Isolation and Characterization of Microvesicles from Peripheral Blood

Menck, Kerstin; Bleckmann, Annalen; Schulz, Matthias; Ries, Lena; Binder, Claudia R.

doi:10.3791/55057

Cited by 44 publications

(43 citation statements)

References 32 publications

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“…“ Fig 1A ” shows the presence of CD9, CD63 and Hsp 70 proteins in exosomes isolated from the plasma of four healthy donors. We also analyzed the exosomes isolated by the above method for the presence of CD41 and CD45 antigens which are reported to be concentrated in microvesicles [ 9 ]. According to “ Fig 1B , detectable amounts of CD41 and CD45 antigens were not found in exosome pellet.…”

Section: Resultsmentioning

confidence: 99%

“…This shows that when blood was drawn, there was very low concentrations of red cell-derived exosomes in blood but increase when blood is stored for longer periods of time. CD41 and CD45 proteins which originate from platelets and leucocytes, respectively are mainly present in microvesicles [ 9 ]. “ Fig 6C ” shows that CD41 and CD45 proteins are absent in exosomes isolated from stored blood samples indicating that exosomes isolated using Invitrogen kit was not contaminated with microvesicles.…”

Section: Resultsmentioning

confidence: 99%

“…According to previously published reports, size of exosomes range from 40–100 nm and has a specific morphology and shape [ 9 , 10 ]. Plasma exosomes isolated using Invitrogen exosome isolation kit showed shape and morphology under electron microscope that are characteristic of exosomes ( Fig 1D ).…”

Section: Discussionmentioning

confidence: 99%

“…When plasma exosomes isolated by Invitrogen kit were analyzed by Western blotting, it showed the presence of cytocolic protein Hsp 70, commonly present in exosomes and membrane-bound tetraspanins, CD9 and CD63 that are known to be enriched in exosomes [ 7 , 9 , 12 ]. Membrane-bound tetraspanins, CD41 and CD45 which are enriched in microvesicles [ 9 ] could not be detected in exosomes isolated by the kit ( Fig 1B ) indicating that exosomes isolated were relatively free of microvesicles. Exosomes were also isolated using a previously described density gradient centrifugation method.…”

Section: Discussionmentioning

confidence: 99%

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New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

et al. 2017

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Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K3EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

et al. 2017

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“…The data revealed that MVs pelleted from EDTA-anticoagulated plasma samples by differential centrifugation were 100-600 nm in diameter. MVs can be distinguished from exosomes by detecting the presence of proteins tubulin, actinin-4, or mitofilin, while antibodies for CD9 and CD81 were used as markers for exosomes [85].…”

Section: Biomarkers On Microvesiclesmentioning

confidence: 99%

Microvesicles Released from Human Red Blood Cells: Properties and Potential Applications

Nguyen¹,

Ly²,

Bernhardt³

2017

Novel Implications of Exosomes in Diagnosis and Treatment of Cancer and Infectious Diseases

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Microvesicles (MVs) are small spherical fragments of plasma membrane between 50 and 1000 nm in diameter. MVs arise through direct outward budding and fission of the plasma membrane. As almost all cells, human red blood cells (RBCs) are able to release MVs into extracellular environment under stimulating or storage conditions. Recently, it has been known that MVs not only play a role in homeostasis but also have diverse functions in cell-cell interactions and in the pathogenesis of diseases. In this chapter, the formation and release of MVs from human RBCs have been described. In addition, MVs have demonstrated to be potential vehicle for transport of nucleic acid and other molecules to the target cells. Although RBC-derived MVs are potential material for the development of delivery systems, it is still a great challenge to the clinical application. Future research should pay more attention to MVs as biological targets for diagnosis and practical therapeutics of cancer and other diseases.

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Exosomal miR‐122, miR‐128, miR‐200, miR‐298, and miR‐342 as novel diagnostic biomarkers in NAFL/NASH: Impact of LPS/TLR‐4/FoxO3 pathway

Samy,

Kandeil,

Sabry

et al. 2024

Archiv der Pharmazie

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Nonalcoholic fatty liver disease (NAFLD) is a common liver disorder affecting a quarter of the global residents. Progression of NAFL into nonalcoholic steatohepatitis (NASH) may cause cirrhosis, liver cancer, and failure. Gut microbiota imbalance causes microbial components translocation into the circulation, triggering liver inflammation and NASH‐related fibrosis. MicroRNAs (miRNAs) regulate gene expression via repressing target genes. Exosomal miRNAs are diagnostic and prognostic biomarkers for NAFL and NASH liver damage. Our work investigated the role of the gut microbiota in NAFLD pathogenesis via the lipopolysaccharide/toll‐like receptor 4/Forkhead box protein O3 (LPS/TLR‐4/FoxO3) pathway and certain miRNAs as noninvasive biomarkers for NAFL or its development to NASH. miRNA expression levels were measured using quantitative reverse transcription polymerase chain reaction (qRT‐PCR) in 50 NAFL patients, 50 NASH patients, and 50 normal controls. Plasma LPS, TLR‐4, adiponectin, peroxisome proliferator‐activated receptor γ (PPAR‐γ), and FoxO3 concentrations were measured using enzyme‐linked immunosorbent assay (ELISA). In NAFL and NASH patients, miR‐122, miR‐128, FoxO3, TLR‐4, LPS, and PPAR‐γ were upregulated while miR‐200, miR‐298, miR‐342, and adiponectin were downregulated compared with the normal control. The examined miRNAs might distinguish NAFL and NASH patients from the normal control using receiver operating characteristic analysis. Our study is the first to examine these miRNAs in NAFLD. Our findings imply that these are potentially promising biomarkers for noninvasive early NAFL diagnosis and NASH progression. Understanding the LPS/TLR‐4/FoxO3 pathway involvement in NAFL/NASH pathogenesis may aid disease management.

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Isolation and Characterization of Microvesicles from Peripheral Blood

Cited by 44 publications

References 32 publications

New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes

Microvesicles Released from Human Red Blood Cells: Properties and Potential Applications

Exosomal miR‐122, miR‐128, miR‐200, miR‐298, and miR‐342 as novel diagnostic biomarkers in NAFL/NASH: Impact of LPS/TLR‐4/FoxO3 pathway

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