Internalization of trophoblastic small extracellular vesicles and detection of their miRNA cargo in P-bodies.
ABSTRACT: Pregnancy is a unique situation, in which placenta-derived small extracellular vesicles (sEVs) may communicate with maternal and foetal tissues. While relevant to homoeostatic and pathological functions, the mechanisms underlying sEV entry and cargo handling in target cells remain largely unknown. Using fluorescently or luminescently labelled sEVs, derived from primary human placental trophoblasts or from a placental cell line, we interrogated the endocytic pathways used by these sEVs to enter relevant target cells, including the neighbouring primary placental fibroblasts and human uterine microvascular endothelial cells. We found that trophoblastic sEVs can enter target cells, where they retain biological activity. Importantly, using a broad series of pharmacological inhibitors and siRNA-dependent silencing approaches, we showed that trophoblastic sEVs enter target cells using macropinocytosis and clathrin-mediated endocytosis pathways, but not caveolin-dependent endocytosis. Tracking their intracellular course, we localized the sEVs to early endosomes, late endosomes, and lysosomes. Finally, we used coimmunoprecipitation to demonstrate the association of the sEV microRNA (miRNA) with the P-body proteins AGO2 and GW182. Together, our data systematically detail endocytic pathways used by placental sEVs to enter relevant fibroblastic and endothelial target cells, and provide support for "endocytic escape" of sEV miRNA to P-bodies, a key site for cytoplasmic RNA regulation.
Project description:Extracellular vesicles (EVs), including exosomes and microvesicles, derived from bone marrow stromal cells (BMSCs) have been demonstrated as key factors in the progression and drug resistance of multiple myeloma (MM). EV uptake involves a variety of mechanisms which largely depend on the vesicle origin and recipient cell type. The aim of the present study was to identify the mechanisms involved in the uptake of BMSC-derived small EVs (sEVs) by MM cells, and to evaluate the anti-MM effect of targeting this process. <b>Methods:</b> Human BMSC-derived sEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blot. The effects of chemical inhibitors and shRNA-mediated knockdown of endocytosis-associated genes on sEV uptake and cell apoptosis were analyzed by flow cytometry. The anti-MM effect of blocking sEV uptake was evaluated <i>in vitro</i> and in a xenograft MM mouse model. <b>Results:</b> sEVs derived from BMSC were taken up by MM cells in a time- and dose-dependent manner, and subsequently promoted MM cell cycling and reduced their chemosensitivity to bortezomib. Chemical endocytosis inhibitors targeting heparin sulphate proteoglycans, actin, tyrosine kinase, dynamin-2, sodium/proton exchangers, or phosphoinositide 3-kinases significantly reduced MM cell internalization of BMSC-derived sEVs. Moreover, shRNA-mediated knockdown of endocytosis-associated proteins, including caveolin-1, flotillin-1, clathrin heavy chain, and dynamin-2 in MM cells suppressed sEV uptake. Furthermore, an endocytosis inhibitor targeting dynamin-2 preferentially suppressed the uptake of sEV by primary MM cells <i>ex vivo</i> and enhanced the anti-MM effects of bortezomib <i>in vitro</i> and in a mouse model. <b>Conclusion:</b> Clathrin- and caveolin-dependent endocytosis and macropinocytosis are the predominant routes of sEV-mediated communication between BMSCs and MM cells, and inhibiting endocytosis attenuates sEV-induced reduction of chemosensitivity to bortezomib, and thus enhances its anti-MM properties.
