Microvesicles Research Articles

Microglial NLRP3 Inflammasome Induces Excitatory Synaptic Loss Through IL-1β-Enriched Microvesicle Release: Implications for Sepsis-Associated Encephalopathy.

Acute cerebral dysfunction is a pathological state common in severe infections and a pivotal determinant of long-term cognitive outcomes. Current evidence indicates that a loss of synaptic contacts orchestrated by microglial activation is central in sepsis-associated encephalopathy. However, the upstream signals that lead to microglial activation and the mechanism involved in microglial-mediated synapse dysfunction in sepsis are poorly understood. This study investigated the involvement of the NLRP3 inflammasome in microglial activation and synaptic loss related to sepsis. We demonstrated that septic insult using the cecal ligation and puncture (CLP) model induced the expression of NLRP3 inflammasome components in the brain, such as NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), caspase-1, and IL-1β. Immunostaining techniques revealed increased expression of the NLRP3 inflammasome in microglial cells in the hippocampus of septic mice. Meanwhile, an in vitro model of primary microglia stimulated with LPS exhibited an increase in mitochondrial reactive oxygen species (ROS) production, NLRP3 complex recruitment, and IL-1β release. Pharmacological inhibition of NLRP3, caspase-1, and mitochondrial ROS all decreased IL-1β secretion by microglial cells. Furthermore, we found that microglial NLRP3 activation is the main pathway for IL-1β-enriched microvesicle (MV) release, which is caspase-1-dependent. MV released from LPS-activated microglia induced neurite suppression and excitatory synaptic loss in neuronal cultures. Moreover, microglial caspase-1 inhibition prevented neurite damage and attenuated synaptic deficits induced by the activated microglial MV. These results suggest that microglial NLRP3 inflammasome activation is the mechanism of IL-1β-enriched MV release and potentially synaptic impairment in sepsis.

Patent foramen ovale (PFO) and migraine: role of platelet Tissue Factor expression and oxidative stress. The LEARNER study

Abstract Background Migraine is a chronic neurovascular disorder with a multifactorial aetiology. A strong relationship links migraine with aura (MHA) and patent foramen ovale (PFO), with migraine regression after PFO closure. Increased platelet aggregation and oxidative stress were documented in migraineurs. Current guidelines suggest the use of aspirin or P2Y12-antagonists. To date, however, no clear mechanisms connecting MHA to PFO has been demonstrated. Purpose To perform a comprehensive analysis of platelet activation, inflammation and oxidative stress status in 78 aspirin-treated MHA-patients before (T0) and 6-months after (T1) PFO closure (LEARNER Study-NCT03521193-clinicaltrials.gov). Primary endpoint was migraine regression rate in relation to these parameters. Methods P-selectinpos-, activated-glycoproteinIIbIIIa (aGPIIbIIIa)pos-, Tissue Factor (TF)pos-, reactive oxygen species (ROS)pos-platelets, platelet-leukocyte aggregates (PLA) and microvesicles (MVs) were evaluated by flow cytometry; thrombin generation (TG) by Calibrated Automated Thrombogram (CAT) assay; oxidative stress status by mass spectrometry. Twelve aspirin-treated-healthy subjects (HS) were enrolled for comparison. To test the effect of PFO patients' plasma on platelet activation, blood from HS was plasma-depleted and replaced with a pool of plasma from PFO patients. Results Migraine resolution occurred in 69.7%, significant reduction in 27%, while no effect was observed in 2 patients (3.2%). Only ROSpos-platelets, and TFpos-platelets and -MVs were significantly higher at T0, sustaining a TG capacity that was associated with an altered blood GSSG/GSH (Oxidized/Reduced Glutathione) ratio. This phenotype reverted to HS levels at T1 (Fig. 1). MHA-PFO plasma, added to HS blood, mirrored the in vivo platelet activation and N-acetylcysteine blunted it. In vitro GSSG treatment of HS platelets reproduced the in vivo condition. Aspirin had little effect on the platelet prothrombotic phenotype which was effectively inhibited by a P2Y12-antagonist (Fig.2). Conclusions MHA-PFO patients show a platelet-associated prothrombotic phenotype, characterized by a marked thrombin generation capacity sustained by an elevated number of platelets and MVs expressing a functionally active Tissue Factor and sustained by altered oxidative stress status. This phenotype, not fully controlled by aspirin but by P2Y12-antagonism, reverted after PFO closure together with a complete migraine remission. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health

