Hydroxytyrosol Enhances Plasma Extracellular Vesicle Secretion, Modulates Their miRNAs Cargo, and Reduces LDL Oxidation in Humans: Postprandial and 1-Week Effects.

Extracellular vesicle (EV) secretion enables cell–cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue‐specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion.

These findings provide a novel mechanism by which IRF-1 regulates Rab27a transcription and EV secretion, leading to OxPL activation of neutrophils and subsequent hepatic IR injury. (Hepatology 2018;67:1056-1070).

Placental extracellular vesicles (EVs) can be found in the maternal circulation throughout gestation, and their concentration, content and bioactivity are associated with pregnancy outcomes, including gestational diabetes mellitus (GDM). However, the effect of changes in the maternal microenvironment on the mechanisms associated with the secretion of EVs from placental cells remains to be fully established. Here, we evaluated the effect of high glucose on proteins associated with the trafficking and release of different populations of EVs from placental cells. BeWo and HTR8/SVneo cells were used as placental models and cultured under 5‐mM D‐glucose (i.e. control) or 25‐mM D‐glucose (high glucose). Cell‐conditioned media (CCM) and cell lysate were collected after 48 h. Different populations of EVs were isolated from CCM by ultracentrifugation (i.e. pellet 2K‐g, pellet 10K‐g, and pellet 100K‐g) and characterised by Nanoparticle Tracking Analysis. Quantitative proteomic analysis (IDA/SWATH) and multiple reaction monitoring protocols at high resolution (MRM HR ) were developed to quantify 37 proteins related to biogenesis, trafficking/release and recognition/uptake of EVs. High glucose increased the secretion of total EVs across the pellets from BeWo cells, an effect driven mainly by changes in the small EVs concentration in the CCM. Interestingly, no effect of high glucose on HTR8/SVneo cells EVs secretion was observed. High glucose induces changes in proteins associated with vesicle trafficking in BeWo cells, including Heat Shock Protein Family A (Hsp70) Member 9 (HSPA9) and Member 8 (HSPA8). For HTR8/SVneo, altered proteins including prostaglandin F2α receptor regulatory protein (FPRP), RAB5A, RAB35, RAB5B, and RB11B, STAM1 and TSG101. These proteins are associated with the secretion and trafficking of EVs, which could explain in part, changes in the levels of circulating EVs in diabetic pregnancies. Further, we identified that proteins RAB11B, PDCD6IP, STAM, HSPA9, HSPA8, SDCBP, RAB5B, RAB5A, RAB7A and ERAP1 regulate EV release in response to high and low glucose when overexpressed in cells. Interestingly, immunohistochemistry analysis of RAB7A revealed distinct changes in placental tissues obtained from women with normal glucose tolerance (NGT, n = 6) and those with GDM ( n = 6), influenced by diet or insulin treatment. High glucose regulation of proteins involved in intercellular dynamics and the trafficking of multivesicular bodies to the plasma membrane in placental cells is relevant in the context of GDM pregnancies.

Targeting Extracellular Vesicle Secretion As a Strategy to Overcome Microenvironment Mediated Drug Resistance in Acute Myeloid Leukemia

Even though cancers have been widely studied and real advances in therapeutic care have been made in the last few decades, relapses are still frequently observed, often due to therapeutic resistance. Cancer Stem Cells (CSCs) are, in part, responsible for this resistance. They are able to survive harsh conditions such as hypoxia or nutrient deprivation. Autophagy and Extracellular Vesicles (EVs) secretion are cellular processes that help CSC survival. Autophagy is a recycling process and EVs secretion is essential for cell-to-cell communication. Their roles in stemness maintenance have been well described. A common pathway involved in these processes is vesicular trafficking, and subsequently, regulation by Rab GTPases. In this review, we analyze the role played by Rab GTPases in stemness status, either directly or through their regulation of autophagy and EVs secretion.

Extracellular vesicle (EV) secretion is a highly conserved evolutionary trait in all organisms in the three domains of life. The packaging and release of EVs appears to be a bulk-flow process which takes place mainly under extreme conditions. EVs participate in horizontal gene transfer, which supports the survival of prokaryotic and eukaryotic microbes. In higher eukaryotes, almost all cells secrete a heterogeneous population of EVs loaded with various biomolecules. EV secretion is typically higher in cancer microenvironments, promoting tumor progression and metastasis. EVs are now recognized as additional mediators of autocrine and paracrine communication in health and disease. In this context, proteins and RNAs have been studied the most, but extracellular vesicle DNA (EV-DNA) has started to gain in importance in the last few years. In this review, we summarize new findings related to the loading mechanism(s), localization, and post-shedding function of EV-DNA. We also discuss the feasibility of using EV-DNA as a biomarker when performing a liquid biopsy, at the same time emphasizing the lack of data from clinical trials in this regard. Finally, we outline the potential of EV-DNA uptake and its interaction with the host genome as a promising tool for understanding the mechanisms of cancer evolution.

