Multiplexed mRNA analysis of brain-derived extracellular vesicles upon experimental stroke in mice reveals increased mRNA content related to inflammation and recovery processes

Extracellular vesicles (EVs) are lipid bilayer enclosed structures that not only represent a newly discovered means for cell-to-cell communication but may also serve as promising disease biomarkers and therapeutic tools. Apart from proteins, lipids, and metabolites, EVs can deliver genetic information such as mRNA eliciting a response in the recipient cells. In the present study, we have analyzed the mRNA content of brain-derived EVs (BDEVs) isolated 72 hours after experimental stroke in mice and compared them to controls (shams) using the nCounter® Nanostring panels, with or without prior RNA isolation from BDEVs. We found that both panels show similar results when comparing upregulated mRNA in stroke. Notably, the higher upregulated mRNAs were related to processes of stress and immune system responses, but also to anatomical structure development, cell differentiation, and extracellular matrix organization, indicating that regenerative mechanisms are already taking place at this time-point. The five top overexpressed mRNAs in stroke mice compared to shams were confirmed by RT-qPCR and, interestingly, were found to be present as full-length open-reading frame in BDEVs. We could reveal that the majority of the mRNA cargo in BDEVs was of microglial origin and probably predominantly present in small BDEVs (≤200 nm in diameter). However, the EV population with the highest increase in the total BDEVs pool at 72 h after stroke was of oligodendrocytic origin. Our study shows that nCounter® panels are a good tool to study mRNA content in tissue-derived EVs as they can be carried out even without previous mRNA isolation and that the mRNA cargo of BDEVs indicates their participation in inflammatory but also recovery processes after stroke.

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Exosomes and Extracellular Vesicles in Myeloid Neoplasia: The Multiple and Complex Roles Played by These “Magic Bullets”

Biology ◽

10.3390/biology10020105 ◽

2021 ◽

Vol 10 (2) ◽

pp. 105

Author(s):

Simona Bernardi ◽

Mirko Farina

Keyword(s):

Extracellular Vesicles ◽

New Technologies ◽

Cell Communication ◽

Response To Treatment ◽

Liquid Biopsies ◽

Disease Biomarkers ◽

Mediate Cell ◽

Therapeutic Tools ◽

Myeloid Neoplasia ◽

Multicellular Organisms

Extracellular vesicles (exosomes, in particular) are essential in multicellular organisms because they mediate cell-to-cell communication via the transfer of secreted molecules. They are able to shuttle different cargo, from nucleic acids to proteins. The role of exosomes has been widely investigated in solid tumors, which gave us surprising results about their potential involvement in pathogenesis and created an opening for liquid biopsies. Less is known about exosomes in oncohematology, particularly concerning the malignancies deriving from myeloid lineage. In this review, we aim to present an overview of immunomodulation and the microenvironment alteration mediated by exosomes released by malicious myeloid cells. Afterwards, we review the studies reporting the use of exosomes as disease biomarkers and their influence in response to treatment, together with the recent experiences that have focused on the use of exosomes as therapeutic tools. The further development of new technologies and the increased knowledge of biological (exosomes) and clinical (myeloid neoplasia) aspects are expected to change the future approaches to these malignancies.

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On the Choice of the Extracellular Vesicles for Therapeutic Purposes

International Journal of Molecular Sciences ◽

10.3390/ijms20020236 ◽

2019 ◽

Vol 20 (2) ◽

pp. 236 ◽

Cited By ~ 29

Author(s):

Claudia Campanella ◽

Celeste Caruso Bavisotto ◽

Mariantonia Logozzi ◽

Antonella Marino Gammazza ◽

Davide Mizzoni ◽

...

Keyword(s):