Long COVID-19 syndrome: association of cardiopulmonary impairment with a persistent platelet activation

Abstract Background A considerable proportion of patients do not fully recover from COVID-19 infection and report symptoms that persist beyond the initial phase of infection: this condition is defined long-COVID-19 syndrome (LCS). LCS can involve lungs as well as several extrapulmonary organs, including the cardiovascular system. The risk and 1-year burden of cardiovascular diseases (CVD) is increased in COVID-19 survivors, even in subjects at low risk of CVD. Recently, we documented that acute COVID-19 infection induces altered platelet activation state characterized by a prothrombotic phenotype and by the formation of platelet-leukocyte aggregates (PLA), that may be involved in the pulmonary microthrombi found in autoptic specimens. No data are yet available on the contribution of platelet activation to residual pulmonary impairment and procoagulant potential in LCS patients. Purpose To study platelet activation status, microvesicle (MV) profile, platelet thrombin generation capacity (pTGC) in LCS patients enrolled at 6 months after resolution of the acute phase (6mo-FU), compared to acute COVID-19 infection patients. Methods 6mo-FU COVID-19 patients (n=24) with established LCS were enrolled at Centro Cardiologico Monzino. Residual pulmonary impairment was assessed by Cardiopulmonary Exercise Testing (CPET) and 64-rows-CT scan evaluation. Platelet activation (P-selectin, Tissue Factor [TF] and PLA) and MV profile were assessed by flow cytometry; pTGC by calibrated automated thrombogram. 46 patients enrolled during acute COVID-19 infection and 46 healthy subjects (HS) were used for comparison. Results Dispnea in LCS patients was confirmed by CPET showing compromised alveolus-capillary membrane diffusion and residual pulmonary impairment. TF+-platelet and -MV levels were 3-fold (1.5% [1.2–2.9] vs 2.4% [1.6–5.7]) and 2-fold (217/μl [137–275] vs 435/μl [275–633]) lower at 6mo-FU compared to acute phase, being comparable to HS. pTGC behaved similarly. At 6mo-FU, the MV profile, in terms of total number and cell origin, returned to physiological levels. Conversely, although lower than that measured in acute phase, a 2.5-fold higher platelet P-selectin expression (6.9% [3–13.5] vs 11.7% [5.2–18.9]) and PLA formation (35.5% [27.4–46.8] vs 67.7% [45.7–85.3]) was observed at 6mo-FU compared to HS. Interestingly, a significant correlation between PLA formation and residual pulmonary impairment was observed (r=−0.423; p=0.02). Conclusion These data strengthen the hypothesis that the presence of PLA in the bloodstream, and thus also in the pulmonary microcirculation, may contribute to support pulmonary dysfunction still observed in LCS patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health (Ricerca Corrente 2020 MPP COVID4)

EXTRACELLULAR VESICLES AS MARKERS OF INFLAMMATION AND HYPERCOAGULABILITY DURING THE FIRST MONTH OF SARS-COV-2 INFECTION IN OUTPATIENTS AND HOSPITALIZED PATIENTS

