Size-Exclusion Chromatography Separation Reveals That Vesicular and Non-Vesicular Small RNA Profiles Differ in Cell Free Urine

Urinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases. We aimed to identify the optimal method for isolating small (<200 nm) EVs from human urine prior to small RNA analysis. EVs from filtered healthy volunteer urine were concentrated using three methods: ultracentrifugation (UC); a precipitation-based kit (PR); and ultrafiltration (UF). EVs were further purified by size-exclusion chromatography (SEC). EV preparations were analysed with transmission electron microscopy (TEM), Western blotting, nanoparticle tracking analysis (NTA) and an Agilent Bioanalyzer Small RNA kit. UF yielded the highest number of particles both before and after SEC. Small RNA analysis from UF-concentrated urine identified two major peaks at 10–40 nucleotides (nt) and 40–80 nt. In contrast, EV preparations obtained after UC, PR or SEC combined with any concentrating method, contained predominantly 40–80 nt sized small RNA. Protein fractions from UF+SEC contained small RNA of 10–40 nt in size (consistent with miRNAs). These data indicate that most of the microRNA-sized RNAs in filtered urine are not associated with small-sized EVs, and highlights the importance of removing non-vesicular proteins and RNA from urine EV preparations prior to small RNA analysis.     

Extracellular Vesicles Secreted by Glioma Stem Cells Are Involved in Radiation Resistance and Glioma Progression

Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs’ specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.     

Differential MicroRNA Expression in Porcine Endometrium Related to Spontaneous Embryo Loss during Early Pregnancy

Litter size is an important indicator to measure the production capacity of commercial pigs. Spontaneous embryo loss is an essential factor in determining sow litter size. In early pregnancy, spontaneous embryo loss in porcine is as high as 20–30% during embryo implantation. However, the specific molecular mechanism underlying spontaneous embryo loss at the end of embryo implantation remains unknown. Therefore, we comprehensively used small RNA sequencing technology, bioinformatics analysis, and molecular experiments to determine the microRNA (miRNA) expression profile in the healthy and arresting embryo implantation site of porcine endometrium on day of gestation (DG) 28. A total of 464 miRNAs were identified in arresting endometrium (AE) and healthy endometrium (HE), and 139 differentially expressed miRNAs (DEMs) were screened. We combined the mRNA sequencing dataset from the SRA database to predict the target genes of these miRNAs. A quantitative real-time PCR assay identified the expression levels of miRNAs and mRNAs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed target genes of DEMs, mainly enriched in epithelial development and amino acids metabolism-related pathways. We performed fluorescence in situ hybridization (FISH) and the dual-luciferase report gene assay to confirm miRNA and predicted target gene binding. miR-205 may inhibit its expression by combining 3′-untranslated regions (3′ UTR) of tubulointerstitial nephritis antigen-like 1 (TINAGL1). The resulting inhibition of angiogenesis in the maternal endometrium ultimately leads to the formation of arresting embryos during the implantation period. This study provides a reference for the effect of miRNA on the successful implantation of pig embryos in early gestation.     

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30–200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.     

Scalable Production of Extracellular Vesicles and Its Therapeutic Values: A Review

Extracellular vesicles (EVs) are minute vesicles with lipid bilayer membranes. EVs are secreted by cells for intercellular communication. Recently, EVs have received much attention, as they are rich in biological components such as nucleic acids, lipids, and proteins that play essential roles in tissue regeneration and disease modification. In addition, EVs can be developed as vaccines against cancer and infectious diseases, as the vesicle membrane has an abundance of antigenic determinants and virulent factors. EVs for therapeutic applications are typically collected from conditioned media of cultured cells. However, the number of EVs secreted by the cells is limited. Thus, it is critical to devise new strategies for the large-scale production of EVs. Here, we discussed the strategies utilized by researchers for the scalable production of EVs. Techniques such as bioreactors, mechanical stimulation, electrical stimulation, thermal stimulation, magnetic field stimulation, topographic clue, hypoxia, serum deprivation, pH modification, exposure to small molecules, exposure to nanoparticles, increasing the intracellular calcium concentration, and genetic modification have been used to improve the secretion of EVs by cultured cells. In addition, nitrogen cavitation, porous membrane extrusion, and sonication have been utilized to prepare EV-mimetic nanovesicles that share many characteristics with naturally secreted EVs. Apart from inducing EV production, these upscaling interventions have also been reported to modify the EVs’ cargo and thus their functionality and therapeutic potential. In summary, it is imperative to identify a reliable upscaling technique that can produce large quantities of EVs consistently. Ideally, the produced EVs should also possess cargo with improved therapeutic potential.     

