Large Extracellular Vesicles Research Articles

Eosinophil, mast cell and basophil microparticle in nasal lavage fluids as potential biomarkers for type 2 inflammation in chronic rhinosinusitis with nasal polyps

Microparticles (MP) are large extracellular vesicles (0.5-1.0μm) that are released from activated cells. Eosinophils, mast cells and basophils play roles in type 2 inflammation in nasal mucosa in chronic rhinosinusitis with nasal polyps (CRSwNP).

Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?

BackgroundLarge extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function.Aim of this studyTo characterize the nature of Mreg-derived L-EV (L-EVMreg) and to gain insights into their role in wound healing and angiogenesis.MethodsMreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EVMreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EVMreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EVMreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays.ResultsMreg and L-EVMreg show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EVMreg regarding their surface marker composition. However, compared to Mreg fewer L-EVMreg were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EVMreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EVMreg positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05).ConclusionL-EVMreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EVMreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Non-contact microfluidic analysis of the stiffness of single large extracellular vesicles from IDH1-mutated glioblastoma cells.

In preparation for leveraging extracellular vesicles (EVs) for disease diagnostics and therapeutics, fundamental research is being done to understand EV biological, chemical, and physical properties. Most published studies have investigated nanoscale EVs and focused on EV biochemical content. There is much less understanding of large microscale EV characteristics and EV mechanical properties. We recently introduced a non-contact microfluidic technique that measures the stiffness of large EVs (>1 μm diameter). This pilot study probes the robustness of the microfluidic technique to distinguish between EV populations by comparing stiffness distributions of large EVs derived from glioblastoma cell lines. EVs derived from cells expressing the IDH1 mutation, a common glioblastoma mutation known to disrupt lipid metabolism, were stiffer than those expressed from wild-type cells in a statistical comparison of sample medians. A supporting lipidomics analysis showed that the IDH1 mutation increased the amount of saturated lipids in EVs. Taken together, these data encourage further investigation into the potential of high-throughput microfluidics to distinguish between large EV populations that differ in biomolecular composition. These findings contribute to the understanding of EV biomechanics, in particular for the less studied microscale EVs.

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma

Extracellular vesicles (EVs) participate in cell-to-cell communication and contribute toward homeostasis under physiological conditions. But EVs can also contribute toward a wide array of pathophysiology like cancer, sepsis, sickle cell disease, and thrombotic disorders. COVID-19 infected patients are at an increased risk of aberrant coagulation, consistent with elevated circulating levels of ultra-high molecular weight VWF multimers, D-dimer and procoagulant EVs. The role of EVs in COVID-19 related hemostasis may depend on cells of origin, vesicular cargo and size, however this is not well defined. We hypothesized that the procoagulant potential of EV isolates from COVID-19 (+) patient plasmas could be defined by thrombin generation assays. Here we isolated small EVs (SEVs) and large EVs (LEVs) from hospitalized COVID-19 (+) patient (n = 21) and healthy donor (n = 20) plasmas. EVs were characterized by flow cytometry, Transmission electron microscopy, nanoparticle tracking analysis, plasma thrombin generation and a multi-omics approach to define coagulation potential. These data were consistent with differences in EV metabolite, lipid, and protein content when compared to healthy donor plasma isolated SEVs and LEVs. Taken together, the effect of EVs on plasma procoagulant potential as defined by thrombin generation and supported by multi-omics is enhanced in COVID-19. Further, we observe that this effect is driven both by EV size and phosphatidyl serine.

Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins.

The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.