IntroductionDuring SARS-CoV-2 infection, a severe hypercoagulability state is observed due to the stimulus of multiple mechanisms of hemostasis, such as coagulation, activation of platelets, endothelial cells, monocytes and neutrophils and impaired fibrinolysis. As a consequence, thrombotic complications are common in the course of COVID-19. Microvesicles (MVs) are intracellular transmitters that participate in pathological conditions, such as inflammatory and infectious processes, and are capable of triggering prothrombotic mechanisms. Since MVs release is potentially associated with COVID-19-induced coagulopathy, our aim was to identify during the course of the disease when the stimulus for MVs release occurs and whether this was associated with adverse outcomes.ObjectiveWe evaluated changes in the levels of MVs markers during the first month of SARS-CoV-2 infection in patients (pts) with severe disease (hospitalized in an Intensive Care Unit ‒ ICU) as compared to outpatients. We also evaluated the association between MVs markers with: inflammatory biomarkers (C-reactive protein, CRP), hypercoagulability (D-dimer) and death.MethodsBlood samples were collected on three occasions: before the 10th day of symptoms, in the 3rd week of symptoms and in the 4th week of symptoms for the quantification of the following MVs markers by flow cytometry: CD41A (platelet activation), CD162 (PSGL-1; leukocyte-platelet interaction), CD31 (endothelium-platelet interaction) and CD142 (tissue factor). Statistical tests of ANOVA with repeated measures, Mann-Whitney and regression methods were used.ResultsThe population studied was 85 pts, being 25 from ICU. Mostly were men (51%), with a median age of 41 years. The concentration of MVs expressing CD31+, CD41+, CD162+ and CD142+ were persistently elevated in pts who required ICU compared to outpatients at the 3 moments studied, except for the levels of MVs-CD31+ and MVs-CD142+ that were similar between ICU and outpatients in the 4th week of symptoms. However, despite the differences between the groups, there were no significant changes in the levels of MVs during the course of the disease within the groups. In subgroup analysis, we observed that increases in the levels of MVs-CD162+ and MVs-CD142+ in the 3rd week of symptoms were associated with the risk of death (p=0.02 and p=0.06, respectively). We also observed that during the course of the disease an association between MVs, coagulability and inflammation was evident. In the 3rd week of symptoms, D-dimer levels were correlated with MV-CD31+ (r=0.52, p<0.0001), MV-CD162+ (r=0.35, p=0.001), MV-CD41A+ (r=0.44, p<0.0001) and MV-CD142+ (r=0.47, p<0.0001) and CRP values were correlated with MV-CD31+ (r=0.56, p=<0.0001), MV-CD162+ (r=0.48, p<0.0001), MV-CD41A+ (r= 0.41, p=0.0001), and MV-CD142+ (r=0.56, p<0.0001). By the 4th week of symptoms, both D-dimers and CRP correlations with the above MVs remained unchanged.ConclusionTo conclude, MVs that express antigens related to platelet activation, leukocyte-platelet interaction and endothelium-platelet interaction, as well as those related to tissue factor are released during the course of COVID-19 in pts with severe disease. After the 4th week of symptoms, the release of these MVs was associated with signs of inflammation and hypercoagulability. Additionally, MVs that express tissue factor and leukocyte-platelet interaction antigens were particularly high among non-survivors, suggesting that these MVs may serve as markers of the risk of death. Finally, these findings suggest the participation of innate immunity and tissue factor pathways in the prognosis of COVID-19, and point towards a possible role of MVs as biomarkers of disease prognosis.

MM-324 Effects of Soluble and Microvesicle-Bound Therapeutic Anti-CD38 Antibodies on Immune Effector Cells in Multiple Myeloma

CD38 is a pleiotropic cell surface molecule with a short cytoplasmic tail. This molecule is highly expressed by multiple myeloma (MM) cells, and it was chosen as the target of therapeutic monoclonal antibodies, such as daratumumab (Dara). This work aims at investigating how Dara interacts with target and immune cells and what effects are induced on natural killer (NK) cells and myeloid-derived suppressor cells (MDSCs). Experiments were conducted on MM-derived cell lines stabilized in our laboratory and fresh human myeloma cells. Microvesicles (MVs) were collected through serial centrifugations. MVs' surface phenotype and internalization were evaluated by flow cytometry. Functional experiments were performed on cells purified from peripheral blood or bone marrow mononuclear cells and analyzed by flow cytometry. As previously reported, CD38 engagement by Dara on MM cells in conditions of membrane fluidity is followed by the release of MVs with peculiar phenotype and cargo. To extend these observations, the activity of both soluble and insolubilized Dara was assessed by exposing NK cells and MDSC to Dara. The results indicate that Dara treatment of NK cells is followed by increased expression of CD69 (activation marker) and CD107 (degranulation marker) and decreased IFNγ production by the latter cells. NK proliferation was reduced by Dara treatment, thus confirming in-vivo observations. On MDSCs, Dara treatment was followed by increased nitrite release and iNos expression. Cell viability was not altered by Dara treatment. MVs were also incubated with dendritic cells, and a high internalization rate was observed, the consequences of which are under investigation. The results of this work indicate that MVs obtained from Dara-treated MM cells may be a particulate system to modulate the immune response, either locally in the bone marrow niche or at distance. Moreover, these observations provide evidence that Dara enhances NK degranulation and activation while paradoxically decreasing their proliferation. These phenomena were observed with both the soluble and insolubilized forms of the antibody. Our data may describe a mechanism underlying Dara effectiveness and may also partially explain the onset of Dara resistance. In conclusion, our results may pave the way for new strategies to improve treatment efficacy and avoid the onset of resistance.