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

Elucidation of the biological functions of extracellular vesicles (EVs) and their potential roles in physiological and pathological processes is an expanding field of research. In this study, we characterized USC–derived EVs and studied their capacity to modulate the human immune response in vitro. We found that the USC–derived EVs are a heterogeneous population, ranging in size from that of micro–vesicles (150 nm–1 μm) down to that of exosomes (60–150 nm). Regarding their immunomodulatory functions, we found that upon isolation, the EVs (60–150 nm) induced B cell proliferation and IgM antibody secretion. Analysis of the EV contents unexpectedly revealed the presence of BAFF, APRIL, IL–6, and CD40L, all known to play a central role in B cell stimulation, differentiation, and humoral immunity. In regard to their effect on T cell functions, they resembled the function of mesenchymal stem cell (MSC)–derived EVs previously described, suppressing T cell response to activation. The finding that USC–derived EVs transport a potent bioactive cargo opens the door to a novel therapeutic avenue for boosting B cell responses in immunodeficiency or cancer.     

Profiling Blood Serum Extracellular Vesicles in Plaque Psoriasis and Psoriatic Arthritis Patients Reveals Potential Disease Biomarkers

Psoriasis vulgaris (PsV) and psoriatic arthritis (PsA) are inflammatory diseases with unresolved pathophysiological aspects. Extracellular vesicles (EVs) play an important role in intercellular communication. We compared the miRNA contents and surface proteome of the EVs in the blood serum of PsV and PsA patients to healthy controls. Size-exclusion chromatography was used to isolate EVs from the blood serum of 12 PsV patients, 12 PsA patients and 12 healthy control subjects. EV samples were characterized and RNA sequencing was used to identify differentially enriched EV-bound miRNAs. We found 212 differentially enriched EV-bound miRNAs present in both PsV and PsA groups—a total of 13 miRNAs at FDR ≤ 0.05. The predicted target genes of these miRNAs were significantly related to lesser known but potentially disease-relevant pathways. The EV array revealed that PsV patient EV samples were significantly enriched with CD9 EV-marker compared to controls. Analysis of EV-bound miRNAs suggests that signaling via EVs in the blood serum could play a role in the pathophysiological processes of PsV and PsA. EVs may be able to fill the void in clinically applicable diagnostic and prognostic biomarkers for PsV and PsA.     

Pregnancy-Related Extracellular Vesicles Revisited

Extracellular vesicles (EVs) are small vesicles ranging from 20–200 nm to 10 μm in diameter that are discharged and taken in by many different types of cells. Depending on the nature and quantity of their content—which generally includes proteins, lipids as well as microRNAs (miRNAs), messenger-RNA (mRNA), and DNA—these particles can bring about functional modifications in the receiving cells. During pregnancy, placenta and/or fetal-derived EVs have recently been isolated, eliciting interest in discovering their clinical significance. To date, various studies have associated variations in the circulating levels of maternal and fetal EVs and their contents, with complications including gestational diabetes and preeclampsia, ultimately leading to adverse pregnancy outcomes. Furthermore, EVs have also been identified as messengers and important players in viral infections during pregnancy, as well as in various congenital malformations. Their presence can be detected in the maternal blood from the first trimester and their level increases towards term, thus acting as liquid biopsies that give invaluable insight into the status of the feto-placental unit. However, their exact roles in the metabolic and vascular adaptations associated with physiological and pathological pregnancy is still under investigation. Analyzing peer-reviewed journal articles available in online databases, the purpose of this review is to synthesize current knowledge regarding the utility of quantification of pregnancy related EVs in general and placental EVs in particular as non-invasive evidence of placental dysfunction and adverse pregnancy outcomes, and to develop the current understanding of these particles and their applicability in clinical practice.     