Role of extracellular vesicles in early synaptic dysfunction in AD

AbstractBackgroundSynaptic dysfunction is an early mechanism in Alzheimer’s Disease (AD) which involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. We hypothesised that large extracellular vesicles (EVs) released by microglia exposed to and carrying Aβ42 (Aβ‐EVs) may be responsible for these early events in AD.MethodCombining optical manipulation with time lapse imaging we placed single Aβ‐EVs and ctrl‐EVs (released by microglia not exposed to Aβ) on axons or dendrites of RFP‐positive cultured neuron to test both the dynamics of their interaction with neurons and their effects on the synapse. Then, EVs were stereotaxically injected into the mouse entorhinal cortex (EC), one of the most vulnerable regions in AD, and long‐term potentiation (LTP) was measured in the EC and in its main target region, the dentate gyrus of the hippocampus (DG), through field potential extracellular recordings in cortico‐hippocampal brain slices. Finally, taking advantage of high‐resolution accurate‐mass spectrometry SWATH™‐MS, we investigated the molecular differences between ctrl‐EVs and Aβ‐EVs.ResultAβ‐EVs were able to move along the axonal surface more efficiently than ctrl‐EVs and predominantly in an anterograde direction. They locally altered dendritic spine morphology in vitro and impaired LTP in vivo. One hour after injection in the EC, LTP was impaired in the EC of brains injected with Aβ‐EVs but not ctrl‐EVs. However, 24h after Aβ‐EV injection, LTP was impaired also in the DG, indicating a spreading of synaptic dysfunction between the two connected regions. Results were reproduced using large EVs released by microglia exposed to naturally secreted Aβ oligomers, whereas neither Aβ alone nor inflammatory EVs devoid of Aβ were able to propagate LTP impairment. Importantly, when Aβ‐EV motility was limited by annexin‐V coating, no propagation of LTP deficit occurred along the EC‐DG circuit. Proteomic analysis provided insights into the possible molecular mechanisms underneath Aβ‐EV extracellular motion at the neuronal surface.ConclusionOur data provide evidence of the involvement of microglial EVs in early synaptic dysfunction, through a new mechanism ‐ extracellular motion at the neuronal surface ‐ not implying EV internalization, paving the way for novel therapeutic strategies.

Large tumour-derived extracellular vesicles as prognostic indicators of metastatic cancer patient survival.

Extracellular vesicles (EVs) are released by all cells and produced at particularly high levels by many cancer cells, often inducing pro-tumorigenic effects. Since these cancer EVs carry tumour proteins and RNAs, they can potentially be used at biomarkers. The heterogeneity of surface markers and cargos carried by EVs, however, presents some challenges to developing such approaches. Nanou et al. [1] found that automated counting of large tumour-derived EVs (tdEVs) performed at least as effectively as counting circulating tumour-derived cells (CTCs) and with higher sensitivity, in distinguishing the survival of patients with castration-resistant prostate cancer (CRPC), metastatic colorectal cancer (mCRC) and metastatic breast cancer (MBC), but not for non-small cell lung cancer (NSCLC). Subsequent work has suggested that these tdEVs may also be used to assess tumour subtype and that the number of large EVs produced by endothelial cells can also be increased in cancer patients. While by itself, the tdEV imaging approach used by Nanou et al. [1] is not specific enough to predict the survival of individual patients, in combination with other EV-associated assays, this test, perhaps enhanced through the inclusion of other tumour antigens, could prove invaluable in predicting cancer survival and other outcomes in the clinic.

Critical Role of Hodgkin Lymphoma Cells-Derived Large and Small Extracellular Vesicles in the Regulation of the Immune-Inflammatory Microenvironment