82P Obtaining microvesicles from T-lymphocytes using various methods of induction

Microvesicles (MVs) are a subset of extracellular vesicles secreted by most body cells. They are being actively studied as cell-free biosimilar drugs. Many ways of inducing MVs are being investigated - natural (passive secretion, cell activation) and stressful (hypoxia, chemical impact, etc.).

Biogenesis and release of endothelial extracellular vesicles: Morphological aspects

BackgroundCell-cell communication through extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies has been shown to be important in physiological homoeostasis as well as pathological processes such as atherosclerosis. However, while the cellular machinery controlling EV formation and composition has been studied during the past decade, less is known about the morphological process of their formation and release. MethodsUsing different electron microscopic approaches including transmission-, scanning-, immun-, and serial block face electron microscopy we studied the morphogenetic events of EV formation and release. We analysed the different steps of EV formation and release in cultured myocardial endothelial (MyEnd) and aortic endothelial (AoEnd) cell lines under serum starvation and under inflammatory conditions. ResultsWe show that in a narrow time frame, the number of active cells and microvesicle (MV) producing cells increased in dependence of time spent in cultivation and additional stimulation by TNF-α. However, MV secretion was a highly heterogeneous process which couldn´t be seen in all cells cultivated under the same conditions. Release of MVs could be observed all over the cells’ surface with no preferred release site. While no single specific microscopic approach applied was sufficient to provide a comprehensive analysis of EV biogenesis, we show that the limitations of one technique could be compensated by the qualities of the respective other applied techniques, thus enabling us to provide a detailed ultrastructural analysis of MV and exosome biogenesis. Surprisingly, exosome release in endothelial cells occurred via a yet undescribed process indicating that MVBs were incorporated into a novel distinct cellular compartment covered by fenestrated endothelium before exosome release. Lastly, we could show that TNF-α stimulation of AoEnd cells leads not only to the upregulation of CD44 in parental cells, but also to incorporation of CD44 into the membranes of generated MVs and exosomes. ConclusionsTaken together, our data contribute to a better understanding of biogenesis and release of EVs. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells at distant sites including vessel wall cells and this could be a mechanism by which MVs may change the and thus contribute to the development and progression of atherosclerotic lesions.

Protective effects of endothelial progenitor cell microvesicles carrying miR‑98‑5p on angiotensin II‑induced rat kidney cell injury

With the increasing number of patients with hypertensive nephropathy worldwide, it has posed a major threat to health and studies on its treatment and pathogenesis are imminent. The present study investigated the mechanism through which microRNA (miR)-98-5p in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) is involved in the repair of angiotensin II (Ang II)-induced injury of rat primary renal kidney cells (PRKs). After isolation of rat renal cortical sections, PRKs were isolated by density gradient centrifugation and identified by immunofluorescence staining. Transmission electron microscopy identifies successful separation of Mvs. An in vitro cell injury model was constructed using Ang II. The Gene Expression Omnibus was used to analyze the differentially expressed genes between diabetic rats and normal rats, and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the signaling pathways involved in these differentially expressed genes. Reverse transcription-quantitative PCR was used to analyze the effect of EPC-MVs on the expression of miRNA induced by Ang II, and the levels of target genes and signaling pathway-related proteins involved were analyzed by western blot. luciferase was used to detect the targeted binding of miR-98-5p to insulin-like growth factor 1 receptor (IGF1R). Enzyme-linked immunosorbent assay was used to analyze the effect of EPC-MVs on Ang II-induced oxidative stress and inflammation levels on PRKs. Cell Counting Kit-8 was used to analyze the effect of EPC-MVs on the cell viability of PRKs induced by Ang II. The results showed that treatment of PRKs with Ang II decreased cell viability, whereas oxidative stress and inflammation were increased. However, EPC-MVs alleviated Ang II-induced damage of the PRKs. During this process, the Ang-II-induced downregulation of miR-98-5p was reversed by EPC-MVs, so miR-98-5p may be a key factor regulating the action of EPC-MVs. Dual-luciferase assay confirmed that miR-98-5p targets IGF1R. It was subsequently demonstrated that EPC-MVs overexpressing miR-98-5p promoted phosphorylation of PI3K/Akt/endothelial nitric oxide synthase (eNOS), and inhibited the oxidative stress and inflammation in PRKs, which were reversed by the overexpression of IGF1R. In conclusion, the results of the present study demonstrated that EPC-MVs with high expression of miR-98-5p can activate the PI3K/Akt/eNOS pathway by regulating IGF1R, as well as protect PRKs from Ang II-induced oxidative stress, inflammation and inhibition of cell viability.

