Phosphatidylserine-deficient small extracellular vesicle is a major somatic cell-derived sEV subpopulation in blood.
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ABSTRACT:
SUBMITTER: Matsumoto A
PROVIDER: S-EPMC8326202 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Small extracellular vesicles (sEVs) are important mediators of intercellular communication with respect to diverse pathophysiological processes. The heterogeneity of sEV populations is a key factor in pursuing the sEV-related translational and basic research into forward but remains challenging for unraveling. Here, we discovered novel phosphatidylserine (PS; marker for sEV uptake by macrophage)-deficient sEV subpopulations which possessed super long blood circulation through escape from the uptake by macrophages (main player of the rapid sEV clearance from blood). PS(-)-sEVs were identified in various cultured-cells as a minor population. Meanwhile, circulating somatic cell-derived sEVs in the blood were found to be majorly PS(-)-sEVs, which were caused by rapid uptake of PS(+)-sEVs by macrophages within 10 min after entry into the circulation. These results suggest endogenous PS(-)-sEVs could truly be the key player of sEV-mediated intracellular communication and that PS(-)-sEVs can be a good target for sEV-based diagnosis as well as a potent candidate for sEV-based delivery carrier. Our findings shift the paradigm for further understanding the biology as well as for the translational applications of sEV.
2021-07-08 | PXD019244 | JPOST Repository
Project description: Not available
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Project description:Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil (4TUc) into 4-thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labelled with streptavidin conjugates or isolated via streptavidin-affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CM UPRT mice) and tested our hypothesis that CM-derived miRNAs (CM miRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PB sEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PB sEV of CM UPRT mice 6 h after 4TUc injection. In PB sEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labelled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a (CM miR-208a) levels peaked 12 h after experimentally induced MI in PB sEV and 24 h after MI in the lung. Notably, miR-208a is expressed from intron 29 of α myosin heavy chain (αMHC), but αMHC transcripts were nearly undetectable in the lung. When PB sEV from mice that underwent MI (MI-PB sEV) or sham surgery (Sham-PB sEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-κB pathway, was lower in the lungs of MI-PB sEV-treated animals than the lungs of animals treated with Sham-PB sEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Thus, CM UPRT mice enables us to track PB sEV-mediated transport of CM miRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.
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Project description:Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30-200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein-Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.
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Project description:Immune disorders caused by sepsis have recently drawn much attention. We sought to dynamically monitor the expression of small extracellular vesicle (sEV) miRNAs in peripheral blood during sepsis to explore these miRNAs as potential biomarkers for monitoring immune function in sepsis patients. This study included patients with sepsis. Blood samples were obtained from 10 patients on the first through 10th days, the 12th day and the 14th day since sepsis onset, resulting in 120 collected samples. Serum sEVs were extracted from peripheral venous blood, and levels of MIR497HG, miR-195, miR-497, and PD-L1 in serum sEVs were detected by qPCR, and clinical information was recorded. Our study revealed that the levels of MIR497HG, miR-195, miR-497 and PD-L1 in serum sEVs showed periodic changes; the time from peak to trough was approximately 4-5 days. The levels of sEV MIR497HG and miR-195 had a positive linear relationship with SOFA score (r values were -0.181 and -0.189; p values were 0.048 and 0.039, respectively). The recorded quantities of sEV MIR497HG, miR-195 and PD-L1 showed a substantial correlation with ARDS. ROC curve analysis revealed that sEV MIR497HG, miR-195 and miR-497 could predict the 28-day mortality of sepsis patients with an AUC of 0.66, 0.68 and 0.72, respectively. Levels of sEVs MIR497HG, miR-195, miR-497 and PD-L1 showed periodic changes with the immune status of sepsis, which provides a new exploration direction for immune function biomarkers and immunotherapy timing in sepsis patients.
| S-EPMC10826429 | biostudies-literature
Project description:With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.
| S-EPMC8750562 | biostudies-literature
Project description:Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) converts 4-thiouracil (4TUc) into 4-thiouridine (4TUd), which is incorporated into nascent RNAs and can be biotinylated, then labeled with streptavidin conjugates or isolated via streptavidin-affinity methods. Here, we generated mice that expressed T. gondii UPRT only in cardiomyocytes (CMUPRT mice) and tested our hypothesis that CM-derived miRNAs (CMmiRs) are transferred into remote organs after myocardial infarction (MI) by small extracellular vesicles (sEV) that are released from the heart into the peripheral blood (PBsEV). We found that 4TUd was incorporated with high specificity and sensitivity into RNAs isolated from the hearts and PBsEV of CMUPRT mice six hours after 4TUc injection. In PBsEV, 4TUd was incorporated into CM-specific/enriched miRs including miR-208a, but not into miRs with other organ or tissue-type specificities. 4TUd-labeled miR208a was also present in lung tissues, especially lung endothelial cells (ECs), and CM-derived miR-208a (CMmiR-208a) levels peaked 12 hours after experimentally induced MI in PBsEV and 24 hours after MI in the lung. Notably, miR-208a is expressed from intron 29 of alpha myosin heavy chain (a-MHC), but a-MHC transcripts were nearly undetectable in the lung. When PBsEV from mice that underwent MI (MI-PBsEV) or sham surgery (Sham-PBsEV) were injected into intact mice, the expression of Tmbim6 and NLK, which are suppressed by miR-208a and cooperatively regulate inflammation via the NF-kappa B pathway, was lower in the lungs of MI-PBsEV-treated animals than the lungs of animals treated with Sham-PBsEV or saline. In MI mice, Tmbim6 and NLK were downregulated, whereas endothelial adhesion molecules and pro-inflammatory cells were upregulated in the lung; these changes were significantly attenuated when the mice were treated with miR-208a antagomirs prior to MI surgery. Thus, CMUPRT mice enables us to track sEV-mediated transport of CMmiRs and identify an miR-208a-mediated mechanism by which myocardial injury alters the expression of genes and inflammatory response in the lung.