Introduction: Classical Hodgkin Lymphoma (cHL) is characterized by a low number of CD30+ malignant cells and an extensive immune-inflammatory microenvironment. However, this unique histopathological picture and its pathogenesis are still partially understood. Extracellular vesicles (EVs) are submicron particles delimited by a lipid bilayer that promote (tumor) intercellular communication and carry bioactive molecules that can influence the immune system. Based on size and biogenesis mechanism, Small (S-) and Large (L-) EVs (30-200 nm and 0.2-10 µm, respectively) have been identified. Extracellular Adenosine Triphosphate (eATP), a key component of the tumor microenvironment, plays a role in biogenesis and dynamics of EVs. Here we aim to elucidate the role of cHL cell-derived EVs in the education of the immune microenvironment. Methods: cHL L-428 cell line was cultured in exosome-free medium for 48 hours at 37°C in the presence or absence of eATP (1 mM). L-428 cell blebbing has been detected by fluorescence microscopy (DAPI and FITC-Cholera Toxin B subunit (CTxB) labeling). L-EVs and S-EVs were isolated from the cell culture supernatant by differential ultracentrifugation (2.800g for 2.8K and 20.000g for 20K L-EVs, and 100.000g for 100K S-EVs) and purified by iodixanol density gradient. L- and S-EVs were then characterized by Tunable Resistive Pulse Sensing (TRPS), single EV-flow cytometry, and MACSPlex (bead-based antigen binding array). Functionally, normal peripheral blood mononuclear cells were in vitro cultured with titrated amount of isolated L- and S-EVs (from 1:5 to 1:20) for 24 hours. The intracytoplasmic interleukin-1β and interferon-γ production by CD14+ monocytes and CD3+/CD4+/CD8+ T cells was assessed by flow cytometry. Results: Our results showed that human cell line L-428 is able to produce L-EVs through membrane blebbing (Figure 1A). According to EV size distributions, both 2.8K and 20K L-EVs displayed a size range from 1 to 4 µm whereas S-EVs were enriched in particles of 150-200 nm diameter. Interestingly, the amounts of S-EVs shed by L-428 cells were significantly higher compared to the number of L-EVs. Considering the EV surface protein phenotype, S-EVs express all the EV-related tetraspanin proteins (CD9, CD63, and CD81). By contrast, L-EV express CD63 and CD81 only. Additionally, in L-EVs we observed an enrichment of cancer-related markers including SSEA-4, CD44, CD24, CD29 and CD133-1 in comparison with the S-EV counterpart. Of note, the expression of selected immune-related molecules such as CD86 and CD40 was higher in the 2.8K and 20K L-EVs as compared with that of the 100K S-EVs. The HLA-II molecules were expressed by all fractions, with the highest expression in 2.8K L-EVs. Along with PD-L1, the CD30 HL-associated marker was expressed by all EV fractions at the same extent. When we tested the effects of eATP on EVs produced by L-428 cells, we found a highly decline in the expression of EV-surface proteins from all EV sub-fractions, being most noticeable in L-EVs. Importantly, only PD-L1 expression was increased in the 20K L-EVs after eATP treatment. Functionally, at variance with S-EVs (Figure 1B), L-EVs highly stimulate the intracytoplasmic production of interleukin-1β by monocytes. Conversely, L-EVs derived from e-ATP treated L-428 cells fail to stimulate the intracytoplasmic production of interleukin-1β by CD14+ monocytes, which instead was promoted by the e-ATP treated S-EV counterparts. Of note, neither L-EVs nor S-EVs stimulated interferon-γ production by CD14+ monocytes. No intracytoplasmic production of the selected inflammatory cytokines has been observed when T-cells treated with EVs were analyzed. Conclusions: These results demonstrate that L-EVs from HL cells display a cancer- and immune-related phenotype and play a role in the modulation of the immune microenvironment by promoting the production of inflammatory cytokines by monocytes. This might be partially due to the high expression of CD44 on the surface of L-EVs, since it has been previously demonstrated that cancer-derived soluble CD44 may trigger monocyte/macrophage interleukin-1β production. Interestingly, eATP plays a role in shaping the immune modulatory ability of L- and S-EVs. These findings shed light on the role of HL-derived EVs in the establishment of the immune-inflammatory microenvironment and provide novel insight into clinically relevant EV driven mechanisms in HL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Large extracellular vesicles in bovine follicular fluid inhibit the apoptosis of granulosa cell and stimulate the production of steroid hormones

The cargo carried by extracellular vesicles (EVs) plays an important physiological role in their corresponding target organs or target tissue cells. Extracellular vesicles are classified into large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs) according to their diameters. Since different subtypes contain different contents, their roles are also different. In this study, the morphology and size of LEVs were analyzed by transmission electron microscopy and nanoparticle size, and the marker proteins of LEVs (CD63, GP96, TSG101, ALB) were identified by western blot, and high-purity LEVs were obtained. Through the uptake of extracellular vesicles by purified ovarian granulosa cells and the determination of granulosa cell viability, cell apoptosis, and steroid hormone production, the result indicated that LEVs significantly enhanced cell viability (P < 0.05), reduced the rate of granulosa cell apoptosis (P < 0.05). Meanwhile, LEVs promoted the secretion of estradiol in granulosa cells (P < 0.05). This study provides a reference for the in-depth study of the function of follicular fluid extracellular vesicle subtypes and the research on the regulation of extracellular vesicles on follicle and oocyte development.

Synovial Extracellular Vesicles: Structure and Role in Synovial Fluid Tribological Performances.