Gingival epithelial cell-derived microvesicles activate mineralization in gingival fibroblasts

Soft tissue calcification occurs in many parts of the body, including the gingival tissue. Epithelial cell-derived MVs can control many functions in fibroblasts but their role in regulating mineralization has not been explored. We hypothesized that microvesicles (MVs) derived from gingival epithelial cells could regulate calcification of gingival fibroblast cultures in osteogenic environment. Human gingival fibroblasts (HGFs) were cultured in osteogenic differentiation medium with or without human gingival epithelial cell-derived MV stimulation. Mineralization of the cultures, localization of the MVs and mineral deposits in the HGF cultures were assessed. Gene expression changes associated with MV exposure were analyzed using gene expression profiling and real-time qPCR. Within a week of exposure, epithelial MVs stimulated robust mineralization of HGF cultures that was further enhanced by four weeks. The MVs taken up by the HGF's did not calcify themselves but induced intracellular accumulation of minerals. HGF gene expression profiling after short exposure to MVs demonstrated relative dominance of inflammation-related genes that showed increases in gene expression. In later cultures, OSX, BSP and MMPs were significantly upregulated by the MVs. These results suggest for the first time that epithelial cells maybe associated with the ectopic mineralization process often observed in the soft tissues.

Engineering Extracellular Vesicles to Modulate Their Innate Mitochondrial Load.

Extracellular vesicles (EVs) are promising carriers for the delivery of biotherapeutic cargo such as RNA and proteins. We have previously demonstrated that the innate EV mitochondria in microvesicles (MVs), but not exosomes (EXOs) can be transferred to recipient BECs and mouse brain slice neurons. Here, we sought to determine if the innate EV mitochondrial load can be further increased via increasing mitochondrial biogenesis in the donor cells. We hypothesized that mitochondria-enriched EVs ("mito-EVs") may increase the recipient BEC ATP levels to a greater extent than naïve MVs. We treated NIH/3T3, a fibroblast cell line and hCMEC/D3, a human brain endothelial cell (BEC) line using resveratrol to activate peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α), the central mediator of mitochondrial biogenesis. Naïve EVs and mito-EVs isolated from the non-activated and activated donor cells were characterized using transmission electron microscopy, dynamic light scattering and nanoparticle tracking analysis. The effect of mito-EVs on resulting ATP levels in the recipient BECs were determined using Cell Titer Glo ATP assay. The uptake of Mitotracker Red-stained EVs into recipient BECs and their colocalization with recipient BEC mitochondria were studied using flow cytometry and fluorescence microscopy. Resveratrol treatment increased PGC-1α expression in the donor cells. Mito-MVs but not mito-EXOs showed increased expression of mitochondrial markers ATP5A and TOMM20 compared to naïve MVs. TEM images showed that a greater number of mito-MVs contained mitochondria compared to naïve MVs. Mito-MVs but not mito-EXOs showed a larger particle diameter compared to their naïve EV counterparts from the non-activated cells suggesting increased mitochondria incorporation. Mito-EVs were generated at higher particle concentrations compared to naïve EVs from non-activated cells. Mito-EVs increased the cellular ATP levels and transferred their mitochondrial load into the recipient BECs. Mito-MV mitochondria also colocalized with recipient BEC mitochondria. Our results suggest that the pharmacological modulation of mitochondrial biogenesis in the donor cells can change the mitochondrial load in the secreted MVs. Outcomes of physicochemical characterization studies and biological assays confirmed the superior effects of mito-MVs compared to naïve MVs-suggesting their potential to improve mitochondrial function in neurovascular and neurodegenerative diseases. The online version contains supplementary material available at 10.1007/s12195-022-00738-8.

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

Characteristics of culture-condition stimulated exosomes or their loaded hydrogels in comparison with other extracellular vesicles or MSC lysates.