2022-10-26 | GSE205405 | GEO
Project description:The clinical benefit of therapies using Mesenchymal Stem Cells (MSCs) is attributable to their pleiotropic effect over cells and tissues, mainly through their secretome. This paracrine effect is mediated by secreted growth factors and extracellular vesicles (EV) including small EV (sEV). sEV are extra-cellular, membrane encompassed vesicles of 40 to 200 nm diameter that can trigger and signal many cellular responses depending on their cargo protein and nucleic acid repertoire. sEV are purified from cell culture conditioned media using several kits and protocols available that can be tedious and time-consuming, involving sequences of ultracentrifugations and density gradient separations, making their production a major challenge under Good Manufacturing Practices (GMP) conditions. We have developed a method to efficiently enrich cell culture media with high concentrations of sEV by encapsulating cells in semipermeable cellulose beads that allows selectively the release of small particles while offering a 3D culture condition. This method is based on the pore size of the capsules, allowing the release of particles of ? 200 nm including sEV. As a proof-of-principle, MSCs were encapsulated and their sEV release rate (sEV-Cap) was monitored throughout the culture and compared to sEV isolated from 2D seeded cells (sEV-2D) by repetitive ultracentrifugation cycles or a commercial kit. The isolated sEV expressed CD63, CD9, and CD81 as confirmed by flow cytometry analysis. Under transmission electron microscopy (TEM), they displayed the similar rounded morphology as sEV-2D. Their corresponding diameter size was validated by nanoparticle tracking analysis (NTA). Interestingly, sEV-Cap retained the expected biological activities of MSCs, including a pro-angiogenic effect over endothelial cells, neuritic outgrowth stimulation in hippocampal neurons and immunosuppression of T cells in vitro. Here, we successfully present a novel, cost, and time-saving method to generate sEV from encapsulated MSCs. Future applications include using encapsulated cells as a retrievable delivery device that can interact with the host niche by releasing active agents in vivo, including sEV, growth factors, hormones, and small molecules, while avoiding cell clearance, and the negative side-effect of releasing undesired components including apoptotic bodies. Finally, particles produced following the encapsulation protocol display beneficial features for their use as drug-loaded delivery vehicles.
| S-EPMC7253686 | biostudies-literature
Project description:Extracellular Vesicles were isolated from primary cultures of neural stem cells (NSCs) and astrocytes by performing serial centrifugation and a 100,000 x g ultracentrifugation step. The goal of this experiment is to compare EV small RNAs from two cell types.
2019-05-13 | GSE121652 | GEO
Project description:The placenta could transmit information to the maternal circulation via the secretion of small extracellular vesicles (sEVs), but little is known about whether and how these sEVs participate in premature labor (PTL). We speculate that miRNA plays an important role in sEV-mediated fetal-maternal information transmission. The present study was aimed at investigating whether the placenta can regulate the contraction of the maternal myometrium via sEV-mediated transmit of miR-25-3p during PTL. The expression of miR-25-3p and its targets Cav3.2 and SERCA2a in clinical samples, cells, and animal specimens was detected. The role of miR-25-3p was observed in the LPS-induced preterm labor mouse model. The sEVs from HTR-8/SVneo cells were characterized by transmission electron microscopy and nanoparticle tracking analysis. The Ca2+ oscillation in HMSMs was analyzed by an intracellular Ca2+ change tracking assay on a confocal microscope. The contraction of HMSMs was detected by a collagen matrix contraction assay. We found that miR-25-3p can directly bind to the 3'UTR of Cav3.2 and SERCA2a. The miR-25-3p level was decreased, and the expression of its targets Cav3.2 and SERCA2a was increased in the placenta and myometrium tissues of PTL patients. Forced upregulation of miR-25-3p reduced the oxidative stress and inflammation responses and the incidence of PTL in LPS-treated mice. The expression of miR-25-3p was not changed in LPS-stimulated human myometrial smooth muscle cells (HMSMs) but was strongly reduced in the trophoblast cell and its sEVs. Additionally, the trophoblast cell line HTR-8/SVneo could transmit miR-25-3p into HMSMs via sEVs. The sEVs derived from LPS-stimulated trophoblasts upregulated the expression of Cav3.2 and SERCA2a and triggered Ca2+ oscillation as well as the contraction of HMSMs; these effects were partially reversed by the overexpression of miR-25-3p in the trophoblasts. Further, the upregulation of miR-25-3p induced changes of Ca2+ oscillation and contraction of HMSMs mediated by sEVs which were also abrogated by the knockdown of miR-25-3p in HMSMs. The results demonstrated that miR-25-3p plays a crucial role in PTL via Cav3.2- and SERCA2a-mediated Ca2+ oscillation and contraction of HMSMs. PTL seems to be related to the decreased exosomal miR-25-3p content transmitted by the trophoblasts under inflammatory conditions.
| S-EPMC8369173 | biostudies-literature