Blood concentrations of small extracellular vesicles are determined by a balance between abundant secretion and rapid clearance.
ABSTRACT: Small extracellular vesicles (sEVs) are important mediators of cell-cell communication with respect to diverse physiological processes. To further understand their physiological roles, understanding blood sEV homoeostasis in a quantitative manner is desired. In this study, we propose novel kinetic approaches to estimate the secretion and clearance of mouse plasma-derived sEVs (MP-sEVs) based on the hypothesis that blood sEV concentrations are determined by a balance between the secretion and clearance of sEVs. Using our specific and sensitive sEV labelling technology, we succeeded in analysing MP-sEV clearance from the blood after intravenous administration into mice. This revealed the rapid disappearance of MP-sEVs with a half-life of approximately 7 min. Moreover, the plasma sEV secretion rate, which is presently impossible to directly evaluate, was calculated as 18 ?g/min in mice based on pharmacokinetic (PK) analysis. Next, macrophage-depleted mice were prepared as a model of disrupted sEV homoeostasis with retarded sEV clearance. MP-sEV concentrations were increased in macrophage-depleted mice, which probably reflected a shift in the balance of secretion and clearance. Moreover, the increased MP-sEV concentration in macrophage-depleted mice was successfully simulated using calculated clearance rate constant, secretion rate constant and volume of distribution, suggesting the validity of our PK approaches. These results demonstrate that blood sEV concentration homoeostasis can be explained by the dynamics of rapid secretion/clearance.
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:Small extracellular vesicles (sEVs) such as exosomes are nanocarriers of proteins, RNAs and DNAs. Isolation of pure sEV populations remains challenging, with reports of protein and lipoprotein contaminants in the isolates. Cellular uptake - a cornerstone for understanding exosome and sEV function - is frequently examined using lipophilic dyes such as PKH67 or CellMask to label the vesicles. In this study, we investigated whether contaminants can confound the outcomes from sEV and exosomes uptake experiments. sEVs were isolated from blood plasma of fasted or non-fasted rats as well as from serum-supplemented or serum-free conditioned cell culture medium using size-exclusion chromatography (SEC). Eluent fractions were characterized using nanoparticle tracking, protein and triglyceride assays and immunoassays. SEC fractions were labelled with different lipophilic dyes and cellular uptake was quantified using endothelial cells or primary cardiomyocytes. We report co-isolation of sEVs with apolipoprotein B-containing lipoproteins. Cellular dye transfer did not correspond to sEV content of the SEC fractions, but was severely affected by lipoprotein and protein content. Overnight fasting of rats decreased lipoprotein content and also decreased dye transfer, while late, sEV-poor/protein-rich fractions demonstrated even greater dye transfer. The potential for dye transfer to occur in the complete absence of sEVs was clearly shown by experiments using staining of sEV-depleted serum or pure protein sample. In conclusion, proteins and lipoproteins can make a substantial contribution to transfer of lipophilic dyes to recipient cells. Considering the likelihood of contamination of sEV and exosome isolates, lipophilic dye staining experiments should be carefully controlled, and conclusions interpreted with caution.
Project description:The ?1 integrins, known to promote cancer progression, are abundant in extracellular vesicles (EVs). We investigated whether prostate cancer (PrCa) EVs affect anchorage-independent growth and whether ?1 integrins are required for this effect. Specifically using a cell-line-based genetic rescue and an in vivo PrCa model, we show that gradient-purified small EVs (sEVs) from either cancer cells or blood from tumor-bearing TRAMP (transgenic adenocarcinoma of the mouse prostate) mice promote anchorage-independent growth of PrCa cells. In contrast, sEVs from cultured PrCa cells harboring a short hairpin RNA to ?1, from wild-type mice or from TRAMP mice carrying a ?1 conditional ablation in the prostatic epithelium (?1pc-/-), do not. We find that sEVs, from cancer cells or TRAMP blood, are functional and co-express ?1 and sEV markers; in contrast, sEVs from ?1pc-/-/TRAMP or wild-type mice lack ?1 and sEV markers. Our results demonstrate that ?1 integrins in tumor-cell-derived sEVs are required for stimulation of anchorage-independent growth.
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: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: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.
Project description:Macrophages play an important role in material-related immune responses and bone formation, but the functionality of macrophage-derived extracellular vesicles (EVs) in material-mediated bone regeneration is still unclear. Here, we evaluated intracellular communication through small extracellular vesicles (sEVs) and its effects on endogenous bone regeneration mediated by biomimetic intrafibrillarly mineralized collagen (IMC). After implantation in the bone defect area, IMC generated more neobone and recruited more mesenchymal stem cells (MSCs) than did extrafibrillarly mineralized collagen (EMC). More CD63<sup>+</sup>CD90<sup>+</sup> and CD63<sup>+</sup>CD163<sup>+</sup> cells were detected in the defect area in the IMC group than in the EMC group. To determine the functional roles of sEVs, extracellular vesicles from macrophages cultured on different mineralized collagen were isolated, and they showed no morphological differences. However, macrophage-derived sEVs in the IMC group showed an enhanced Young's modulus and exerted beneficial effects on the osteogenic differentiation of bone marrow MSCs by increasing the expression of the osteoblastic differentiation markers BMP2, BGLAP, COL1, and OSX and calcium nodule formation. Mechanistically, sEVs from IMC-treated macrophages facilitated MSC osteogenesis through the BMP2/Smad5 pathway, and blocking sEV secretion with GW4869 significantly impaired MSC proliferative, immunomodulative and osteogenic potential. Taken together, these findings show that macrophage-derived sEVs may serve as an emerging functional tool in biomaterial-mediated endogenous bone regeneration.
Project description:Exosomes are important mediators for cell-cell communication. In previous study, the clearance of exosomes were markedly delayed in systemic circulation of macrophage-depleted mice. To reveal the possible molecular regulation involved in exosome clearance, we performed exosomal proteomic analysis on MP-Exo from macrophage depleted mice and control ones.
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:Small extracellular vesicles (sEVs) mediate cell-to-cell communication. We recently reported that circulating sEVs regulate systolic blood pressure in an animal model of human systemic hypertension. However, the underlying mechanisms still remain to be elucidated. As the first step for detailed analyses, we sought to increase the yield and purity of sEVs isolated from rat plasma. We compared the concentration and size distribution of sEVs as well as protein expression of the sEV marker and contaminants among plasma sEVs isolated by the ultracentrifugation (UC) method, the precipitation with polyethylene-glycol and ultracentrifugation (PEG-UC) method, or the precipitation with polyethylene-glycol (PEG) method. Effects of anticoagulants were also examined. The total concentration of plasma sEVs isolated by the PEG or PEG-UC method was much higher than that of the UC method. In the plasma sEVs isolated by the PEG-UC method, contaminating proteins were lower, while the protein expression of certain sEV markers was higher than that of the PEG method. There was no significant difference in total concentration or protein expression of sEV markers in sEVs isolated from rat plasma treated with three different anticoagulants (heparin, ethylenediaminetetraacetic acid, or acid citrate dextrose buffer) by the PEG-UC method. We, for the first time, determined that the PEG-UC method was optimal for sEV isolation from rat plasma.