Project description:Intercellular communication is critical for organismal homeostasis, and defects can contribute to human disease states. Polarized epithelial cells execute distinct signaling agendas via apical and basolateral surfaces to communicate with different cell types. Small extracellular vesicles (sEVs), including exosomes and small microvesicles, represent an understudied form of intercellular communication in polarized cells. Human cholangiocytes, epithelial cells lining bile ducts, were cultured as polarized epithelia in a Transwell system as a model with which to study polarized sEV communication. Characterization of isolated apically and basolaterally released EVs revealed enrichment in sEVs. However, differences in apical and basolateral sEV composition and numbers were observed. Genetic or pharmacological perturbation of cellular machinery involved in the biogenesis of intralumenal vesicles at endosomes (the source of exosomes) revealed general and domain-specific effects on sEV biogenesis/release. Additionally, analyses of signaling revealed distinct profiles of activation depending on sEV population, target cell, and the function of the endosomal sorting complex required for transport (ESCRT)-associated factor ALG-2-interacting protein X (ALIX) within the donor cells. These results support the conclusion that polarized cholangiocytes release distinct sEV pools to mediate communication via their apical and basolateral domains and suggest that defective ESCRT function may contribute to disease states through altered sEV signaling.
Project description:Macromolecules enter cells by endocytosis and are sorted to different cellular destinations in early/sorting endosomes. The mechanism and regulation of sorting are poorly understood, although transitions between vesicular and tubular endosomes are important. We found that the antihypertensive drug Prazosin inhibits endocytic sorting by an off-target perturbation of the G protein-coupled receptor dopamine receptor D(3) (DRD3). Prazosin is also a potent cytokinesis inhibitor, likely as a consequence of its effects on endosomes. Prazosin stabilizes a normally transient interaction between DRD3 and the coatomer COPI, a complex involved in membrane transport, and shifts endosomal morphology entirely to tubules, disrupting cargo sorting. RNAi depletion of DRD3 alone also inhibits endocytic sorting, indicating a noncanonical role for a G protein-coupled receptor. Prazosin is a powerful tool for rapid and reversible perturbation of endocytic dynamics.
Project description:Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB+ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium.
Project description:Serum small extracellular vesicles (sEVs) have recently drawn considerable interest because of the diagnostic and therapeutic potential of their miRNAs content. However, the characteristics of human, mouse and rat serum sEVs and their differences in small RNA contents are still unknown. In this study, through nanoparticle tracking analysis and small RNA sequencing, we found that human, rat, and mouse serum sEVs exhibited distinct sizes and particle numbers as well as small RNA contents. Serum sEVs contained not only abundant miRNAs but also a large number of tRNA fragments. Most serum miRNAs existed both inside and outside of sEVs but were enriched in sEVs. Common serum sEV miRNAs (188 miRNAs) and species-specific serum sEV miRNAs (265, 58, and 159 miRNAs, respectively) were identified in humans, rats, or mice. The serum sEVs contained miRNAs from tissues and organs throughout the body, with blood cells as the main contributors. In conclusion, our findings confirmed the rationality of exploring serum sEV miRNAs as noninvasive diagnostic markers and revealed great differences in serum sEV small RNAs between humans, rats, and mice. Inadequate attention to these differences and the contribution of blood cells to serum sEV miRNAs could hinder the clinical translation of basic studies.
Project description:Studies of small extracellular vesicles (sEVs), known as exosomes, have been flourishing in the last decade with several achievements, from advancing biochemical knowledge to use in biomedical applications. Physiological changes of sEVs due to the variety of cargos they carry undoubtedly leave an impression that affects the understanding of the mechanism underlying disease and the development of sEV-based shuttles used for treatments and non-invasive diagnostic tools. Indeed, the remarkable properties of sEVs are based on their nature, which helps shield them from recognition by the immune system, protects their payload from biochemical degradation, and contributes to their ability to translocate and convey information between cells and their inherent ability to target disease sites such as tumors that is valid for sEVs derived from cancer cells. However, their transport, biogenesis, and secretion mechanisms are still not thoroughly clear, and many ongoing investigations seek to determine how these processes occur. On the other hand, lead compounds have been playing critical roles in the drug discovery process and have been recently employed in studies of the biogenesis and secretion of sEVs as external agents, affecting sEV release and serving as drug payloads in sEV drug delivery systems. This article gives readers an overview of the roles of lead compounds in these two research areas of sEVs, the rising star in studies of nanoscale medicine.