Recently, it has become popular to study the use of extracellular vesicles (EVs) secreted by stem cells to repair damaged tissues or lost cells. Various cell types and physiological fluids release EVs, and they play an important role in cell-to-cell communication. Moreover, EVs have been implicated in important processes, such as immune responses, homeostasis maintenance, coagulation, inflammation, cancer progression, angiogenesis, and antigen presentation. Thus, EVs participate in both physiological and pathological progression. The main classes of EVs include exosomes, microvesicles (MVs), and apoptotic bodies (ApoBDs). Exosomes, which carry a mass of signal molecules such as RNA, DNA, proteins, and lipids, are the most important of these EVs subsets. Currently, exosomes are generating substantial interest in the scientific community. Exosomes loaded hydrogels or under different cultural environments exhibit different properties and functions. Therefore, the exosomes obtained from different sources and conditions are worth reviewing. More importantly, no review article has compared the different EVs, such as exosomes, MVs, ApoBDs, and mesenchymal stem cell (MSC) lysates, which are special soluble substances. The differentiation between EVs and MSC lysates is a logical approach. Accordingly, this review provides an update on the latest progress in studying the roles of culture-condition stimulated exosomes or their loaded hydrogels and the differentiation between exosomes, MVs, ApoBDs, and MSC lysates. Published studies were retrieved from the PubMed® database for review.

Circulating Microvesicles in Association with the NLRP3 Inflammasome in Coronary Thrombi from STEMI Patients.

Microvesicles (MVs) are actively secreted by cells. The NLRP3-inflammasome and the interleukin 6 (IL-6)-pathways are central in cardiovascular disease. Knowledge of how the inflammasome influences the MVs is limited. In a cross-sectional study, we assessed whether MVs in plasma associate with genes encoding inflammasome signalling in coronary thrombi. Moreover, any relationships between inflammasome activation and phosphatidylserine (PS) externalization, determined through Annexin V (AV+) labelling, and myocardial injury, assessed by cardiac troponin T (cTnT), were analysed. Intracoronary thrombi and blood samples from STEMI patients (n = 33) were investigated. mRNA of NLRP3, caspase-1, interleukin-1β (IL-1β), interleukin-18 (IL-18), IL-6, soluble IL-6-receptor (sIL-6R), and glycoprotein-130 (gp130) were isolated from the thrombi and relatively quantified by RT-PCR. MVs were analysed by flow cytometry. Total AV+ MVs, mainly reflecting hypercoagulability, correlated positively to NLRP3 gene expression (r = 0.545, p = 0.009). A similar pattern was seen for platelet, endothelial and leukocyte derived MVs, separately. The majority of the MVs were AV− (96%). Total and AV− MVs correlated inversely with IL-1β (r = −0.399 and −0.438, respectively, p < 0.05, both) and gp130 (r = −0.457 and −0.502, respectively, p < 0.05, both). No correlations between MVs and cTnT were observed. Our findings indicate an association between NLRP3-inflammasome in coronary thrombi and procoagulant AV+ MVs in STEMI patients. The inverse relationships between AV− MVs and the gene expression of inflammasome activation may indicate an immuno-dampening role of this subpopulation.

Artificial Microvesicles: New Perspective on Healing Tendon Wounds

Tendons have a limited capacity to repair both naturally and following clinical interventions. Damaged tissue often presents with structural and functional differences, adversely affecting animal performance, mobility, health, and welfare. Advances in cell therapies have started to overcome some of these issues, however complications such as the formation of ectopic bone remain a complication of this technique. Regenerative medicine is therefore looking toward future therapies such as the introduction of microvesicles (MVs) derived from stem cells (SCs). The aim of the present study was to assess the characteristics of artificially derived MVs, from equine mesenchymal stem cells (MSCs), when delivered to rat tendon cells in vitro and damaged tendons in vivo. The initial stages of extracting MVs from equine MSCs and identifying and characterizing the cultured tendon stem/progenitor cells (TSCs) from rat Achilles tendons were undertaken successfully. The horse MSCs and the rat tendon cells were both capable of differentiating in 3 directions: adipogenic, osteogenic, and chondrogenic pathways. The artificially derived equine MVs successfully fused with the TSC membranes, and no cytotoxic or cytostimulating effects were observed. In addition, co-cultivation of TSCs with MVs led to stimulation of cell proliferation and migration, and cytokine VEGF and fractalkine expression levels were significantly increased. These experiments are the first to show that artificially derived MVs exhibited regeneration-stimulating effects in vitro, and that fusion of cytoplasmic membranes from diploid cell lines originating from different species was possible. The experiment in vivo demonstrated the influence of MVs on synthesis of collagen I and III types in damaged tendons of rats. Explorations in vivo showed accelerated regeneration of injured tendons after introduction of the MVs into damaged areas. The results from the studies performed indicated obvious positive modifying effects following the administration of MVs. This represents the initial successful step required prior to translating this regenerative medicine technique into clinical trials, such as for tendon repair in injured horses.