Background: Vascular amyloidosis, caused when peptide monomers aggregate into insoluble amyloid, is a prevalent age-associated pathology. Aortic medial amyloid (AMA) is the most common human amyloid and is composed of medin, a 50-amino acid fragment of milk fat globule EGF-factor 8 (MFGE8). Emerging evidence has implicated extracellular vesicles (EVs) as mediators of pathological amyloid metabolism and accumulation in the extracellular matrix (ECM). MFGE8 has been identified as EV cargo in several cell types, including vascular smooth muscle cells (VSMCs). To determine the mechanisms of AMA formation with age, we explored the impact of VSMC senescence, EV secretion and ECM remodelling on medin accumulation. Methods: Primary human VSMCs were used for in vitro experiments, including EV isolation and synthesis of decellularised ECM for Western blotting, immunofluorescence and mass spectrometry analysis. Results: Medin was detected in EVs secreted from VSMCs. Small, round medin aggregates colocalised with EV markers in the ECM and medin was shown on the surface of EVs deposited in the ECM. Decreasing VSMC EV secretion with an inhibitor attenuated aggregation and deposition of medin in the ECM. Medin accumulation in the aortic wall of human subjects was strongly correlated with age. VSMC senescence increased EV secretion, increased EV medin loading and triggered extracellular deposition of medin in fibrillar form. Proteomic analysis showed VSMC senescence induced changes in EV cargo and ECM composition. These changes led to enhanced EV-ECM binding in the senescent ECM and accelerated aggregation of medin into amyloid fibrils. Abundance of the proteoglycan, HSPG2, was increased in the senescent ECM and colocalised with EVs and medin. Knock-down of HSPG2 decreased medin fibril formation in the senescent ECM. Conclusions: Medin is secreted into the ECM by VSMC-derived EVs, which enhance its aggregation. Senescence induces changes in EV secretion and cargo and ECM composition. Together, these changes trigger accumulation of fibrillar medin, contributing to the age-associated development of AMA.

Exposure of Cord Blood Hematopoietic Stem Cells to Bone Marrow Mesenchimal Cells-Derived Microvesicles Induces Cell Survival and Inhibition of Differentiation

Extracellular vesicles (EVs) and their microRNA (miRNA) cargo have been proposed as possible mammary gland health biomarkers in cattle. However, throughout the day, the biologically active milk components, such as miRNAs, may change due to the dynamic nature of milk. The current study aimed to evaluate the circadian fluctuation of milk EVs miRNA cargo to assess the feasibility of milk EVs as future biomarkers for mammary gland health management. Milk from four healthy dairy cows was collected for four consecutive days in the two daily milking sessions in the morning and the evening. The isolated EVs were heterogeneous, intact, and carried the EV protein markers CD9, CD81, and TSG101, as shown by transmission electron microscopy and western blot. The miRNA sequencing results demonstrate that the abundance of miRNA cargo in milk EVs remained stable, unlike other milk components, such as somatic cells, that changed during milking sessions. These findings indicated that the miRNA cargo within milk EVs remains stable irrespective of the time of day, suggesting their potential utility as diagnostic markers for mammary gland health.

According to clinical studies, statins improve the efficacy of programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) blockade therapy for breast cancer; however, the underlying mechanisms are unclear. Herein, we showed that atorvastatin (ATO) decreased the content of PD-L1 in extracellular vesicles (EVs) by reducing cellular PD-L1 expression and inhibiting EV secretion in breast cancer cells, thereby enhancing the efficacy of anti-PD-L1 therapy. ATO reduced EV secretion by regulating the Rab proteins involved in EV biogenesis and secretion. ATO-mediated inhibition of the Ras-activated MAPK signaling pathway downregulated PD-L1 expression. In addition, ATO strongly promoted antitumor efficacy by inducing T cell-mediated tumor destruction when combined with an anti-PD-L1 antibody. Moreover, suppression of EV PD-L1 by ATO improved the reactivity of anti-PD-L1 therapy by enhancing T-cell activity in draining lymph nodes of EMT6-bearing immunocompetent mice. Therefore, ATO is a potential therapeutic drug that improves antitumor immunity by inhibiting EV PD-L1, particularly in response to immune escape during cancer.