Project description:Cancer-derived small extracellular vesicles (sEVs) induce stromal cells to become permissive for tumor growth. However, it is unclear whether this induction solely occurs through transfer of vesicular cargo into recipient cells. Here we show that cancer-derived sEVs can stimulate endothelial cell migration and tube formation independently of uptake. These responses were mediated by the 189 amino acid isoform of vascular endothelial growth factor (VEGF) on the surface of sEVs. Unlike other common VEGF isoforms, VEGF189 preferentially localized to sEVs through its high affinity for heparin. Interaction of VEGF189 with the surface of sEVs profoundly increased ligand half-life and reduced its recognition by the therapeutic VEGF antibody bevacizumab. sEV-associated VEGF (sEV-VEGF) stimulated tumor xenograft growth but was not neutralized by bevacizumab. Furthermore, high levels of sEV-VEGF were associated with disease progression in bevacizumab-treated cancer patients, raising the possibility that resistance to bevacizumab might stem in part from elevated levels of sEV-VEGF.
Project description:Most patients with acute myeloid leukaemia (AML) experience disease recurrence after chemotherapy largely due to the development of drug resistance. Small extracellular vesicles (sEVs) are known to play a significant role in leukaemia drug resistance by delivery of anti-apoptotic proteins and genes conferring resistance to recipient cells. sEV levels are elevated in AML patients' plasma at the time of diagnosis and remain elevated in complete remission after chemotherapy. The mechanism of enhanced sEV secretion in AML is unknown. We speculated that cholesterol synthesis by AML blasts may be related to elevated sEV secretion. Intracellular levels of cholesterol and of HMGCR (3-hydroxy-3-methyl-glutaryl-coenzyme A reductase), the rate-limiting enzyme in cholesterol synthesizing mevalonate pathway, significantly increased in cultured AML cells or primary human non-malignant cells treated with cytarabine or decitabine. Concomitantly, levels of sEVs produced by these cells also increased. Treatment with an HMGCR inhibitor, Simvastatin, or siRNAs targeting HMGCR blocked the chemotherapy-induced enhancement of sEV secretion in AML cells. sEVs carry HMGCR and chemotherapy enhances HMGCR levels in sEVs. HMGCR+ sEVs upregulate intracellular cholesterol and promote AML cell proliferation. A pharmacologic blockade of HMGCR emerges as a potential future therapeutic option for disrupting sEV signalling leading to cholesterol-driven chemo-resistance in AML.
Project description:Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy.
Project description:Activated osteoclasts release large amounts of small extracellular vesicles (sEVs) during bone remodeling. However, little is known about whether osteoclast-derived sEVs affect surrounding cells. In this study, osteoclasts were generated by stimulating bone marrow macrophages (BMMs) with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear actor ?B ligand (RANKL). We performed microarray analysis of sEV-microRNAs (miRNAs)s secreted from osteoclast at different stages and identified four miRNAs that were highly expressed in mature osteoclast-derived sEVs. One of these miRNAs, miR-324, significantly induced osteogenic differentiation and mineralization of primary mesenchymal stem cells (MSCs) <i>in vitro</i> by targeting <i>ARHGAP1</i>, a negative regulator of osteogenic differentiation. We next fabricated an sEV-modified scaffold by coating decalcified bone matrix (DBM) with osteoclast-derived sEVs, and the pro-osteogenic regeneration activities of the sEV-modified scaffold were validated in a mouse calvarial defect model. Notably, miR-324-enriched sEV-modified scaffold showed the highest capacity on bone regeneration, whereas inhibition of miR-324 in sEVs abrogated these effects. Taken together, our findings suggest that miR-324-contained sEVs released from mature osteoclast play an essential role in the regulation of osteogenic differentiation and potentially bridge the coupling between osteoclasts and MSCs.