Regenerative Medicine ◽

Extracellular Vesicles ◽

Lipid Membrane ◽

Cell Communication ◽

Membrane Vesicles ◽

Free Form ◽

Target Cells ◽

Disease Biomarkers ◽

Cellular Source ◽

Therapeutic Molecules

Extracellular vesicles (EVs) are lipid membrane vesicles released by all human cells and are widely recognized to be involved in many cellular processes, both in physiological and pathological conditions. They are mediators of cell-cell communication, at both paracrine and systemic levels, and therefore they are active players in cell differentiation, tissue homeostasis, and organ remodeling. Due to their ability to serve as a cargo for proteins, lipids, and nucleic acids, which often reflects the cellular source, they should be considered the future of the natural nanodelivery of bio-compounds. To date, natural nanovesicles, such as exosomes, have been shown to represent a source of disease biomarkers and have high potential benefits in regenerative medicine. Indeed, they deliver both chemical and bio-molecules in a way that within exosomes drugs are more effective that in their exosome-free form. Thus, to date, we know that exosomes are shuttle disease biomarkers and probably the most effective way to deliver therapeutic molecules within target cells. However, we do not know exactly which exosomes may be used in therapy in avoiding side effects as well. In regenerative medicine, it will be ideal to use autologous exosomes, but it seems not ideal to use plasma-derived exosomes, as they may contain potentially dangerous molecules. Here, we want to present and discuss a contradictory relatively unmet issue that is the lack of a general agreement on the choice for the source of extracellular vesicles for therapeutic use.

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Why the need and how to approach the functional diversity of extracellular vesicles

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2016.0479 ◽

2017 ◽

Vol 373 (1737) ◽

pp. 20160479 ◽

Cited By ~ 116

Author(s):

Mercedes Tkach ◽

Joanna Kowal ◽

Clotilde Théry

Keyword(s):

Plasma Membrane ◽

Extracellular Vesicles ◽

Cell Communication ◽

Membrane Vesicles ◽

Tumour Microenvironment ◽

Efficient Separation ◽

The Past ◽

Therapeutic Tools ◽

Opinion Article ◽

Current Stage

In the past decade, cell-to-cell communication mediated by exosomes has attracted growing attention from biomedical scientists and physicians, leading to several recent publications in top-tier journals. Exosomes are generally defined as secreted membrane vesicles, or extracellular vesicles (EVs), corresponding to the intraluminal vesicles of late endosomal compartments, which are secreted upon fusion of multi-vesicular endosomes with the cell's plasma membrane. Cells, however, were shown to release other types of EVs, for instance, by direct budding off their plasma membrane. Some of these EVs share with exosomes major biophysical and biochemical characteristics, such as size, density and membrane orientation, which impose difficulties in their efficient separation. Despite frequent claims in the literature, whether exosomes really display more important patho/physiological functions, or are endowed with higher potential as diagnostic or therapeutic tools than other EVs, is not yet convincingly demonstrated. In this opinion article, we describe reasons for this lack of precision knowledge in the current stage of the EV field, we review recently described approaches to overcome these caveats, and we propose ways to improve our knowledge on the respective functions of distinct EVs, which will be crucial for future development of well-designed EV-based clinical applications. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’.

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Extracellular vesicles in malaria: an agglomeration of two decades of research

Malaria Journal ◽

10.1186/s12936-021-03969-8 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Tosin Opadokun ◽

Petra Rohrbach

Keyword(s):

Extracellular Vesicles ◽

Public Health Problem ◽

Malaria Prevention ◽

Host Cells ◽

Parasitic Disease ◽

Global Public Health ◽

Disease Biomarkers ◽

Therapeutic Tools ◽

And Control ◽

Global Public Health Problem

AbstractMalaria is a complex parasitic disease, caused by Plasmodium spp. More than a century after the discovery of malaria parasites, this disease continues to pose a global public health problem and the pathogenesis of the severe forms of malaria remains incompletely understood. Extracellular vesicles (EVs), including exosomes and microvesicles, have been increasingly researched in the field of malaria in a bid to fill these knowledge gaps. EVs released from Plasmodium-infected red blood cells and other host cells during malaria infection are now believed to play key roles in disease pathogenesis and are suggested as vital components of the biology of Plasmodium spp. Malaria-derived EVs have been identified as potential disease biomarkers and therapeutic tools. In this review, key findings of malaria EV studies over the last 20 years are summarized and critically analysed. Outstanding areas of research into EV biology are identified. Unexplored EV research foci for the future that will contribute to consolidating the potential for EVs as agents in malaria prevention and control are proposed.

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Extracellular Vesicles in Multiple Sclerosis: Role in the Pathogenesis and Potential Usefulness as Biomarkers and Therapeutic Tools

Cells ◽

10.3390/cells10071733 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1733

Author(s):

Marianna D’Anca ◽

Chiara Fenoglio ◽

Francesca Romana Buccellato ◽

Caterina Visconte ◽

Daniela Galimberti ◽

...

