The fluorochrome-to-protein ratio is crucial for the flow cytometric detection of tissue factor on extracellular vesicles.

Extracellular vesicles (EVs) have crucial roles in hemostasis and coagulation. They sustain coagulation by exposing phosphatidylserine and initiate clotting by surface expression of tissue factor (TF) under inflammatory conditions. As their relevance as biomarkers of coagulopathy is increasingly recognized, there is a need for the sensitive and reliable detection of TF+ EVs, but their flow cytometric analysis is challenging and has yielded controversial findings for TF expression on EVs in the vascular system. We investigated the effect of different fluorochrome-to-protein (F/P) ratios of anti-TF-fluorochrome conjugates on the flow cytometric detection of TF+ EVs from activated monocytes, mesenchymal stem cells (MSCs), and in COVID-19 plasma. Using a FITC-labeled anti-TF antibody (clone VD8), we show that the percentage of TF+ EVs declined with decreasing F/P ratios. TF was detected on 7.6%, 5.4%, and 1.1% of all EVs derived from activated monocytes at F/P ratios of 7.7:1, 6.6:1, and 5.2:1. A similar decline was observed for EVs from MSCs and for EVs in plasma, whereas the detection of TF on cells remained unaffected by different F/P ratios. We provide clear evidence that next to the antibody clone, the F/P ratio affects the flow cytometric detection of TF+ EVs and should be carefully controlled.

Dynamic cultivation of human mesenchymal stem/stromal cells for the production of extracellular vesicles in a 3D bioreactor system.

PURPOSE: 3D cell culture and hypoxia have been demonstrated to increase the therapeutic effects of mesenchymal stem/stromal cells (MSCs)-derived extracellular vesicles (EVs). In this study, a process for the production of MSC-EVs in a novel 3D bioreactor system under normoxic and hypoxic conditions was established and the resulting EVs were characterized. METHODS: Human adipose-derived MSCs were seeded and cultured on a 3D membrane in the VITVO® bioreactor system for 7 days. Afterwards, MSC-EVs were isolated and characterized via fluorescence nanoparticle tracking analysis, flow cytometry with staining against annexin V (Anx5) as a marker for EVs exposing phosphatidylserine, as well as CD73 and CD90 as MSC surface markers. RESULTS: Cultivation of MSC in the VITVO® bioreactor system demonstrated a higher concentration of MSC-EVs from the 3D bioreactor (9.1 × 109 ± 1.5 × 109 and 9.7 × 109 ± 3.1 × 109 particles/mL) compared to static 2D culture (4.2 × 109 ± 7.5 × 108 and 3.9 × 109 ± 3.0 × 108 particles/mL) under normoxic and hypoxic conditions, respectively. Also, the particle-to-protein ratio as a measure for the purity of EVs increased from 3.3 × 107 ± 1.1 × 107 particles/µg protein in 2D to 1.6 × 108 ± 8.3 × 106 particles/µg protein in 3D. Total MSC-EVs as well as CD73-CD90+ MSC-EVs were elevated in 2D normoxic conditions. The EV concentration and size did not differ significantly between normoxic and hypoxic conditions. CONCLUSION: The production of MSC-EVs in a 3D bioreactor system under hypoxic conditions resulted in increased EV concentration and purity. This system could be especially useful in screening culture conditions for the production of 3D-derived MSC-EVs.

Expression of Tissue Factor and Platelet/Leukocyte Markers on Extracellular Vesicles Reflect Platelet-Leukocyte Interaction in Severe COVID-19.

Severe COVID-19 is frequently associated with thromboembolic complications. Increased platelet activation and platelet-leukocyte aggregate formation can amplify thrombotic responses by inducing tissue factor (TF) expression on leukocytes. Here, we characterized TF-positive extracellular vesicles (EVs) and their cellular origin in 12 patients suffering from severe COVID-19 (time course, 134 samples overall) and 25 healthy controls. EVs exposing phosphatidylserine (PS) were characterized by flow cytometry. Their cellular origin was determined by staining with anti-CD41, anti-CD45, anti-CD235a, and anti-CD105 as platelet, leukocyte, red blood cell, and endothelial markers. We further investigated the association of EVs with TF, platelet factor 4 (PF4), C-reactive protein (CRP), and high mobility group box-1 protein (HMGB-1). COVID-19 patients showed higher levels of PS-exposing EVs compared to controls. The majority of these EVs originated from platelets. A higher amount of EVs in patient samples was associated with CRP, HMGB-1, PF4, and TF as compared to EVs from healthy donors. In COVID-19 samples, 16.5% of all CD41+ EVs displayed the leukocyte marker CD45, and 55.5% of all EV aggregates (CD41+CD45+) co-expressed TF, which reflects the interaction of platelets and leukocytes in COVID-19 on an EV level.