The quality of the lubricant between cartilaginous joint surfaces impacts the joint’s mechanistic properties. In this study, we define the biochemical, ultrastructural, and tribological signatures of synovial fluids (SF) from patients with degenerative (osteoarthritis-OA) or inflammatory (rheumatoid arthritis-RA) joint pathologies in comparison with SF from healthy subjects. Phospholipid (PL) concentration in SF increased in pathological contexts, but the proportion PL relative to the overall lipids decreased. Subtle changes in PL chain composition were attributed to the inflammatory state. Transmission electron microscopy showed the occurrence of large multilamellar synovial extracellular vesicles (EV) filled with glycoprotein gel in healthy subjects. Synovial extracellular vesicle structure was altered in SF from OA and RA patients. RA samples systematically showed lower viscosity than healthy samples under a hydrodynamic lubricating regimen whereas OA samples showed higher viscosity. In turn, under a boundary regimen, cartilage surfaces in both pathological situations showed high wear and friction coefficients. Thus, we found a difference in the biochemical, tribological, and ultrastructural properties of synovial fluid in healthy people and patients with osteoarthritis and arthritis of the joints, and that large, multilamellar vesicles are essential for good boundary lubrication by ensuring a ball-bearing effect and limiting the destruction of lipid layers at the cartilage surface.

HK1 from hepatic stellate cell–derived extracellular vesicles promotes progression of hepatocellular carcinoma

Extracellular vesicles play crucial roles in intercellular communication in the tumor microenvironment. Here we demonstrate that in hepatic fibrosis, TGF-β stimulates the palmitoylation of hexokinase 1 (HK1) in hepatic stellate cells (HSCs), which facilitates the secretion of HK1 via large extracellular vesicles in a TSG101-dependent manner. The large extracellular vesicle HK1 is hijacked by hepatocellular carcinoma (HCC) cells, leading to accelerated glycolysis and HCC progression. In HSCs, the nuclear receptor Nur77 transcriptionally activates the expression of depalmitoylase ABHD17B to inhibit HK1 palmitoylation, consequently attenuating HK1 release. However, TGF-β-activated Akt functionally represses Nur77 by inducing Nur77 phosphorylation and degradation. We identify the small molecule PDNPA that binds Nur77 to generate steric hindrance to block Akt targeting, thereby disrupting Akt-mediated Nur77 degradation and preserving Nur77 inhibition of HK1 release. Together, this study demonstrates an overlooked function of HK1 in HCC upon its release from HSCs and highlights PDNPA as a candidate compound for inhibiting HCC progression.

Multianalyte liquid biopsy to aid the diagnostic workup of breast cancer

Breast cancer (BC) affects 1 in every 8 women in the United States and is currently the most prevalent cancer worldwide. Precise staging at diagnosis and prognosis are essential components for the clinical management of BC patients. In this study, we set out to evaluate the feasibility of the high-definition single cell (HDSCA) liquid biopsy (LBx) platform to stratify late-stage BC, early-stage BC, and normal donors using peripheral blood samples. Utilizing 5 biomarkers, we identified rare circulating events with epithelial, mesenchymal, endothelial and hematological origin. We detected a higher level of CTCs in late-stage patients, compared to the early-stage and normal donors. Additionally, we observed more tumor-associated large extracellular vesicles (LEVs) in the early-stage, compared to late-stage and the normal donor groups. Overall, we were able to detect reproducible patterns in the enumeration of rare cells and LEVs of cancer vs. normal donors and early-stage vs. late-stage BC with high accuracy, allowing for robust stratification. Our findings illustrate the feasibility of the LBx assay to provide robust detection of rare circulating events in peripheral blood draws and to stratify late-stage BC, early-stage BC, and normal donor samples.

The profile of inflammatory extracellular vesicles in intracerebral hemorrhage patients

