Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles

Sean M Davidson¹, Chantal M Boulanger², Elena Aikawa³, Lina Badimon⁴, Lucio Barile⁵, Christoph J Binder⁶, Alain Brisson⁷, Edit Buzas⁸, Costanza Emanueli⁹, Felix Jansen¹⁰, Miroslava Katsur¹, Romaric Lacroix^{11

12}, Sai Kiang Lim^{13

14}, Nigel Mackman¹⁵, Manuel Mayr¹⁶, Philippe Menasché^{17

18}, Rienk Nieuwland^{19

20}, Susmita Sahoo²¹, Kaloyan Takov¹⁶, Thomas Thum^{22

23}, Pieter Vader^{2

24}, Marca H M Wauben²⁵, Kenneth Witwer^{26

27}, Joost P G Sluijter¹⁸

Affiliations

¹ The Hatter Cardiovascular Institute, University College London, WC1E 6HX London, UK.
² Université Paris Cité, Paris-Cardiovascular Research Center, INSERM, Paris, France.
³ Department of Medicine, Center for Excellence in Vascular Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ Cardiovascular Science Program-ICCC, IR-Hospital de la Santa Creu i Santa Pau-IIBSantPau, CiberCV, Autonomous University of Barcelona, Barcelona, Spain.
⁵ Laboratory for Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale and Faculty of Biomedical Sciences, Università Svizzera italiana, 6900 Lugano, Switzerland.
⁶ Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
⁷ Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN, CNRS-University of Bordeaux-IPB, Bat. B14, Allée Geoffroy Saint-Hilaire, 33600 Pessac, France.
⁸ Department of Genetics, Cell- and Immunobiology, Semmelweis University, HCEMM-SU and ELKH-SE Immune Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary.
⁹ National Heart and Lung Institute, Imperial College London, Hammersmith Campus, London W12 0NN, UK.
¹⁰ Department of Internal Medicine II, Heart Center, University Hospital Bonn, Bonn, Germany.
¹¹ Aix Marseille University, INSERM 1263, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Centre de Recherche en CardioVasculaire et Nutrition (C2VN), Marseille, France.
¹² Department of Haematology and Vascular Biology, CHU La Conception, APHM, Marseille, France.
¹³ Institute of Medical Biology and Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.
¹⁴ Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
¹⁵ Department of Medicine, UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹⁶ King's College London British Heart Foundation Centre, School of Cardiovascular Medicine and Sciences, London, UK.
¹⁷ Department of Cardiovascular Surgery, Hôpital Européen Georges Pompidou, Paris, France.
¹⁸ Laboratory of Experimental Cardiology, Department of Cardiology, UMC Utrecht Regenerative Medicine Center and Circulatory Health Laboratory, Utrecht University, University Medical Center Utrecht, Utrecht, The Netherlands.
¹⁹ Vesicle Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
²⁰ Laboratory of Experimental Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
²¹ Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
²² Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Hannover, Germany.
²³ Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
²⁴ CDL Research, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
²⁵ Faculty of Veterinary Medicine, Department of Biomolecular Health Sciences, Utrecht University, Yalelaan 2, Utrecht, The Netherlands.
²⁶ Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
²⁷ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

PMID: 35325061
PMCID: PMC10233250
DOI: 10.1093/cvr/cvac031

Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles

Sean M Davidson et al. Cardiovasc Res. 2023.

. 2023 Mar 17;119(1):45-63.

doi: 10.1093/cvr/cvac031.

Authors

Affiliations

¹ The Hatter Cardiovascular Institute, University College London, WC1E 6HX London, UK.
² Université Paris Cité, Paris-Cardiovascular Research Center, INSERM, Paris, France.
³ Department of Medicine, Center for Excellence in Vascular Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁴ Cardiovascular Science Program-ICCC, IR-Hospital de la Santa Creu i Santa Pau-IIBSantPau, CiberCV, Autonomous University of Barcelona, Barcelona, Spain.
⁵ Laboratory for Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale and Faculty of Biomedical Sciences, Università Svizzera italiana, 6900 Lugano, Switzerland.
⁶ Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
⁷ Molecular Imaging and NanoBioTechnology, UMR-5248-CBMN, CNRS-University of Bordeaux-IPB, Bat. B14, Allée Geoffroy Saint-Hilaire, 33600 Pessac, France.
⁸ Department of Genetics, Cell- and Immunobiology, Semmelweis University, HCEMM-SU and ELKH-SE Immune Proteogenomics Extracellular Vesicle Research Group, Budapest, Hungary.
⁹ National Heart and Lung Institute, Imperial College London, Hammersmith Campus, London W12 0NN, UK.
¹⁰ Department of Internal Medicine II, Heart Center, University Hospital Bonn, Bonn, Germany.
¹¹ Aix Marseille University, INSERM 1263, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement (INRAE), Centre de Recherche en CardioVasculaire et Nutrition (C2VN), Marseille, France.
¹² Department of Haematology and Vascular Biology, CHU La Conception, APHM, Marseille, France.
¹³ Institute of Medical Biology and Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore.
¹⁴ Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
¹⁵ Department of Medicine, UNC Blood Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
¹⁶ King's College London British Heart Foundation Centre, School of Cardiovascular Medicine and Sciences, London, UK.
¹⁷ Department of Cardiovascular Surgery, Hôpital Européen Georges Pompidou, Paris, France.
¹⁸ Laboratory of Experimental Cardiology, Department of Cardiology, UMC Utrecht Regenerative Medicine Center and Circulatory Health Laboratory, Utrecht University, University Medical Center Utrecht, Utrecht, The Netherlands.
¹⁹ Vesicle Observation Center, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
²⁰ Laboratory of Experimental Clinical Chemistry, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands.
²¹ Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
²² Institute of Molecular and Translational Therapeutic Strategies, Hannover Medical School, Hannover, Germany.
²³ Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.
²⁴ CDL Research, University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
²⁵ Faculty of Veterinary Medicine, Department of Biomolecular Health Sciences, Utrecht University, Yalelaan 2, Utrecht, The Netherlands.
²⁶ Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
²⁷ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

