دورية أكاديمية
Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis.
| العنوان: | Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis. |
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| المؤلفون: | Merij LB; Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; Programa de Pós-Graduação em Ciências Biológicas, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil., da Silva LR; Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil., Palhinha L; Laboratory of Immunopharmacology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil., Gomes MT; Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; Programa de Pós-Graduação em Ciências Biológicas, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil., Dib PRB; Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; Programa de Pós-Graduação em Ciências Biológicas, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil., Martins-Gonçalves R; Laboratory of Immunopharmacology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil., Toledo-Quiroga K; Laboratory of Toxinology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil., Raposo-Nunes MA; Laboratory of Immunopharmacology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil., Andrade FB; Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; Programa de Pós-Graduação em Ciências Biológicas, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil., de Toledo Martins S; Gene Expression Regulation Laboratory, Carlos Chagas Institute, ICC-Fiocruz, Curitiba, Paraná, Brazil., Nascimento ALR; Laboratory of Ultrastructure and Tissue, Department of Histology and Embryology, State University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil., Rocha VN; Laboratory of Veterinary Pathology and Histology, Department of Veterinary Medicine, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil., Alves LR; Gene Expression Regulation Laboratory, Carlos Chagas Institute, ICC-Fiocruz, Curitiba, Paraná, Brazil., Bozza PT; Gene Expression Regulation Laboratory, Carlos Chagas Institute, ICC-Fiocruz, Curitiba, Paraná, Brazil., de Oliveira Trugilho MR; Laboratory of Immunopharmacology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil; Center for Technological Development in Health, Fiocruz, Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: mrotrugilho@hotmail.com., Hottz ED; Laboratory of Immunothrombosis, Department of Biochemistry, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil; Programa de Pós-Graduação em Ciências Biológicas, Institute of Biological Sciences, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil. Electronic address: eugeniohottz@gmail.com. |
| المصدر: | Journal of thrombosis and haemostasis : JTH [J Thromb Haemost] 2024 May; Vol. 22 (5), pp. 1372-1388. Date of Electronic Publication: 2024 Jan 24. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Elsevier Country of Publication: England NLM ID: 101170508 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1538-7836 (Electronic) Linking ISSN: 15387836 NLM ISO Abbreviation: J Thromb Haemost Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: 2023- : [New York] : Elsevier Original Publication: Oxford : Blackwell Pub. |
| مواضيع طبية MeSH: | Extracellular Vesicles*/metabolism , Proteomics*/methods , Lipoproteins*/blood , Chromatography, Gel*, Humans ; Blood Platelets/metabolism ; Centrifugation, Density Gradient ; Inflammation/blood ; Proteome ; Monocytes/metabolism |
| مستخلص: | Background: Blood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in inflammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboinflammation. Objectives: Here, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboinflammatory analysis. Methods: Density-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics. Results: G-UC efficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quantification, and apoB-100 detection confirmed the significant reduction in lipoproteins from isolated EVs. Proteomic analysis identified similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboinflammatory responses of platelets and monocytes stimulated in vitro with EV isolates. Conclusion: Combination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses. Competing Interests: Declaration of competing interests There are no conflicting interests to disclose. (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.) |
| فهرسة مساهمة: | Keywords: EV isolation; EV proteomics; blood plasma; extracellular vesicles; lipoproteins |
| المشرفين على المادة: | 0 (Lipoproteins) 0 (Proteome) |
| تواريخ الأحداث: | Date Created: 20240126 Date Completed: 20240426 Latest Revision: 20240426 |
| رمز التحديث: | 20240427 |
| DOI: | 10.1016/j.jtha.2024.01.010 |
| PMID: | 38278418 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1538-7836 |
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| DOI: | 10.1016/j.jtha.2024.01.010 |