Keyword(s):

Multiple Sclerosis ◽

Extracellular Vesicles ◽

Current Knowledge ◽

Recipient Cell ◽

Bioactive Molecules ◽

Disease Biomarkers ◽

The Central Nervous System ◽

Membrane Bound ◽

Therapeutic Tools ◽

Vast Range

Although extracellular vesicles (EVs) were initially relegated to a waste disposal role, nowadays, they have gained multiple fundamental functions working as messengers in intercellular communication as well as exerting active roles in physiological and pathological processes. Accumulating evidence proves the involvement of EVs in many diseases, including those of the central nervous system (CNS), such as multiple sclerosis (MS). Indeed, these membrane-bound particles, produced in any type of cell, carry and release a vast range of bioactive molecules (nucleic acids, proteins, and lipids), conferring genotypic and phenotypic changes to the recipient cell. This means that not only EVs per se but their content, especially, could reveal new candidate disease biomarkers and/or therapeutic agents. This review is intended to provide an overview regarding current knowledge about EVs’ involvement in MS, analyzing the potential versatility of EVs as a new therapeutic tool and source of biomarkers.

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Endothelial Extracellular Vesicles: From Keepers of Health to Messengers of Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22094640 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4640

Author(s):

Allison Mathiesen ◽

Tyree Hamilton ◽

Nigeste Carter ◽

Michael Brown ◽

William McPheat ◽

...

Keyword(s):

Endothelial Cells ◽

Extracellular Vesicles ◽

Stromal Cells ◽

Molecular Heterogeneity ◽

Major Contributor ◽

Diagnostic Biomarkers ◽

Disease Biomarkers ◽

Therapeutic Tools ◽

Parenchymal Cells

Endothelium has a rich vesicular network that allows the exchange of macromolecules between blood and parenchymal cells. This feature of endothelial cells, along with their polarized secretory machinery, makes them the second major contributor, after platelets, to the particulate secretome in circulation. Extracellular vesicles (EVs) produced by the endothelial cells mirror the remarkable molecular heterogeneity of their parent cells. Cargo molecules carried by EVs were shown to contribute to the physiological functions of endothelium and may support the plasticity and adaptation of endothelial cells in a paracrine manner. Endothelium-derived vesicles can also contribute to the pathogenesis of cardiovascular disease or can serve as prognostic or diagnostic biomarkers. Finally, endothelium-derived EVs can be used as therapeutic tools to target endothelium for drug delivery or target stromal cells via the endothelial cells. In this review we revisit the recent evidence on the heterogeneity and plasticity of endothelial cells and their EVs. We discuss the role of endothelial EVs in the maintenance of vascular homeostasis along with their contributions to endothelial adaptation and dysfunction. Finally, we evaluate the potential of endothelial EVs as disease biomarkers and their leverage as therapeutic tools.

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Separation of Extracellular Vesicles by DNA-Directed Immunocapturing Followed by Enzymatic Release

10.26434/chemrxiv.12234683 ◽

2020 ◽

Author(s):

Dario Brambilla ◽

Laura Sola ◽

Elisa Chiodi ◽

Natasa Zarovni ◽

Diogo Fortunato ◽

...

Keyword(s):

Cell Culture ◽

Extracellular Vesicles ◽

Culture Media ◽

Biological Fluids ◽

Imaging Techniques ◽

Cell Communication ◽

Disease Diagnosis ◽

Cell Culture Supernatant ◽

Transmission Electron ◽

Downstream Analysis

Extracellular vesicles (EVs) have attracted great interest among researchers due to their role in cell-cell communication, disease diagnosis, and drug delivery. In spite of their potential in the medical field, there is no consensus on the best method for separating microvesicles from cell culture supernatant and complex biological fluids. Obtaining a good recovery yield and preserving physical characteristics is critical for the diagnostic and therapeutic use of EVs. The separation is made complex by the fact that blood and cell culture media, contain a large number of nanoparticles in the same size range. Methods that exploit immunoaffinity capture provide high purity samples and overcome the issues of currently used separation methods. However, the release of captured nanovesicles requires harsh conditions that hinder their use in certain types of downstream analysis. Herein, a novel capture and release approach for small extracellular vesicles (sEVs), based on DNAdirected immobilization of antiCD63 antibody is presented. The flexible DNAlinker increases the capture efficiency and allows releasing of EVs by exploiting the endonucleasic activity of DNAse I. This separation protocol works under mild conditions, enabling the release of intact vesicles that can be successfully analyzed by imaging techniques. In this article sEVs recovered from plasma were characterized by established techniques for EVs analysis including nanoparticle tracking and transmission electron microscopy.<br>

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Multiple Roles of Exosomal Long Noncoding RNAs in Cancers