Role of Extracellular Vesicles in Epithelial Ovarian Cancer: A Systematic Review

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived submicron vesicles released under physiological or pathological conditions. EVs mediate the cellular crosstalk, thus contributing to defining the tumor microenvironment, including in epithelial ovarian cancer (EOC). The available literature investigating the role of EVs in EOC has been reviewed following PRISMA guidelines, focusing on the role of EVs in early disease diagnosis, metastatic spread, and the development of chemoresistance in EOC. Data were identified from searches of Medline, Current Contents, PubMed, and from references in relevant articles from 2010 to 1 April 2020. The research yielded 194 results. Of these, a total of 36 papers, 9 reviews, and 27 original types of research were retained and analyzed. The literature findings demonstrate that a panel of EV-derived circulating miRNAs may be useful for early diagnosis of EOC. Furthermore, it appears clear that EVs are involved in mediating two crucial processes for metastatic and chemoresistance development: the epithelial–mesenchymal transition, and tumor escape from the immune system response. Further studies, more focused on in vivo evidence, are urgently needed to clarify the role of EV assessment in the clinical management of EOC patients.     

Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems.     

Diagnostic Performance of Circulating miRNAs and Extracellular Vesicles in Acute Ischemic Stroke

Background: Increased inflammation activates blood coagulation system, higher platelet activation plays a key role in the pathophysiology of ischemic stroke (IS). During platelet activation and aggregation process, platelets may cause increased release of several proinflammatory, and prothrombotic mediators, including microRNAs (miRNAs) and extracellular vesicles (EVs). In the current study we aimed to assess circulating miRNAs profile related to platelet function and inflammation and circulating EVs from platelets, leukocytes, and endothelial cells to analyse their diagnostic and predictive utility in patients with acute IS. Methods: The study population consisted of 28 patients with the diagnosis of the acute IS. The control group consisted of 35 age- and gender-matched patients on acetylsalicylic acid (ASA) therapy without history of stroke and/or TIA with established stable coronary artery disease (CAD) and concomitant cardiovascular risk factors. Venous blood samples were collected from the control group and patients with IS on ASA therapy (a) 24 h after onset of acute IS, (b) 7-days following index hospitalization. Flow cytometry was used to determine the concentration of circulating EVs subtypes (from platelets, leukocytes, and endothelial cells) in platelet-depleted plasma and qRT-PCR was used to determine several circulating plasma miRNAs (miR-19a-3p, miR-186-5p and let-7f). Results: Patients with high platelet reactivity (HPR, based on arachidonic acid-induced platelet aggregometry) had significantly elevated platelet-EVs (CD62+) and leukocyte-EVs (CD45+) concentration compared to patients with normal platelet reactivity at the day of 1 acute-stroke (p = 0.012, p = 0.002, respectively). Diagnostic values of baseline miRNAs and EVs were evaluated with receiver operating characteristic (ROC) curve analysis. The area under the ROC curve for miR-19a-3p was 0.755 (95% CI, 0.63–0.88) p = 0.004, for let-7f, it was 0.874 (95% CI, 0.76–0.99) p = 0.0001; platelet-EVs was 0.776 (95% CI, 0.65–0.90) p = 0.001, whereas for leukocyte-EVs, it was 0.715 (95% CI, 0.57–0.87) p = 0.008. ROC curve showed that pooling the miR-19a-3p expressions, platelet-EVs, and leukocyte-EVs concentration yielded a higher AUC than the value of each individual biomarker as AUC was 0.893 (95% CI, 0.79–0.99). Patients with moderate stroke had significantly elevated miR-19a-3p expression levels compared to patients with minor stroke at the first day of IS. (AUC: 0.867, (95% CI, 0.74–0.10) p = 0.001). Conclusion: Combining different biomarkers of processes underlying IS pathophysiology might be beneficial for early diagnosis of ischemic events. Thus, we believe that in the future circulating biomarkers might be used in the prehospital phase of IS. In particular, circulating plasma EVs and non-coding RNAs including miRNAs are interesting candidates as bearers of circulating biomarkers due to their high stability in the blood and making them highly relevant biomarkers for IS diagnostics.     

