CD146 is an adhesion molecule present on many tumors (melanoma, kidney, pancreas, breast, ...). In addition, it has been shown to be expressed on vascular endothelial and smooth muscle cells. Generating an antibody able to specifically recognize CD146 in cancer cells (designated as tumor CD146), but not in normal cells, would thus be of major interest for targeting tumor CD146 without affecting the vascular system. We thus generated antibodies against the extracellular domain of the molecule produced in cancer cells and selected an antibody that specifically recognizes tumor CD146. This antibody (TsCD146 mAb) was able to detect CD146-positive tumors in human biopsies and , by PET imaging, in a murine xenograft model. In addition, TsCD146 mAb antibody was able to specifically detect CD146-positive cancer microparticles in the plasma of patients. TsCD146 mAb displayed also therapeutic effects since it was able to reduce the growth of human CD146-positive cancer cells xenografted in nude mice. This effect was due to a decrease in the proliferation and an increase in the apoptosis of CD146-positive cancer cells after TsCD146-mediated internalization of the cell surface CD146. Thus, TsCD146 mAb could be of major interest for diagnostic and therapeutic strategies against CD146-positive tumors in a context of personalized medicine.
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http://dx.doi.org/10.18632/oncotarget.22736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5762510PMC
December 2017

Biogenesis of Pro-senescent Microparticles by Endothelial Colony Forming Cells from Premature Neonates is driven by SIRT1-Dependent Epigenetic Regulation of MKK6.

Sci Rep 2017 08 15;7(1):8277. Epub 2017 Aug 15.

Aix Marseille Univ, INSERM, VRCM, Marseille, France.

Senescent cells may exert detrimental effect on microenvironment through the secretion of soluble factors and the release of extracellular vesicles, such as microparticles, key actors in ageing and cardiovascular diseases. We previously reported that sirtuin-1 (SIRT1) deficiency drives accelerated senescence and dysfunction of endothelial colony-forming cells (ECFC) in PT neonates. Because preterm birth (PT) increases the risk for cardiovascular diseases during neonatal period as well as at adulthood, we hypothesized that SIRT1 deficiency could control the biogenesis of microparticles as part of a senescence-associated secretory phenotype (SASP) of PT-ECFC and investigated the related molecular mechanisms. Compared to control ECFC, PT-ECFC displayed a SASP associated with increased release of endothelial microparticles (EMP), mediating a paracrine induction of senescence in naïve endothelial cells. SIRT1 level inversely correlated with EMP release and drives PT-ECFC vesiculation. Global transcriptomic analysis revealed changes in stress response pathways, specifically the MAPK pathway. We delineate a new epigenetic mechanism by which SIRT1 deficiency regulates MKK6/p38/Hsp27 pathway to promote EMP biogenesis in senescent ECFC. These findings deepen our understanding of the role of ECFC senescence in the disruption of endothelial homeostasis and provide potential new targets towards the control of cardiovascular risk in individuals born preterm.
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http://dx.doi.org/10.1038/s41598-017-08883-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557933PMC
August 2017

Platelet function and microparticle levels in atrial fibrillation: Changes during the acute episode.

Int J Cardiol 2017 Sep;243:216-222

IHU Liryc, Electrophysiology and Heart Modeling Institute, fondation Bordeaux Université, 33600, Pessac, France. Electronic address:

Background: Thrombotic risk constitutes a major complication of atrial fibrillation (AF). Platelets and microparticles (MPs) are important for hemostasis and thrombosis, however their participation during AF is not well known. The aim of this study was to characterize platelet function and MPs procoagulant and fibrinolytic activity in AF patients and to determine the effects of an acute-AF episode.

Methods: Blood was collected from paroxysmal (21) and persistent (16) AF patients referred for AF catheter ablation. Ten patients in sinus rhythm for 10days were induced in AF allowing comparisons of left atrium samples before and after induction. Platelet aggregation with ADP, TRAP, collagen, and ristocetin was studied. Platelet surface expression of PAR-1, αIIbβ3, GPIb and P-selectin were evaluated by flow cytometry, and MPs-associated procoagulant and fibrinolytic activity levels were determined by functional assays.

Results: A specific reduction in platelet aggregation to TRAP, activating the thrombin receptor PAR-1, was found in all AF patients. No differences in platelet receptor expression were found. Yet, after acute-induced AF, the platelet response was improved. Furthermore, a significant decrease of left atrium tissue factor-dependent procoagulant activity of MPs was observed.