BioMed Research International ◽

10.1155/2019/1460572 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Wenyuan Zhao ◽

Yuanqi Liu ◽

Chunfang Zhang ◽

Chaojun Duan

Keyword(s):

Extracellular Vesicles ◽

Cancer Diagnosis ◽

Tumor Angiogenesis ◽

Cell Communication ◽

Noncoding Rnas ◽

Multiple Roles ◽

Long Noncoding Rnas ◽

Cancer Therapies ◽

Transcriptional Noise ◽

Intercellular Exchange

Long noncoding RNAs (lncRNAs) are not transcriptional noise, as previously understood, but are currently considered to be multifunctional. Exosomes are derived from the internal multivesicular compartment and are extracellular vesicles (EVs) with diameters of 30–100 nm. Exosomes play significant roles in the intercellular exchange of information and material. Exosomal lncRNAs may be promising biomarkers for cancer diagnosis and potential targets for cancer therapies, since they are increasingly understood to be involved in tumorigenesis, tumor angiogenesis, and chemoresistance. This review mainly focuses on the roles of emerging exosomal lncRNAs in cancer. In addition, the biogenesis of exosomes, the functions of lncRNAs, and the mechanisms of lncRNAs in exosome-mediated cell-cell communication are also summarized.

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CSF extracellular vesicles and risk of disease activity after a first demyelinating event

Multiple Sclerosis Journal ◽

10.1177/1352458520987542 ◽

2021 ◽

pp. 135245852098754

Author(s):

Gloria Dalla Costa ◽

Tommaso Croese ◽

Marco Pisa ◽

Annamaria Finardi ◽

Lorena Fabbella ◽

...

Keyword(s):

Disease Activity ◽

Extracellular Vesicles ◽

Prognostic Markers ◽

Cell Communication ◽

Extracellular Vesicle ◽

Intervention Strategies ◽

Targeted Intervention ◽

Improve Risk Stratification ◽

Risk Of Disease ◽

In Cis

Background: Extracellular vesicles (EVs), a recently described mechanism of cell communication, are released from activated microglial cells and macrophages and are a candidate biomarker in diseases characterized by chronic inflammatory process such as multiple sclerosis (MS). Methods: We explored cerebrospinal fluid extracellular vesicle (CSF EV) of myeloid origin (MEVs), cytokine and chemokine levels in patients with clinically isolated syndrome (CIS). Results: We found that CSF MEVs were significantly higher in CIS patients than in controls and were inversely correlated to CSF CCL2 levels. MEVs level were significantly associated with an shorter time to evidence of disease activity (hazard ratio: 1.01, 95% confidence interval: 1.00–1.02, p < 0.01) independently from other known prognostic markers. Conclusion: After a first demyelinating event, CSF EVs may improve risk stratification of these patients and allow more targeted intervention strategies.

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Glycosylation of Cancer Extracellular Vesicles: Capture Strategies, Functional Roles and Potential Clinical Applications

Cells ◽

10.3390/cells10010109 ◽

2021 ◽

Vol 10 (1) ◽

pp. 109

Author(s):

Álvaro M. Martins ◽

Cátia C. Ramos ◽

Daniela Freitas ◽

Celso A. Reis

Keyword(s):

Extracellular Vesicles ◽

Biomarker Discovery ◽

De Novo ◽

Recipient Cell ◽

Cell Communication ◽

Cancer Biomarkers ◽

Functional Roles ◽

Molecular Information ◽

Glycosylation Pathway ◽

Organ Tropism

Glycans are major constituents of extracellular vesicles (EVs). Alterations in the glycosylation pathway are a common feature of cancer cells, which gives rise to de novo or increased synthesis of particular glycans. Therefore, glycans and glycoproteins have been widely used in the clinic as both stratification and prognosis cancer biomarkers. Interestingly, several of the known tumor-associated glycans have already been identified in cancer EVs, highlighting EV glycosylation as a potential source of circulating cancer biomarkers. These particles are crucial vehicles of cell–cell communication, being able to transfer molecular information and to modulate the recipient cell behavior. The presence of particular glycoconjugates has been described to be important for EV protein sorting, uptake and organ-tropism. Furthermore, specific EV glycans or glycoproteins have been described to be able to distinguish tumor EVs from benign EVs. In this review, the application of EV glycosylation in the development of novel EV detection and capture methodologies is discussed. In addition, we highlight the potential of EV glycosylation in the clinical setting for both cancer biomarker discovery and EV therapeutic delivery strategies.

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