Driving the quadricyclane-to-norbornadiene isomerization by charge separation with perylenediimide as electron acceptor.

Through comprehensive photo-assays, this study investigates the reaction coordinate governing the interconversion between quadricyclane (QC) and norbornadiene (NBD) upon photo-irradiation up to a wavelength of 550 nm. To harness this spectroscopic range for energy release, we link the NBD-core with a highly electron-accepting perylenediimide (PDI) with broad absorption, achieving strong electronic coupling between them. We detail the successful synthesis and present extensive DFT calculations to determine the amount of stored energy. By means of transient absorption spectroscopy, an oxidative electron transfer is observed during the QC-to-NBD isomerization following the initial PDI photoexcitation. This charge-separated state is key to triggering the back-isomerization with visible light excitation.

Tetracene Dimers: A Platform for Intramolecular Down- and Up-conversion.

Photon energy conversion can be accomplished in many different ways, including the two opposing manners, down-conversion (i.e., singlet fission, SF) and up-conversion (i.e., triplet-triplet annihilation up-conversion, TTA-UC). Both processes have the potential to help overcome the detailed balance limit of single-junction solar cells. Tetracene, in which the energies of the lowest singlet excited state and twice the triplet excited state are comparable, exhibits both down- and up-conversion. Here, we have designed meta-diethynylphenylene- and 1,3-diethynyladamantyl-linked tetracene dimers, which feature different electronic coupling, to characterize the interplay between intramolecular SF (intra-SF) and intramolecular TTA-UC (intra-TTA-UC) via steady-state and time-resolved absorption and fluorescence spectroscopy. Furthermore, we have used Pd-phthalocyanine as a sensitizer to enable intra-TTA-UC in the two dimers via indirect photoexcitation in the near-infrared part of the solar spectrum. The work is rounded off by temperature-dependent measurements, which outline key aspects of how thermal effects impact intra-SF and intra-TTA-UC in different dimers.

Infection with SARS-CoV-2 Is Associated with Elevated Levels of IP-10, MCP-1, and IL-13 in Sepsis Patients.

Immunothrombosis, an excessive inflammatory response with simultaneous overactivation of the coagulation system, is a central pathomechanism in sepsis and COVID-19. It is associated with cellular activation, vascular damage, and microvascular thrombosis, which can lead to multiple organ failure and death. Here, we characterized factors related to immunothrombosis in plasma samples from 78 sepsis patients. In the course of routine clinical testing, SARS-CoV-2 was detected in 14 of these patients. Viral infection was associated with a higher mortality. Both, COVID-19 negative and COVID-19 positive sepsis patients showed increased levels of effectors of immunothrombosis, including platelet factor 4, D-dimer, nucleosomes, citrullinated histone H3, high mobility group box-1 protein, as well as phosphatidylserine-expressing platelet-derived extracellular vesicles, compared to healthy controls (n = 25). Using a 27-plex cytokine bead array, we found that Interleukin (IL)-1ra, IL-6, IL-8, IL-13, tumor necrosis factor (TNF)-α, interferon inducible protein (IP)-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, and granulocyte-colony stimulating factor (G-CSF) were elevated in both, COVID-19 negative and COVID-19 positive sepsis patients, as compared to healthy controls. SARS-CoV-2 infection was associated with elevated levels of IP-10, MCP-1, and IL-13, while all other mediators widely overlapped between COVID-19 negative and COVID-19 positive patients.

Polyzwitterionic Coating of Porous Adsorbents for Therapeutic Apheresis.