BackgroundIntracerebral hemorrhage (ICH) is one of the leading life-threatening types of strokes with high mortality. A prominent feature of ICH is neuroinflammation involving leukocytes, such as neutrophils and macrophages. Large extracellular vesicles (lEV) and small extracellular vesicles (sEV) released from various cells are used as biomarkers for different diseases. Here, we aimed to determine the concentration/population of lEV and sEV from different leukocytes in ICH patients and analyze the correlation of these lEV/sEV with clinical parameters.MethodslEV and sEV were isolated from the plasma of ICH patients (n = 39) by using the serial centrifuge methods. Nanoparticle tracking analysis (NTA, NS300) was used to determine the type and concentration of different leukocytes-released lEV/sEV. Specific antibodies, CD66b, P2RY12, and CD80 were used for different leukocyte types.ResultsA predictive relationship between both hospital length of stay (R2 = 0.83) and Intensive care units (ICU) length of stay (R2 = 0.88) was found with lEV and sEV and patient data [including low-density lipoprotein (LDL), ICH volume, etc.]. Further predictive multiple linear regression relationship was seen between lEV and sEV concentrations and MRSV3 (Modified Rankin Scale at 90 days) (R2 = 0.46) and MRSV5 (modified Rankin Scale at 180 days) (R2 = 0.51). Additionally, a slight, but statistically significant (p = 0.0151), multiple linear regression relationship was seen between lEV and sEV concentrations and ICU length of stay (R2 = 0.26).ConclusionThis study found predictive relationships between patient outcomes and lEV and sEV. When combined with generally collected patient data (LDL, etc.), measurements of lEV and sEV are strongly predictive of overall patient outcome. Further, larger studies should investigate these effects.

Host Cell Signatures of the Envelopment Site within Beta-Herpes Virions.

Beta-herpesvirus infection completely reorganizes the membrane system of the cell. This system is maintained by the spatiotemporal arrangement of more than 3000 cellular proteins that continuously adapt the configuration of membrane organelles according to cellular needs. Beta-herpesvirus infection establishes a new configuration known as the assembly compartment (AC). The AC membranes are loaded with virus-encoded proteins during the long replication cycle and used for the final envelopment of the newly formed capsids to form infectious virions. The identity of the envelopment membranes is still largely unknown. Electron microscopy and immunofluorescence studies suggest that the envelopment occurs as a membrane wrapping around the capsids, similar to the growth of phagophores, in the area of the AC with the membrane identities of early/recycling endosomes and the trans-Golgi network. During wrapping, host cell proteins that define the identity and shape of these membranes are captured along with the capsids and incorporated into the virions as host cell signatures. In this report, we reviewed the existing information on host cell signatures in human cytomegalovirus (HCMV) virions. We analyzed the published proteomes of the HCMV virion preparations that identified a large number of host cell proteins. Virion purification methods are not yet advanced enough to separate all of the components of the rich extracellular material, including the large amounts of non-vesicular extracellular particles (NVEPs). Therefore, we used the proteomic data from large and small extracellular vesicles (lEVs and sEVs) and NVEPs to filter out the host cell proteins identified in the viral proteomes. Using these filters, we were able to narrow down the analysis of the host cell signatures within the virions and determine that envelopment likely occurs at the membranes derived from the tubular recycling endosomes. Many of these signatures were also found at the autophagosomes, suggesting that the CMV-infected cell forms membrane organelles with phagophore growth properties using early endosomal host cell machinery that coordinates endosomal recycling.

Large extracellular vesicles secreted by human iPSC-derived MSCs ameliorate tendinopathy via regulating macrophage heterogeneity.

Tendinopathy is a common musculoskeletal disorder which results in chronic pain and reduced performance. The therapeutic effect of stem cell derived-small extracellular vesicles (sEVs) for tendinopathy has been validated in recent years. However, whether large extracellular vesicles (lEVs), another subset of extracellular vesicles, possesses the ability for the improvement of tendinopathy remains unknown. Here, we showed that lEVs secreted from iPSC-derived MSCs (iMSC-lEVs) significantly mitigated pain derived from tendinopathy in rats. Immunohistochemical analysis showed that iMSC-lEVs regulated the heterogeneity of infiltrated macrophages and several inflammatory cytokines in rat tendon tissue. Meanwhile, in vitro experiments revealed that the M1 pro-inflammatory macrophages were repolarized towards M2 anti-inflammatory macrophages by iMSC-lEVs, and this effect was mediated by regulating p38 MAPK pathway. Moreover, liquid chromatography-tandem mass spectrometry analysis identified 2208 proteins encapsulated in iMSC-lEVs, including 134 new-found proteins beyond current Vesiclepedia database. By bioinformatics and Western blot analyses, we showed that DUSP2 and DUSP3, the negative regulator of p38 phosphorylation, were enriched in iMSC-lEVs and could be transported to macrophages. Further, the immunomodulatory effect of iMSC-lEVs on macrophages was validated in explant tendon tissue from tendinopathy patients. Taken together, our results demonstrate that iMSC-lEVs could reduce inflammation in tendinopathy by regulating macrophage heterogeneity, which is mediated via the p38 MAPK pathway by delivery of DUSP2 and DUSP3, and might be a promising candidate for tendinopathy therapy.