Conclusion: Acute episodes of AF results in a decrease in MPs-associated tissue factor activity, possibly corresponding to consumption, which in turn favors coagulation and the local production of thrombin. A decreased platelet basal aggregation to TRAP may result from PAR1 desensitization, whereas the improved response after an induced episode of AF suggests activation of coagulation and PAR1 re-sensitization.
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http://dx.doi.org/10.1016/j.ijcard.2017.03.068DOI Listing
September 2017

Microvesicles in vascular homeostasis and diseases. Position Paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis and Vascular Biology.

Thromb Haemost 2017 06 1;117(7):1296-1316. Epub 2017 Jun 1.

Victoria Ridger, PhD, Department of Infection, Immunity and Cardiovascular Disease, Faculty of Medicine, Dentistry and Health, University of Sheffield, Sheffield, UK, E-mail: or, Chantal M. Boulanger, PhD, INSERM UMR-S 970, Paris Cardiovascular Research Center - PARCC, 56 rue Leblanc, 75015 Paris, France, E-mail:

Microvesicles are members of the family of extracellular vesicles shed from the plasma membrane of activated or apoptotic cells. Microvesicles were initially characterised by their pro-coagulant activity and described as "microparticles". There is mounting evidence revealing a role for microvesicles in intercellular communication, with particular relevance to hemostasis and vascular biology. Coupled with this, the potential of microvesicles as meaningful biomarkers is under intense investigation. This Position Paper will summarise the current knowledge on the mechanisms of formation and composition of microvesicles of endothelial, platelet, red blood cell and leukocyte origin. This paper will also review and discuss the different methods used for their analysis and quantification, will underline the potential biological roles of these vesicles with respect to vascular homeostasis and thrombosis and define important themes for future research.
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http://dx.doi.org/10.1160/TH16-12-0943DOI Listing
June 2017

Extracellular Vesicles in Angiogenesis.

Circ Res 2017 May;120(10):1658-1673

From the Aix-Marseille Univ, INSERM, VRCM, UMR_S 1076, Marseille, France (D.T., S.S., R.L., F.S., F.D.-G.); APHM, CHU de la Conception, Service d'Hématologie, Marseille, France (R.L., F.D.-G.); and APHM, CHU de la Conception, Laboratoire de Culture et Thérapie Cellulaire, INSERM, UMR_S 1076, CBT1409, Marseille, France (F.S.).

During the past decade, extracellular vesicles (EVs), which include apoptotic bodies, microvesicles, and exosomes, have emerged as important players in cell-to-cell communication in normal physiology and pathological conditions. EVs encapsulate and convey various bioactive molecules that are further transmitted to neighboring or more distant cells, where they induce various signaling cascades. The message delivered to the target cells is dependent on EV composition, which, in turn, is determined by the cell of origin and the surrounding microenvironment during EV biogenesis. Among their multifaceted role in the modulation of biological responses, the involvement of EVs in vascular development, growth, and maturation has been widely documented and their potential therapeutic application in regenerative medicine or angiogenesis-related diseases is drawing increasing interest. EVs derived from various cell types have the potential to deliver complex information to endothelial cells and to induce either pro- or antiangiogenic signaling. As dynamic systems, in response to changes in the microenvironment, EVs adapt their cargo composition to fine-tune the process of blood vessel formation. This article reviews the current knowledge on the role of microvesicles and exosomes from various cellular origins in angiogenesis, with a particular emphasis on the underlying mechanisms, and discusses the main challenges and prerequisites for their therapeutic applications.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.309681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426696PMC
May 2017

Methodological Guidelines to Study Extracellular Vesicles.

Circ Res 2017 May;120(10):1632-1648

From the Biomedical Engineering and Physics (F.A.W.C., T.G.v.L., E.v.d.P.), Vesicle Observation Centre (F.A.W.C., A.G., T.G.v.L., E.v.d.P., G.S., R.N.), and Laboratory of Experimental Clinical Chemistry (A.G., G.S., R.N.), Academic Medical Center, University of Amsterdam, The Netherlands; Extracellular Vesicles and Membrane Repair, UMR-5248-CBMN CNRS, University of Bordeaux, IPB, Pessac, France (A.R.B.); Department of Genetics, Cell- and Immunobology, Semmelweis University, Budapest, Hungary (E.I.B.); VRCM, UMRS-1076, INSERM, Aix-Marseille University, UFR de Pharmacie, Marseille, France (F.D.-G., R.L.); Haematology and vascular biology department, CHU La Conception, APHM, Marseille, France (F.D.-G., R.L.); Exosomes Research Group, Department of Pathology, VU University Medical Center, De Boelelaan 1117, Amsterdam, The Netherlands (E.E.E.D., D.M.P.); Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden (S.E.-A., Y.L.); Department of Physiology, Anatomy and Genetics, University of Oxford, United Kingdom (S.E.-A., I.M.); Bristol Heart Institute, University of Bristol, United Kingdom (C.E.); National Heart & Lung Institute, Imperial College London, United Kingdom (C.E.); 1st Chair and Department of Cardiology, Medical University of Warsaw, Poland (A.G.); Laboratory of Experimental Cancer Research, Department of Radiation Oncology and Experimental Cancer Research, Ghent University, Belgium (A.H., O.d.W.); Cancer Research Institute Ghent, Belgium (A.H., O.d.W.); Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia (A.F.H.); Department of Medicine, University of North Carolina at Chapel Hill (N.M.); Institute of Technology, University of Tartu, Estonia (I.M.); Scintillon Institute, San Diego, CA (J.P.N.); Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY (S.S.); and EV Core Facility, University of Helsinki and EV-Group, Division of Biochemistry and Biotechnology, Department of Biosciences, University of Helsinki, Finland (P.R.M.S.).