Adsorbents for whole blood apheresis need to be highly blood compatible to minimize the activation of blood cells on the biomaterial surface. Here, we developed blood-compatible matrices by surface modification with polyzwitterionic polysulfobetainic and polycarboxybetainic coatings. Photoreactive zwitterionic terpolymers were synthesized by free-radical polymerization of zwitterionic, photoreactive, and fluorescent monomers. Upon UV irradiation, the terpolymers were photodeposited and mutually crosslinked on the surface of hydrophobic polystyrene-co-divinylbenzene and hydrophilic polyacrylamide-co-polyacrylate (DALI) beads. Fluorescent microscopy revealed coatings with an average thickness of 5 µm, which were limited to the bead surface. Blood compatibility was assessed based on polymer-induced hemolysis, coagulation parameters, and in vitro tests. The maintenance of the adsorption capacity after coating was studied in human whole blood with cytokines for polystyrene beads (remained capacity 25-67%) and with low-density lipoprotein (remained capacity 80%) for polyacrylate beads. Coating enhanced the blood compatibility of hydrophobic, but not of hydrophilic adsorbents. The most prominent effect was observed on coagulation parameters (e.g., PT, aPTT, TT, and protein C) and neutrophil count. Polycarboxybetaine with a charge spacer of five carbons was the most promising polyzwitterion for the coating of adsorbents for whole blood apheresis.

Targeting the Structural Integrity of Extracellular Vesicles via Nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analysis (nES GEMMA).

Extracellular vesicles (EVs) are in the scientific spotlight due to their potential application in the medical field, ranging from medical diagnosis to therapy. These applications rely on EV stability during isolation and purification-ideally, these steps should not impact vesicle integrity. In this context, we investigated EV stability and particle numbers via nano electrospray gas-phase electrophoretic mobility molecular analysis (nES GEMMA) and nanoparticle tracking analysis (NTA). In nES GEMMA, native, surface-dry analytes are separated in the gas-phase according to the particle size. Besides information on size and particle heterogeneity, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU, 18 October 2011). Likewise, and in contrast to NTA, nES GEMMA enables detection of co-purified proteins. On the other hand, NTA, yielding data on hydrodynamic size distributions, is able to relate particle concentrations, omitting electrolyte exchange (and resulting EV loss), which is prerequisite for nES GEMMA. Focusing on EVs of different origin, we compared vesicles concentrations and stability, especially after electrolyte exchange and size exclusion chromatography (SEC). Co-isolated proteins were detected in most samples, and the vesicle amount varied in dependence on the EV source. We found that depletion of co-purified proteins was achievable via SEC, but was associated with a loss of EVs and-most importantly-with decreased vesicle stability, as detected via a reduced nES GEMMA measurement repeatability. Ultimately, we propose the repeatability of nES GEMMA to yield information on EV stability, and, as a result, we propose that nES GEMMA can yield additional valuable information in EV research.

Activated Platelets and Platelet-Derived Extracellular Vesicles Mediate COVID-19-Associated Immunothrombosis.

Activated platelets and platelet-derived extracellular vesicles (EVs) have emerged as central players in thromboembolic complications associated with severe coronavirus disease 2019 (COVID-19). Platelets bridge hemostatic, inflammatory, and immune responses by their ability to sense pathogens via various pattern recognition receptors, and they respond to infection through a diverse repertoire of mechanisms. Dysregulated platelet activation, however, can lead to immunothrombosis, a simultaneous overactivation of blood coagulation and the innate immune response. Mediators released by activated platelets in response to infection, such as antimicrobial peptides, high mobility group box 1 protein, platelet factor 4 (PF4), and PF4+ extracellular vesicles promote neutrophil activation, resulting in the release of neutrophil extracellular traps and histones. Many of the factors released during platelet and neutrophil activation are positively charged and interact with endogenous heparan sulfate or exogenously administered heparin via electrostatic interactions or via specific binding sites. Here, we review the current state of knowledge regarding the involvement of platelets and platelet-derived EVs in the pathogenesis of immunothrombosis, and we discuss the potential of extracorporeal therapies using adsorbents functionalized with heparin to deplete platelet-derived and neutrophil-derived mediators of immunothrombosis.

Heparin-Functionalized Adsorbents Eliminate Central Effectors of Immunothrombosis, including Platelet Factor 4, High-Mobility Group Box 1 Protein and Histones.