PMID: 35325061
PMCID: PMC10233250
DOI: 10.1093/cvr/cvac031

Abstract

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are released from cells of the cardiovascular system, and are considered important mediators of intercellular and extracellular communications. Two types of EVs of particular interest are exosomes and microvesicles, which have been identified in all tissue and body fluids and carry a variety of molecules including RNAs, proteins, and lipids. EVs have potential for use in the diagnosis and prognosis of cardiovascular diseases and as new therapeutic agents, particularly in the setting of myocardial infarction and heart failure. Despite their promise, technical challenges related to their small size make it challenging to accurately identify and characterize them, and to study EV-mediated processes. Here, we aim to provide the reader with an overview of the techniques and technologies available for the separation and characterization of EVs from different sources. Methods for determining the protein, RNA, and lipid content of EVs are discussed. The aim of this document is to provide guidance on critical methodological issues and highlight key points for consideration for the investigation of EVs in cardiovascular studies.

Keywords: Biodistribution; Blood; Cardiovascular diseases; Exosomes; Extracellular vesicle composition; Heart; Microvesicles; Therapeutics.

PubMed Disclaimer

Conflict of interest statement

Conflicts of interest: L.B. has performed advisory board work and received speaker fees from Sanofi and Novartis, and is founder and shareholder of Glycardial Diagnosis SL and Ivestatin Therapeutics, SL (all outside of this work); C.J.B. is a board member of Technoclone. A.B. is the founder and CEO of Exo-Analysis. T.T. has filed and licensed patents in the field of non-coding RNAs and targeted delivery strategies and is the founder and shareholder of Cardior Pharmaceuticals GmbH (outside of the topic of this review). R.L. discloses grants from Stago and a patent on microvesicle fibrinolytic activity licensed to Stago. E.I.B. is a member of the Advisory Board of Sphere Gene Therapeutics Inc. (Boston, USA). M.H.M.W. discloses a collaborative research agreement with BD Biosciences Europe, Erembodegem, Belgium to optimize flow cytometric analysis of EVs. “This manuscript was handled by Reviews Deputy Editor Dr Ali J. Marian”.

Figures

**Figure 1**
The typical size range of the major lipid-bilayer EVs up to 1000 nm diameter. ^aAs reported by Jeppesen *et al*., ^bthe size of apoptotic vesicles/bodies can range up to 5 μm in diameter. Please be aware that the diameter of EVs depends on the detection method used.

**Figure 2**
Representative images of different techniques of EV characterization. (A) Transmission electron micrography (TEM) of multi-vesicular body (MVB) containing exosomes (arrows) in primary HUVECs. (B) Transmission electron micrography (TEM) of negative-stained EVs isolated from HUVECs (sEV = small EVs, lEV = large EVs). (C) Cryo-TEM of a single CD81 + EV from iPS-derived cardiovascular progenitor cells. The lipid bilayer is clearly resolved (arrow). (D) Fractionation of sEVs (purple) from proteins (green, blue) by size-exclusion chromatography. (E) Single frame from NTA of an sEV sample under constant flow, showing particle tracks (red) and particle size distribution (blue). (F) Representative trace of EV sample obtained using resistive pulse sensing (RPS). (G) Individual particles detected by RPS, with size determined relative to calibration beads of a known size. (H) Size distribution of EVs obtained by RPS.

**Figure 3**
Steps towards EV characterization, adapted from MISEV2018 guidelines. (i) Determine the quantity of EVs obtained, relative to the amount of starting material. (ii) Verify the presence of at least three positive protein markers of small EVs, including one transmembrane or GPI-anchored protein (e.g. CD9, CD63, CD81, NT5E/CD73), and one cytosolic, luminal protein (e.g. ALIX/PDCD6IP, HSC70). For large EVs, a wide range of surface markers such as integrins from the cell of origin may be used. (iii) Preferably, demonstrate the relative abundance of significant contamination by non-vesicular, co-isolated components such as lipoproteins (APOB, APOA1, APOA2) or albumin. (iv) Characterize individual EVs, with images of single EVs (both wide-field and close-up).

See this image and copyright information in PMC

References

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Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles

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Methods for the identification and characterization of extracellular vesicles in cardiovascular studies: from exosomes to microvesicles

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Conflict of interest statement

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