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus–oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT–qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8–16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5–50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.     

hnRNPA2B1-Mediated Extracellular Vesicles Sorting of miR-122-5p Potentially Promotes Lung Cancer Progression

Extracellular vesicles (EVs) released by tumor cells play important roles on the remodeling of the tumor–stromal environment and on promoting tumor metastasis. Our earlier studies revealed that miR-122-5p, a type of small non-coding RNA, was dysregulated in non-small cell lung cancer (NSCLC) cell-derived EVs. In this study, we found that miR-122-5p was selectively sorted and secreted into lung cancer EVs through binding to RNA-binding protein hnRNPA2B1. In addition, we found that hnRNPA2B1 interacted with miR-122-5p through the EXO-motif. The delivering of lung cancer EVs-miR-122-5p promoted the migration of liver cells, which may play roles in establishing a pre-metastatic micro-environment and hepatic metastasis of lung cancer. Importantly, our findings revealed the molecular mechanism that RNA-binding protein controls the selective sorting of tumor-derived EV miR-122-5p, which potentially promotes lung cancer progression.     

Glioblastoma Extracellular Vesicle-Specific Peptides Inhibit EV-Induced Neuronal Cytotoxicity

WHO Grade 4 IDH-wild type astrocytoma (GBM) is the deadliest brain tumor with a poor prognosis. Meningioma (MMA) is a more common “benign” central nervous system tumor but with significant recurrence rates. There is an urgent need for brain tumor biomarkers for early diagnosis and effective treatment options. Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles that play essential functions in cell-to-cell communications among tumor cells. We aimed to identify epitopes of brain tumor EVs by phage peptide libraries. EVs from GBM plasma, MMA plasma, or brain tumor cell lines were used to screen phage-displayed random peptide libraries to identify high-affinity peptides. We purified EVs from three GBM plasma pools (23 patients), one MMA pool (10 patients), and four brain tumor cell lines. We identified a total of 21 high-affinity phage peptides (12 unique) specific to brain tumor EVs. The peptides shared high sequence homologies among those selected by the same EVs. Dose–response ELISA demonstrated that phage peptides were specific to brain tumor EVs compared to controls. Peptide affinity purification identified unique brain tumor EV subpopulations. Significantly, GBM EV peptides inhibit brain tumor EV-induced complement-dependent cytotoxicity (necrosis) in neurons. We conclude that phage display technology could identify specific peptides to isolate and characterize tumor EVs.     

Therapeutic Effects of Platelet-Derived Extracellular Vesicles in a Bioengineered Tendon Disease Model

Tendon injuries represent over 30–50% of musculoskeletal disorders worldwide, yet the available therapies do not provide complete tendon repair/regeneration and full functionality restoring. Extracellular vesicles (EVs), membrane-enclosed nanoparticles, have emerged as the next breakthrough in tissue engineering and regenerative medicine to promote endogenous tissue regeneration. Here, we developed a 3D human in vitro model mimicking the signature of pathological tendon and used it to evaluate the influence that different platelet-derived EVs might have in tendon tissue repair mechanisms. For this, different EV populations isolated from platelets, small EVs (sEVs) and medium EVs (mEVs), were added to the culture media of human tendon-derived cells (hTDCs) cultured on isotropic nanofibrous scaffolds. The platelet-derived EVs increased the expression of tenogenic markers, promoted a healthy extracellular matrix (ECM) remodeling, and the synthesis of anti-inflammatory mediators. These findings suggest that platelet EVs provided relevant biochemical cues that potentiated a recovery of hTDCs phenotype from a diseased to a healthy state. Thus, this study opens new perspectives for the translation of platelet-derived EVs as therapeutics.