Inflammation and thrombosis are closely intertwined in numerous disorders, including ischemic events and sepsis, as well as coronavirus disease 2019 (COVID-19). Thrombotic complications are markers of disease severity in both sepsis and COVID-19 and are associated with multiorgan failure and increased mortality. Immunothrombosis is driven by the complement/tissue factor/neutrophil axis, as well as by activated platelets, which can trigger the release of neutrophil extracellular traps (NETs) and release further effectors of immunothrombosis, including platelet factor 4 (PF4/CXCL4) and high-mobility box 1 protein (HMGB1). Many of the central effectors of deregulated immunothrombosis, including activated platelets and platelet-derived extracellular vesicles (pEVs) expressing PF4, soluble PF4, HMGB1, histones, as well as histone-decorated NETs, are positively charged and thus bind to heparin. Here, we provide evidence that adsorbents functionalized with endpoint-attached heparin efficiently deplete activated platelets, pEVs, PF4, HMGB1 and histones/nucleosomes. We propose that this elimination of central effectors of immunothrombosis, rather than direct binding of pathogens, could be of clinical relevance for mitigating thrombotic complications in sepsis or COVID-19 using heparin-functionalized adsorbents.

A possible role of gas-phase electrophoretic mobility molecular analysis (nES GEMMA) in extracellular vesicle research.

The emerging role of extracellular vesicles (EVs) as biomarkers and their envisioned therapeutic use require advanced techniques for their detailed characterization. In this context, we investigated gas-phase electrophoresis on a nano electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA, aka nES differential mobility analyzer, nES DMA) as an alternative to standard analytical techniques. In gas-phase electrophoresis, single-charged, surface-dry, native, polydisperse, and aerosolized analytes, e.g., proteins or bio-nanoparticles, are separated according to their electrophoretic mobility diameter, i.e., globular size. Subsequently, monodisperse particles are counted after a nucleation step in a supersaturated atmosphere as they pass a focused laser beam. Hence, particle number concentrations are obtained in accordance with recommendations of the European Commission for nanoparticle characterization (2011/696/EU from October 18th, 2011). Smaller sample constituents (e.g., co-purified proteins) can be detected next to larger ones (e.g., vesicles). Focusing on platelet-derived EVs, we compared different vesicle isolation techniques. In all cases, nanoparticle tracking analysis (NTA) confirmed the presence of vesicles. However, nES GEMMA often revealed a significant co-purification of proteins from the sample matrix, precluding gas-phase electrophoresis of less-diluted samples containing higher vesicle concentrations. Therefore, mainly peaks in the protein size range were detected. Mass spectrometry revealed that these main contaminants belonged to the group of globulins and coagulation-related components. An additional size exclusion chromatography (SEC) step enabled the depletion of co-purified, proteinaceous matrix components, while a label-free quantitative proteomics approach revealed no significant differences in the detected EV core proteome. Hence, the future in-depth analysis of EVs via gas-phase electrophoresis appears feasible. Platelet-derived extracellular vesicles (EVs)with/without additional size exclusion chromatographic (SEC) purification were subjected to nanoparticle tracking analysis (NTA) and gas-phase electrophoresis (nES GEMMA). The latter revealed presence of co-purified proteins, targetable via mass spectrometry (MS). MS also revealed that SEC did not influence EV protein content. To conclude, nES GEMMA is a valuable tool for quality control of EV-containing samples under native conditions allowing for detection of co-purified proteins from complex matrices.

Analysis of Inflammatory Mediator Profiles in Sepsis Patients Reveals That Extracellular Histones Are Strongly Elevated in Nonsurvivors.

The timely recognition of sepsis and the prediction of its clinical course are challenging due to the complex molecular mechanisms leading to organ failure and to the heterogeneity of sepsis patients. Treatment strategies relying on a "one-fits-all" approach have failed to reduce mortality, suggesting that therapeutic targets differ between patient subgroups and highlighting the need for accurate analysis of the molecular cascades to assess the highly variable host response. Here, we characterized a panel of 44 inflammatory mediators, including cytokines, chemokines, damage-associated molecular patterns, and coagulation-related factors, as well as markers of endothelial activation in 30 patients suffering from renal failure in the course of sepsis. All patients received continuous veno-venous hemodialysis with either high cut-off filters or with standard filters, and mediators were quantified for all patients at the initiation of dialysis and after 24 h and 48 h. Mediator concentrations in individual patients ranged widely, demonstrating the heterogeneity of sepsis patients. None of the mediators correlated with SAPS III or TISS scores. The overall in-hospital mortality of the study population was 56.7% (57.1% vs. 56.3% for high cut-off vs. standard filter). The two filter groups differed regarding most of the mediator levels at baseline, prohibiting conclusions regarding the effect of standard filters versus high cut-off filters on mediator depletion. The elevation and correlation of damage-associated molecular patterns and markers of endothelial activation gave evidence of severe tissue damage. In particular, extracellular histones were strongly increased and were almost 30-fold higher in nonsurvivors as compared to survivors, indicating their diagnostic and prognostic potential.