Background and Aims Acute Kidney Injury (AKI) significantly worsens the prognosis of hospitalized patients. In recent years, cell-based strategies have been established as reliable option for improving AKI outcomes in experimental AKI. Own studies focused on so-called Proangiogenic Cells (PACs). Mechanisms that contribute to PAC-mediated AKI protection include production / secretion of extracellular vesicles (EV). In addition, the cells most likely act by paracrinic processes (secretome). The current study evaluated whether AKI may be preventable by the administration of either PAC-derived EV and / or the secretome alone. Method AKI was induced in male C57/Bl6N mice (8-12 weeks) by bilateral renal ischemia (IRI - 40 minutes). Syngeneic murine PACs were stimulated with either melatonine, Angiopoietin-1 or -2, or with Bone Morphogenetic Protein-5 (BMP-5) for one hour, respectively. The four mediators were chosen since previous own studies showed improved PAC-mediated AKI protection after cell preconditioning with these substances. PAC-derived EV and the vesicle-depleted supernatant were subsequently collected and i.v. injected post-ischemia. Mice were analyzed 48 hours later. Results IRI induced significant kidney excretory dysfunction as reflected by higher serum cystatin C levels. The only measure that improved AKI was the injection of EV, collected from native PACs. The following conditions worsened post-ischemic renal function even further: EV+Ang-1, EV+BMP-5, EV+melatonin, and EV+secretome+Ang-1. Conclusion Together, our data show that PAC-mediated AKI protection substantially depends on the availability of cell-derived EV. The secretome, either collected from native or preconditioned cells does not prevent mice from ischemia-induced dysfunction. However, since previous data showed improved AKI-protection by PACs after cell preconditioning with certain mediators (Ang-1 and -2, melatonine, BMP-5), other than exclusively vesicle-dependent mechanisms must be involved in PAC-mediated AKI protection. We suggest, that the mere presence of intact cells in the post-ischemic tissue is necessary for improving functional and structural outcome parameters under certain conditions.

BackgroundMicroRNAs (miRNAs) are abundant in tumor-derived extracellular vesicles (EVs) and the functions of extracellular miRNA to recipient cells have been extensively studied with tumorigenesis. However, the role of miRNA in EV secretion from cancer cells remains unknown.MethodsqPCR and bioinformatics analysis were applied for determining extracellular let-7a expression from CRC patient serum and cells. Nanosight particle tracking analysis was performed for investigating the effect of let-7a on EV secretion. Luciferase reporter assays was used for identifying targeted genes synaptosome-associated protein 23 (SNAP23). In vitro and in vivo assays were used for exploring the function of let-7a/SNAP23 axis in CRC progression. Bioenergetic assays were performed for investigating the role of let-7a/SNAP23 in cellular metabolic reprogramming.Resultslet-7a miRNA was elevated in serum EVs from CRC patients and was enriched in CRC cell-derived EVs. We determined that let-7a could suppress EV secretion directly targeting SNAP23. In turn, SNAP23 promotes EV secretion of let-7a to downregulate the intracellular let-7a expression. In addition, we found a novel mechanism of let-7a/SNAP23 axis by regulating mitochondrial oxidative phosphorylation (OXPHOS) through Lin28a/SDHA signaling pathway.ConclusionsLet-7a plays an essential role in not only inhibiting EV secretion, but also suppressing OXPHOS through SNAP23, resulting in the suppression of CRC progression, suggesting that let-7a/SNAP23 axis could provide not only effective tumor biomarkers but also novel targets for tumor therapeutic strategies.

Rab3a attenuates spinal cord injury by mediating vesicle release

Extracellular vesicles (EVs) are key players in cancer development and drug resistance. For example, heat shock protein 90 (HSP90) carried via EVs from secreting cancer cells to distant recipient cells mediates apoptosis and metastasis. Here, we study EV secretion from individual breast cancer cells and the changes under treatment with the HSP90-inhibiting cancer drug tanespimycin (17AAG). We introduce a two-layer microfluidic platform with an array of microchambers that coencapsulate single cancer cells with functionalized beads for the capturing and immunostaining of EVs. Microchambers are created by pressurizing a microfluidic layer with densely packed, round openings, which are aligned with cell- and bead-traps. This new design facilitates the isolation of cells in over 5100 microchambers and efficient EV capture. We characterize the EV secretion of two breast cancer cell lines: triple-negative MDA-MB-231 cells and HER2-positive SkBr3 cells secrete EVs that carry distinguishable levels of tetraspanins (CD9, CD63, and CD81) and HSPs (-90 and -70). Upon drug treatment, the signals for HSP90- and HSP70-positive EVs increase for both cell lines. However, analysis of protein colocalization on the EV surface revealed a significant difference in EV subpopulations: while MDA-MB-231 cells have no HSP90 on CD63-positive EVs, these two markers are colocalized on SkBr3-derived EVs, indicating different intracellular biogenesis pathways for HSP90-loaded EVs. Moreover, our results emphasize that using CD63 as the sole EV capture protein may hide important EV subpopulations. Overall, our platform may support future choices of EV biomarkers for diagnostic and biomedical purposes and help in understanding the heterogeneous drug response of cancer cells.

Well-orchestrated physico-chemical and biological factors for enhanced secretion of osteogenic and angiogenic extracellular vesicles by mesenchymal stem cells in a 3D culture format.