Abstract P252: Differential Expression Of ACE2, Neprilysin And ACE In Extracellular Vesicles Isolated From Human Urine

The Ang II convertase ACE2 is present in urine and may correlate with various renal pathologies. We reported functional ACE2 in urinary exosomes and now extend our studies to distinguish exosomes (EXOs) and microvesicles (MVs) containing ACE2, as well as neprilysin (NEP) and ACE. EXO purification was modified by an intermediate centrifugation step (20k xg, 60 mins) to pellet MVs followed by 100k xg to pellet EXOs. The MV, EXO, and 100k supernatant (soluble, SOL) fractions were assessed for ACE2 activity with MCA-APK-DNP, and NEP and ACE activities with MCA-RPPGFSAFK-Dnp with selective inhibitors. Peptidase activities were expressed as relative fluorescent units (RFU x10 6 /min/mL), and data are means ± SEM (*P&lt;0.05; **P&lt;0.01, N=4). Morning collections from male subjects [51 to 65 years of age, non-smokers] were collected, treated with Cibacron blue to bind albumin, 0.2 μM filtration, and 100 kDa Amicon concentration prior to high speed and ultracentrifugation steps. ACE2 activity was &gt;3-fold higher in the EXO versus the MV fractions with minor activity in the SOL fraction. In contrast, NEP activity was highest in the MV fraction [~2-fold &gt; EXO] while endopeptidase activity was barely detectable in the SOL fraction. ACE activity was similar in MVs and EXOs, but NEP activity was 40-fold higher than ACE in both the MV and EXO fractions. Differential centrifugation of the 100-kDa urine concentrate reveals a unique profile of ACE2, NEP and ACE expression in MVs and EXOs. The functional significance of these peptidase activities in different vesicular populations is currently unknown; however, their origin may reflect the distinct regulation and release of ACE2 and NEP/ACE in urinary vesicles.

Comparison of Bone Marrow Mesenchymal Stem Cells-Derived Exosomes, Microvesicles, and Soluble Proteins on Their Chondroprotective Effects and Therapeutic Efficacy of Osteoarthritis Treatment

As a disabling joint disorder, osteoarthritis (OA) is characterized by the degeneration of articular cartilage. Bone marrow mesenchymal stem cells (BMSCs) is considered and used as a tool that is regulated by paracrine for the treatment of OA. Herein, we explored the potential of exosomes (Exos), microvesicles (MVs), and soluble proteins (SPs) produced and isolated from rat BMSCs. First, we observed the biological effects of Exos, MVs, and SPs on IL-1β treated rat chondrocytes. Then, we intra-articularly injected Exos, MVs, SPs, and BMSCs in anterior cruciate ligament transection (ACLT)-induced rat OA model, and observed their therapeutic efficacy. SPs rather than Exos and MVs isolated from rat BMSCs exerted a chondroprotective effect in vitro; MVs and SPs showed comparable effects to the intra-articular injection of BMSCs, whereas the chondroprotective effects of Exos were less potent in vivo. In conclusion, the BMSCs secretome might have inconsistent effects in vivo with in vitro, and Exos might not be superior than other secretome in posttraumatic OA prevention.

Pathophysiology of Coagulation and Emerging Roles for Extracellular Vesicles in Coagulation Cascades and Disorders.

