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In chronic kidney disease (CKD), uremic solutes accumulate in blood and tissues. These compounds probably contribute to the marked increase in cardiovascular risk during the progression of CKD. The uremic solutes indoxyl sulfate and indole-3-acetic acid (IAA) are particularly deleterious for endothelial cells. Here we performed microarray and comparative PCR analyses to identify genes in endothelial cells targeted by these two uremic solutes. We found an increase in endothelial expression of tissue factor in response to indoxyl sulfate and IAA and upregulation of eight genes regulated by the transcription factor aryl hydrocarbon receptor (AHR). The suggestion by microarray analysis of an involvement of AHR in tissue factor production was confirmed by siRNA inhibition and the indirect AHR inhibitor geldanamycin. These observations were extended to peripheral blood mononuclear cells. Tissue factor expression and activity were also increased by AHR agonist dioxin. Finally, we measured circulating tissue factor concentration and activity in healthy control subjects and in patients with CKD (stages 3–5d), and found that each was elevated in patients with CKD. Circulating tissue factor levels were positively correlated with plasma indoxyl sulfate and IAA. Thus, indolic uremic solutes increase tissue factor production in endothelial and peripheral blood mononuclear cells by AHR activation, evoking a ‘dioxin-like’ effect. This newly described mechanism of uremic solute toxicity may help understand the high cardiovascular risk of CKD patients.

Background Ultra-lung-protective ventilation may be useful during veno-venous extracorporeal membrane oxygenation (vv-ECMO) for severe acute respiratory distress syndrome (ARDS) to minimize ventilator-induced lung injury and to facilitate lung recovery. The objective was to compare pulmonary and systemic biotrauma evaluated by numerous biomarkers of inflammation, epithelial, endothelial injuries, and lung repair according to two ventilator strategies on vv-ECMO. Methods This is a prospective randomized controlled study. Patients were randomized to receive during 48 h either ultra-lung-protective ventilation combining very low tidal volume (1–2 mL/kg of predicted body weight), low respiratory rate (5–10 cycles per minute), positive expiratory transpulmonary pressure, and 16 h of prone position or lung-protective-ventilation which followed the ECMO arm of the EOLIA trial (control group). Results The primary outcome was the alveolar concentrations of interleukin-1-beta, interleukin-6, interleukin-8, surfactant protein D, and blood concentrations of serum advanced glycation end products and angiopoietin-2 48 h after randomization. Enrollment was stopped for futility after the inclusion of 39 patients. Tidal volume, respiratory rate, minute ventilation, plateau pressure, and mechanical power were significantly lower in the ultra-lung-protective group. None of the concentrations of the pre-specified biomarkers differed between the two groups 48 h after randomization. However, a trend to higher 60-day mortality was observed in the ultra-lung-protective group compared to the control group (45 vs 17%, p  = 0.06). Conclusions Despite a significant reduction in the mechanical power, ultra-lung-protective ventilation during 48 h did not reduce biotrauma in patients with vv-ECMO-supported ARDS. The impact of this ventilation strategy on clinical outcomes warrants further investigation. Trial registration Clinical trial registered with www.clinicaltrials.gov ( NCT03918603 ). Registered 17 April 2019.

Nanofat is a relatively recent fat grafting technique obtained involving the mechanical emulsification of adipose tissue whose preparation is produced at the patient’s bedside. Although it was initially reported to improve skin quality in intradermal applications, it is now increasingly used in regenerative medicine. However, the absence of standardized protocols and the diversity of commercial devices result in nanofat products of variable quality. This study presents the first comprehensive comparison of nanofat obtained from different commercially available preparation systems, combining both their technical performance and biological characterization. Lipoaspirates from five healthy donors were processed using eight commercially available devices for nanofat production using emulsification or micronization techniques. The technical parameters included preparation time, ease of preparation and injection, volumetric yield, and residual aqueous fraction. Biological analyses included stromal vascular fraction isolation with evaluation of cell viability, viable nucleated cell yield, immunophenotypic cell subtype characterization and clonogenic capacity. These parameters were compared using a scoring model that enabled inter-kit ranking, integrating both a technical performance score and a biological quality score. Additionally, nanofat-conditioned media were collected for extracellular vesicles (EVs) quantification and subtyping by flow cytometry, and confocal microscopy was performed to evaluate the preservation of mature adipocytes, capillary networks, and the extracellular matrix. All devices demonstrated satisfactory technical performance, with Puregraft Boost V2 and Emulsfat achieving the highest overall technical scores. Cell viability was consistently high, with median values above 85% across all devices. Adinizer provided the greatest proportion of adipose-derived stromal/stem cells and achieved the highest overall biological score. In contrast, Hy-Tissue Nanofat produced the lowest cell yields together with the highest leukocyte proportions. All nanofats contained clonogenic progenitors. Extracellular vesicles concentrations were comparable between devices, and were mainly influenced by donor variability, although Emulsfat was enriched in adipocyte-derived EVs. Microscopic analysis revealed preservation of adipocytes, vascular networks, and the extracellular matrix across devices, challenging the assumption that emulsification or micronization completely disrupts tissue architecture. Nanofat properties are strongly device dependent, with possible dissociation between technical ease and biological quality. This first comparative study highlights the need for standardized preparation methods and qualification criteria, and provides guidance for selecting devices aligned with specific clinical objectives to optimize regenerative outcomes.