Large extracellular vesicles do not mitigate the harmful effect of hyperglycemia on endothelial cell mobility

Extracellular vesicles, especially the larger fraction (LEVs – large extracellular vesicles), are believed to be an important means of intercellular communication. Earlier studies on LEVs have shown their healing properties, especially in the vascular cells of diabetic patients. Uptake of LEVs by endothelial cells and internalization of their cargo have also been demonstrated. Endothelial cells change their properties under hyperglycemic conditions (HGC), which reduces their activity and is the cause of endothelial dysfunction. The aim of our study was to investigate how human umbilical vein endothelial cells (HUVECs) change their biological properties: shape, mobility, cell surface stiffness, as well as describe the activation of metabolic pathways after exposure to the harmful effects of HGC and the administration of LEVs released by endothelial cells. We obtained LEVs from HUVEC cultures in HGC and normoglycemia (NGC) using the filtration and ultracentrifugation methods. We assessed the size of LEVs and the presence of biomarkers such as phosphatidylserine, CD63, beta-actin and HSP70. We analyzed the LEVs uptake efficiency by HUVECs, HUVEC shape, actin cytoskeleton remodeling, surface stiffness and finally gene expression by mRNA analysis. Under HGC conditions, HUVECs were larger and had a stiffened surface and a strengthened actin cortex compared to cells under NGC condition. HGC also altered the activation of metabolic pathways, especially those related to intracellular transport, metabolism, and organization of cellular components. The most interesting observation in our study is that LEVs did not restore cell motility disturbed by HGC. Although, LEVs were not able to reverse this deleterious effect of HGC, they activated transcription of genes involved in protein synthesis and vesicle trafficking in HUVECs.

Quantitative Proteomics Identifies Proteins Enriched in Large and Small Extracellular Vesicles

There is a long-held consensus that several proteins are unique to small extracellular vesicles (EVs), such as exosomes. However, recent studies have shown that several of these markers can also be present in other subpopulations of EVs to a similar degree. Furthermore, few markers have been identified as enriched or uniquely present in larger EVs, such as microvesicles. The aim of this study was to address these issues by conducting an in-depth comparison of the proteome of large and small EVs. Large (16,500g) and small EVs (118,000g) were isolated from three cell lines using a combination of differential ultracentrifugation and a density cushion and quantitative mass spectrometry (tandem mass tag–liquid chromatography–tandem mass spectrometry) was used to identify differently enriched proteins in large and small EVs. In total, 6493 proteins were quantified, with 818 and 1567 proteins significantly enriched in small and large EVs, respectively. Tetraspanins, ADAMs and ESCRT proteins, as well as SNAREs and Rab proteins associated with endosomes were enriched in small EVs compared with large EVs, whereas ribosomal, mitochondrial, and nuclear proteins, as well as proteins involved in cytokinesis, were enriched in large EVs compared with small EVs. However, Flotillin-1 was not differently expressed in large and small EVs. In conclusion, our study shows that the proteome of large and small EVs are substantially dissimilar. We validated several proteins previously suggested to be enriched in either small or large EVs (e.g., ADAM10 and Mitofilin, respectively), and we suggest several additional novel protein markers.

A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications.

Simple SummaryDuring tumorigenesis, communication among cells is fundamental. Tumor cells can communicate through the release of extracellular vesicles (EVs), carrying active mediators, that are able to move in the extracellular space and in the body fluids, thereby reaching cells different from the ones of origin. Tumor-derived EVs, upon interaction with tumor target cells, can profoundly change cell characteristics and behavior. Here, we isolated EVs released from human teratocarcinoma cells and we uncovered their ability to inhibit the cell migration of glioblastoma (GBM) cells. Teratocarcinoma EVs contain the oncofetal protein CRIPTO, that is involved in the observed reduction of GBM cell migration. Our results suggest a novel localization and function for CRIPTO in large EVs, and give precious hints for the development of novel therapeutic approaches, based on the control of tumor cell migration, to ultimately improve prognosis and quality of life of GBM patients.Tumor growth and metastasis strongly rely on cell–cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Mesenchymal Stem Cell-Derived Secretome: A Potential Therapeutic Option for Autoimmune and Immune-Mediated Inflammatory Diseases.