The notion of blood coagulation dates back to the ancient Greek civilization. However, the emergence of innovative scientific discoveries that started in the seventeenth century formulated the fundamentals of blood coagulation. Our understanding of key coagulation processes continues to evolve, as novel homeostatic and pathophysiological aspects of hemostasis are revealed. Hemostasis is a dynamic physiological process, which stops bleeding at the site of injury while maintaining normal blood flow within the body. Intrinsic and extrinsic coagulation pathways culminate in the homeostatic cessation of blood loss, through the sequential activation of the coagulation factors. Recently, the cell-based theory, which combines these two pathways, along with newly discovered mechanisms, emerged to holistically describe intricate in vivo coagulation mechanisms. The complexity of these mechanisms becomes evident in coagulation diseases such as hemophilia, Von Willebrand disease, thrombophilia, and vitamin K deficiency, in which excessive bleeding, thrombosis, or unnecessary clotting, drive the development and progression of diseases. Accumulating evidence implicates cell-derived and platelet-derived extracellular vesicles (EVs), which comprise microvesicles (MVs), exosomes, and apoptotic bodies, in the modulation of the coagulation cascade in hemostasis and thrombosis. As these EVs are associated with intercellular communication, molecular recycling, and metastatic niche creation, emerging evidence explores EVs as valuable diagnostic and therapeutic approaches in thrombotic and prothrombotic diseases.

CD169+ subcapsular sinus macrophage-derived microvesicles are associated with light zone follicular dendritic cells.

Follicular dendritic cells (FDCs) are a specialized type of stromal cells that exclusively reside in B-cell follicles. When inflammation occurs, the FDC network is reorganized to support germinal center (GC) polarization into the light zone (LZ) and dark zone (DZ). Despite the indispensable role of FDCs in supporting humoral responses, the FDC regulatory requirements remain incompletely defined. In this study, we unexpectedly observed an accumulation of CD169+ subcapsular sinus macrophage (SSM)-derived microvesicles (MVs) in the B-cell zone, which were tightly associated with the FDC network. Interestingly, a selective deposition of CD169+ MVs was detected in both GC LZ FDCs in secondary follicles and on predetermined LZ FDCs in primary follicles. The ablation of CD169+ MVs, resulting from SSM depletion, resulted in significantly decreased expression of LZ-related genes in FDCs. In addition, we found that CD169+ MVs could colocalize with fluorescently tagged antigen-containing immune complexes (ICs), supporting a possible role of CD169+ MVs in transporting antigens to the FDC network. Thus, our data reveal intimate crosstalk between FDCs and SSMs located outside B-cell follicles via SSM-released MVs, providing a novel perspective on the mechanisms underlying the regulation of FDC maturation and polarization.

Circulating microvesicles in association with the NLRP3 inflammasome in coronary thrombi from STEMI patients

Background and Aims : Circulating microvesicles (MVs) are membrane-enclosed particles shed from activated cells. The NLRP3-inflammasome and the interleukin 6 (IL-6)-pathways are essential in cardiovascular disease. Knowledge of how inflammasome-related inflammation impacts secretion of MVs is limited. We aimed to investigate whether MVs in plasma associate with genes encoding inflammasome-signaling in coronary thrombi. Moreover, any relationships between inflammasome-activation, phosphatidylserine (PS) externalization, determined through Annexin V (AV+) labelling, and the degree of myocardial injury, assessed by peak troponin T (TnT), were analyzed.

Microvesicles and Microvesicle-Associated microRNAs Reflect Glioblastoma Regression: Microvesicle-Associated miR-625-5p Has Biomarker Potential

Glioblastoma (GB) is the most aggressive and recurrent form of brain cancer in adults. We hypothesized that the identification of biomarkers such as certain microRNAs (miRNAs) and the circulating microvesicles (MVs) that transport them could be key to establishing GB progression, recurrence and therapeutic response. For this purpose, circulating MVs were isolated from the plasma of GB patients (before and after surgery) and of healthy subjects and characterized by flow cytometry. OpenArray profiling and the individual quantification of selected miRNAs in plasma and MVs was performed, followed by target genes’ prediction and in silico survival analysis. It was found that MVs’ parameters (number, EGFRvIII and EpCAM) decreased after the surgical resection of GB tumors, but the inter-patient variability was high. The expression of miR-106b-5p, miR-486-3p, miR-766-3p and miR-30d-5p in GB patients’ MVs was restored to control-like levels after surgery: miR-106b-5p, miR-486-3p and miR-766-3p were upregulated, while miR-30d-5p levels were downregulated after surgical resection. MiR-625-5p was only identified in MVs isolated from GB patients before surgery and was not detected in plasma. Target prediction and pathway analysis showed that the selected miRNAs regulate genes involved in cancer pathways, including glioma. In conclusion, miR-625-5p shows potential as a biomarker for GB regression or recurrence, but further in-depth studies are needed.