Comparative Analysis of Platelet-Derived Extracellular Vesicles Using Flow Cytometry and Nanoparticle Tracking Analysis.

Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers (Gallios and CytoFLEX LX) and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA+CD41+ EVs compared to Gallios. NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.

Extracellular vesicles are associated with C-reactive protein in sepsis.

There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP+ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP+ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP+ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP+ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.

Desialylation of platelet surface glycans enhances platelet adhesion to adsorbent polymers for lipoprotein apheresis.

BACKGROUND: Lipoprotein apheresis is an important therapeutic option in homozygous familial hypercholesterolemia, progressive atherosclerosis, or when depletion of lipoprotein(a) is indicated. It is generally regarded as safe, but drops in platelet counts as well as sporadic episodes of thrombocytopenia have been reported. We assessed the influence of platelet desialylation, which may be induced by endogenous or pathogen-derived neuraminidases, on platelet adhesion to polyacrylate-based adsorbents for whole blood lipoprotein apheresis. METHODS: Medical grade platelet concentrates were incubated with neuraminidase in vitro and were circulated over adsorbent columns downscaled from clinical application. RESULTS: Cleavage of terminal sialic residues resulted in platelet activation with significantly elevated expression of platelet factor 4 (PF4) and in enhanced platelet adhesion to the adsorbent, accompanied by a pronounced drop in platelet counts in the column flow-through. CONCLUSION: Alterations in endogenous neuraminidase activity or exogenous (pathogen-derived) neuraminidase may trigger enhanced platelet adhesion in whole blood lipoprotein apheresis.

Characterization and Chondroprotective Effects of Extracellular Vesicles From Plasma- and Serum-Based Autologous Blood-Derived Products for Osteoarthritis Therapy.

Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA.

Extracellular Vesicles Derived From Platelets, Red Blood Cells, and Monocyte-Like Cells Differ Regarding Their Ability to Induce Factor XII-Dependent Thrombin Generation.

As transmitters of biological information, extracellular vesicles (EVs) are crucial for the maintenance of physiological homeostasis, but also contribute to pathological conditions, such as thrombotic disorders. The ability of EVs to support thrombin generation has been linked to their exposure of phosphatidylserine, an anionic phospholipid that is normally restricted to the inner leaflet of the plasma membrane but exposed on the outer leaflet during EV biogenesis. Here, we investigated whether EVs of different cellular origin and from different settings, namely platelets and red blood cells from blood bank units and a monocyte-like cell line (THP-1), differ regarding their potential to support factor XII-dependent thrombin generation. EVs were isolated from blood products or THP-1 cell culture supernatants using differential centrifugation and characterized by a combination of flow cytometry, nanoparticle tracking analysis, and Western blotting. Soluble factors co-enriched during the isolation of EVs were depleted from blood-cell derived EV fractions using size exclusion chromatography, while proteins bound to the surface of EVs were degraded by mild protease treatment. We found that platelet-derived and red blood cell-derived EVs supported factor XII-dependent thrombin generation to comparable extents, while monocytic EVs failed to support thrombin generation when added to EV-depleted human plasma. We excluded a major contribution of co-enriched soluble proteins or of proteins bound to the EV surface to the thrombogenicity of blood cell-derived EVs. Our data suggest that the enhanced potential of blood cell-derived EVs to support thrombin generation is rather due to enhanced exposure of phosphatidylserine on the surface of blood cell-derived EVs. Extending these investigations to EVs from other cell types, such as mesenchymal stromal cells, will be crucial for their future therapeutic applications.

Influence of hemoadsorption during cardiopulmonary bypass on blood vesicle count and function.