Immune-mediated inflammatory diseases (IMIDs) encompass several entities such as “classic” autoimmune disorders or immune-mediated diseases with autoinflammatory characteristics. Adult stem cells including mesenchymal stem cells (MSCs) are by far the most commonly used type in clinical practice. However, due to the possible side effects of MSC-based treatments, there is an increase in interest in the MSC-secretome (containing large extracellular vesicles, microvesicles, and exosomes) as an alternative therapeutic option in IMIDs. A wide spectrum of MSC-secretome-related biological activities has been proven thus far including anti-inflammatory, anti-apoptotic, and immunomodulatory properties. In comparison with MSCs, the secretome is less immunogenic but exerts similar biological actions, so it can be considered as an ideal cell-free therapeutic alternative. Additionally, since the composition of the MSC-secretome can be engineered, for a future perspective, it could also be viewed as part of a potential delivery system within nanomedicine, allowing us to specifically target dysfunctional cells or tissues. Although many encouraging results from pre-clinical studies have recently been obtained that strongly support the application of the MSC-secretome in IMIDs, human studies with MSC-secretome administration are still in their infancy. This article reviews the immunomodulatory effects of the MSC-secretome in IMIDs and provides insight into the interpretation of its beneficial biological actions.

Insulin infusion decreases medium-sized extracellular vesicles in adults with metabolic syndrome.

Elevated extracellular vesicles (EVs) are associated with glucose dysmetabolism. However, the effects of insulin on EVs and subsequent relationships with insulin sensitivity, substrate oxidation, and inflammation are unknown. We tested the hypothesis that insulin would lower EVs and relate to insulin action. Fifty-one sedentary adults (54.8 ± 1.0 yr; V̇o2peak : 22.1 ± 0.6 mL/kg/min) with metabolic syndrome (MetS) and obesity (36.4 ± 0.65 kg/m2) underwent a 2-h euglycemic-hyperinsulinemic clamp (5 mmol/L; 40 mU/m2/min). Count and size (medium: 200-624 nm; larger: 625-1,000 nm) for total particle count, endothelial- (CD105+), leukocyte- (CD45+), platelet- (CD41+), and tetraspanin- (TX+: CD9/CD81/CD63), as well as platelet endothelial cell adhesion molecule- (CD31+) derived EVs were determined before and following the clamp using Full Spectrum Profiling (FSPM). Size and MESF (molecules of equivalent soluble fluorochrome) data were generated using FCMPASS Software. Fat and carbohydrate oxidation, in addition to high-sensitivity c-reactive protein (hsCRP), were measured to understand insulin effects and associations between EVs, metabolic flexibility, and inflammation. Despite low metabolic insulin sensitivity (M-Value = 2.56 ± 0.17 mg/kg/min), insulin increased carbohydrate (P = 0.015) and decreased fat oxidation (P = 0.048) and hsCRP (P = 0.016) compared with fasting. Insulin also decreased total particle count (P < 0.001), attributable to decreased medium-sized CD105+ (P = 0.052) and CD45+ EVs (P < 0.001). Elevated fasting insulin was associated with reduced insulin-stimulated changes in all EVs phenotypes (P < 0.001). Interestingly, fasting EVs were associated with increased fasting carbohydrate oxidation (all P < 0.05). These findings suggest that insulin decreases medium-sized EVs in conjunction with metabolic flexibility under euglycemic conditions in adults with MetS. More research is needed to determine how therapies alter EV phenotype/size and consequent cardiometabolic risk.NEW & NOTEWORTHY This study is one of the first to investigate the effects of insulin on medium and larger extracellular vesicles (EVs) in relation to metabolic insulin sensitivity and fuel use in adults with metabolic syndrome. Our data suggest that insulin infusion decreases the concentration of total particle counts, mainly due to reductions in medium-sized EVs. Furthermore, EVs, predominantly medium-sized, are inversely associated with metabolic flexibility.