Rationale COVID-19-associated acute-respiratory distress syndrome (C-ARDS) results from a direct viral injury associated with host excessive innate immune response mainly affecting the lungs. However, cytokine profile in the lung compartment of C-ARDS patients has not been widely studied, nor compared to non-COVID related ARDS (NC-ARDS). Objectives To evaluate caspase-1 activation, IL-1 signature, and other inflammatory cytokine pathways associated with tissue damage using post-mortem lung tissues, bronchoalveolar lavage fluids (BALF), and serum across the spectrum of COVID-19 severity. Methods Histological features were described and activated-caspase-1 labeling was performed in 40 post-mortem biopsies. Inflammatory cytokines were quantified in BALF and serum from 19 steroid-treated-C-ARDSand compared to 19 NC-ARDS. Cytokine concentrations were also measured in serum from 128 COVID-19 patients at different severity stages. Measurements and main results Typical “diffuse alveolar damage” in lung biopsies were associated with activated caspase-1 expression and vascular lesions. Soluble Caspase-1p20, IL-1β, IL-1Ra, IL-6 and at lower level IFNγ and CXCL-10, were highly elevated in BALF from steroid-treated-C-ARDS as well as in NC-ARDS. IL-1β appeared concentrated in BALF, whereas circulating IL-6 and IL-1Ra concentrations were comparable to those in BALF and correlated with severity. TNFα, TNFR1 and CXCL8 however, were significantly higher in NC-ARDS compared to C-ARDS, treated by steroid. Conclusions In the lungs of C-ARDS, both caspase-1 activation with a predominant IL-1β/IL-6 signature and IFNγ -associated chemokines are elevated despite steroid treatment. These pathways may be specifically targeted in ARDS to improve response to treatment and to limit alveolar and vascular lung damage.

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 MAPK /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.

Septic shock is characterised by abnormal coagulation activation with defective fibrinolysis, leading to a high mortality rate. Cellular activation triggers the release of extracellular vesicles (EVs) conveying both procoagulant and fibrinolytic activities. We investigated whether the balance between these activities, termed EV‐coagulolytic balance (EV‐CLB), predicts day‐90 mortality in 225 septic shock patients included in a multicentre prospective study. EV‐CLB, quantified as a ratio of TF‐dependent thrombin generation to uPA‐dependent plasmin generation, was higher in non‐survivors than in survivors at 24 h (2.78 [0.86–16.1] a.u. vs. 0.97 [0.34–2.18] a.u., p < 0.001). Moreover, survivors showed a significant decrease in EV‐CLB from H0 to H48 in contrast to non‐survivors. EV‐CLB was a better predictor than EV‐associated–procoagulant and ‐fibrinolytic activities taken individually and better correlated with sepsis severity markers such as SAPS II and lactate levels. Multivariate Cox regression models including severity markers and comorbidities confirmed EV‐CLB as an independent predictor of mortality in septic shock patients. Interestingly, subgroup analysis revealed EV‐CLB's strong prognostic value in peritonitis, biliary and urinary tract infections and Gram‐negative sepsis. Despite challenges in EV measurement requiring technical advancement for clinical translation, EV‐CLB represents a potential novel biomarker to guide individualised therapy targeting coagulation/fibrinolysis imbalance in septic shock. Trial Registration: This trial was registered at ClinicalTrials.gov identifier: NCT02062970

The concentration of cells is a key component of modern blood tests. Given the biomarker potential of extracellular vesicles (EVs) in blood, we aimed to establish reference ranges for blood cell‐derived EVs using flow cytometry. To address the orders‐of‐magnitude variability in reported EV concentrations between different flow cytometers (FCMs), we first validated a calibration methodology to enable reproducible EV concentration measurements. The methodology was evaluated in an interlaboratory comparison study and shows that calibration reduces the median absolute deviation of EV concentrations measured on 25 different FCMs from 67 % to 25 %–31 %. The calibration methodology was then used to determine reference ranges of erythrocyte‐, leukocyte‐, and platelet‐derived EVs in human blood plasma in a cohort of healthy individuals (n = 224). This study demonstrates that calibration enables comparable concentration measurements of blood cell‐derived EVs, thereby bringing EVs one step closer to clinical applications.

Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta.

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] vs 3.1 [1.7-18] A 405  × 10 −3 /min, p = 0.02; 1.4 [1-1.6] vs 5.2 [2.2-16] A 405  × 10 −3 /min, p = 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.

Flow cytometry (FCM) offers a multiparametric technology capable of characterizing single extracellular vesicles (EVs). However, most flow cytometers are designed to detect cells, which are larger than EVs. Whereas cells exceed the background noise, signals originating from EVs partly overlap with the background noise, thereby making EVs more difficult to detect than cells. This technical mismatch together with complexity of EV‐containing fluids causes limitations and challenges with conducting, interpreting and reproducing EV FCM experiments. To address and overcome these challenges, researchers from the International Society for Extracellular Vesicles (ISEV), International Society for Advancement of Cytometry (ISAC), and the International Society on Thrombosis and Haemostasis (ISTH) joined forces and initiated the EV FCM working group. To improve the interpretation, reporting, and reproducibility of future EV FCM data, the EV FCM working group published an ISEV position manuscript outlining a framework of minimum information that should be reported about an FCM experiment on single EVs (MIFlowCyt‐EV). However, the framework contains limited background information. Therefore, the goal of this compendium is to provide the background information necessary to design and conduct reproducible EV FCM experiments. This compendium contains background information on EVs, the interaction between light and EVs, FCM hardware, experimental design and preanalytical procedures, sample preparation, assay controls, instrument data acquisition and calibration, EV characterization, and data reporting. Although this compendium focuses on EVs, many concepts and explanations could also be applied to FCM detection of other particles within the EV size range, such as bacteria, lipoprotein particles, milk fat globules, and viruses.