BACKGROUND: Extracorporeal circulation during major cardiac surgery triggers a systemic inflammatory response affecting the clinical course and outcome. Recently, extracellular vesicle (EV) research has shed light onto a novel cellular communication network during inflammation. Hemoadsorption (HA) systems have shown divergent results in modulating the systemic inflammatory response during cardiopulmonary bypass (CPB) surgery. To date, the effect of HA on circulating microvesicles (MVs) in patients undergoing CPB surgery is unknown. METHODS: Count and function of MVs, as part of the extracellular vesicle fraction, were assessed in a subcohort of a single-center, blinded, controlled study investigating the effect of the CytoSorb device during CPB. A total of 18 patients undergoing elective CPB surgery with (n = 9) and without (n = 9) HA device were included in the study. MV phenotyping and counting was conducted via flow cytometry and procoagulatory potential was measured by tissue factor-dependent MV assays. RESULTS: Both study groups exhibited comparable counts and post-operative kinetics in MV subsets. Tissue factor-dependent procoagulatory potential was not detectable in plasma at any timepoint. Post-operative course and laboratory parameters showed no correlation with MV counts in patients undergoing CPB surgery. CONCLUSION: Additional artificial surfaces to the CPB-circuit introduced by the use of the HA device showed no effect on circulating MV count and function in these patients. Larger studies are needed to assess and clarify the effect of HA on circulating vesicle counts and function. Trial registration ClinicalTrials.Gov Identifier: NCT01879176; registration date: June 17, 2013; https://clinicaltrials.gov/ct2/show/NCT01879176.

Hypoxia Conditioned Mesenchymal Stem Cell-Derived Extracellular Vesicles Induce Increased Vascular Tube Formation in vitro.

Mesenchymal stem/stromal cells (MSCs) display a variety of therapeutically relevant effects, such as the induction of angiogenesis, particularly under hypoxic conditions. It is generally recognized that MSCs exert their effects by secretion of paracrine factors and by stimulation of host cells. Furthermore, there is increasing evidence that some therapeutically relevant effects of MSCs are mediated by MSC-derived extracellular vesicles (EVs). Since our current knowledge on MSC-derived EVs released under hypoxic conditions is very limited, we aimed to characterize MSC-derived EVs from normoxic vs. hypoxic conditions (5% O2). Adipose-derived MSCs were grown under normoxic and hypoxic conditions, and EVs were analyzed by flow cytometry using lactadherin as a marker for EVs exposing phosphatidylserine, CD63 and CD81 as EV markers, as well as CD73 and CD90 as MSC surface markers. Particle concentration and size distribution were measured by nanoparticle tracking analysis (NTA), and the EV surface antigen signature was characterized using bead-based multiplex flow cytometry. Furthermore, we evaluated the potential of MSC-derived EVs obtained under hypoxic conditions to support angiogenesis using an in vitro assay with an hTERT-immortalized human umbilical vein endothelial cell (HUVEC) line. Proliferation and viability of MSCs were increased under hypoxic conditions. EV concentration, size, and surface signature did not differ significantly between normoxic and hypoxic conditions, with the exception of CD44, which was significantly upregulated on normoxic EVs. EVs from hypoxic conditions exhibited increased tube formation as compared to normoxic EVs or to the corresponding supernatants from both groups, indicating that tube formation is facilitated by EVs rather than by soluble factors. In conclusion, hypoxia conditioned MSC-derived EVs appear to be functionally more potent than normoxic MSC-derived EVs regarding the induction of angiogenesis.

Storage of human whole blood, but not isolated monocytes, preserves the distribution of monocyte subsets.

Human monocytes include CD14++CD16- (classical), CD14++CD16+ (intermediate), and CD14+CD16++ (non-classical) subsets with divergent roles in immune regulation and inflammation. Since the functional characterization of monocyte subsets is most commonly performed using isolated monocytes, we investigated the influence of different monocyte isolation protocols on the relative abundance of monocyte subsets. Using flow cytometric subset characterization directly in whole blood as a reference, we found that monocyte isolation by enrichment of peripheral blood mononuclear cells and subsequent depletion of non-monocytes by magnetic labeling did not alter the distribution of monocyte subsets. Particularly, we failed to detect a loss of CD16+ subsets upon monocyte isolation, although one of the negative depletion protocols used contained an anti-CD16 antibody to label granulocytes. Overnight storage of isolated monocytes induced a significant repartition of monocyte subsets towards CD14++CD16+ intermediate monocytes, which was barely seen in stored whole blood. We identified intermediate monocytes as main binding partners of platelet-derived extracellular vesicles (EVs) and propose that residual platelets contained in isolated monocyte preparations release EVs that induce the expression of the IgG receptor FcγRIII (CD16) on monocytes.