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.309417DOI Listing
May 2017

Increased serum levels of fractalkine and mobilisation of CD34CD45 endothelial progenitor cells in systemic sclerosis.

Arthritis Res Ther 2017 03 20;19(1):60. Epub 2017 Mar 20.

Internal Medicine Department, Assistance Publique-Hôpitaux de Marseille (APHM), CHU Nord, 13015, Marseilles, France.

Background: The disruption of endothelial homeostasis is a major determinant in the pathogenesis of systemic sclerosis (SSc) and is reflected by soluble and cellular markers of activation, injury and repair. We aimed to provide a combined assessment of endothelial markers to delineate specific profiles associated with SSc disease and its severity.

Methods: We conducted an observational, single-centre study comprising 45 patients with SSc and 41 healthy control subjects. Flow cytometry was used to quantify circulating endothelial microparticles (EMPs) and CD34 progenitor cell subsets. Colony-forming unit-endothelial cells (CFU-ECs) were counted by culture assay. Circulating endothelial cells were enumerated using anti-CD146-based immunomagnetic separation. Blood levels of endothelin-1, vascular endothelial growth factor (VEGF) and soluble fractalkine (s-Fractalkine) were evaluated by enzyme-linked immunosorbent assay. Disease-associated markers were identified using univariate, correlation and multivariate analyses.

Results: Enhanced numbers of EMPs, CFU-ECs and non-haematopoietic CD34CD45 endothelial progenitor cells (EPCs) were observed in patients with SSc. Patients with SSc also displayed higher serum levels of VEGF, endothelin-1 and s-Fractalkine. s-Fractalkine levels positively correlated with CD34CD45 EPC numbers. EMPs, s-Fractalkine and endothelin-1 were independent factors associated with SSc. Patients with high CD34CD45 EPC numbers had lower forced vital capacity values. Elevated s-Fractalkine levels were associated with disease severity, a higher frequency of pulmonary fibrosis and altered carbon monoxide diffusion.

Conclusions: This study identifies the mobilisation of CD34CD45 EPCs and high levels of s-Fractalkine as specific features of SSc-associated vascular activation and disease severity. This signature may provide novel insights linking endothelial inflammation and defective repair processes in the pathogenesis of SSc.
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http://dx.doi.org/10.1186/s13075-017-1271-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359964PMC
March 2017

Microparticles and Fibrinolysis.

Semin Thromb Hemost 2017 Mar 6;43(2):129-134. Epub 2016 Dec 6.

Division of Hematology/Oncology, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Microparticles (MPs) are submicronic vesicles which are formed by budding of the cellular membrane of virtually any cell type in response to cell activation or apoptosis. Both circulating MPs and MPs generated within tissues harbor molecules with a large repertoire of biological activities and transfer material to target cells. Depending on their cellular origin, the stimuli triggering their formation, or their localization, they may participate in the maintenance of organ or vascular homeostasis as well as inducing dysfunction. MPs have mostly been described as having procoagulant properties. However, the fact that some MP subsets are able to efficiently generate plasmin suggests that the role of MPs in hemostasis is more complex than initially thought. In this review, we summarize key findings showing that MPs provide a heterogeneous catalytic surface for plasmin generation, according to their cellular origin. We further address the specific features of the MP-dependent fibrinolytic system. Potential consequences of this MP-associated fibrinolytic activity in pathology are illustrated in cancer.
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http://dx.doi.org/10.1055/s-0036-1592301DOI Listing
March 2017

Detection of EpCAM-positive microparticles in pleural fluid: A new approach to mini-invasively identify patients with malignant pleural effusions.

Oncotarget 2016 Jan;7(3):3357-66

VRCM, UMR-S1076, Aix-Marseille Université, INSERM, Faculté de Pharmacie, Marseille, France.

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.
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http://dx.doi.org/10.18632/oncotarget.6581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823111PMC
January 2016
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