Biology, Pathophysiological Role, and Clinical Implications of Exosomes: A Critical Appraisal

Exosomes are membrane-enclosed entities of endocytic origin, which are generated during the fusion of multivesicular bodies (MVBs) and plasma membranes. Exosomes are released into the extracellular milieu or body fluids; this process was reported for mesenchymal, epithelial, endothelial, and different immune cells (B-cells and dendritic cells), and was reported to be correlated with normal physiological processes. The compositions and abundances of exosomes depend on their tissue origins and cell types. Exosomes range in size between 30 and 100 nm, and shuttle nucleic acids (DNA, messenger RNAs (mRNAs), microRNAs), proteins, and lipids between donor and target cells. Pathogenic microorganisms also secrete exosomes that modulate the host immune system and influence the fate of infections. Such immune-modulatory effect of exosomes can serve as a diagnostic biomarker of disease. On the other hand, the antigen-presenting and immune-stimulatory properties of exosomes enable them to trigger anti-tumor responses, and exosome release from cancerous cells suggests they contribute to the recruitment and reconstitution of components of tumor microenvironments. Furthermore, their modulation of physiological and pathological processes suggests they contribute to the developmental program, infections, and human diseases. Despite significant advances, our understanding of exosomes is far from complete, particularly regarding our understanding of the molecular mechanisms that subserve exosome formation, cargo packaging, and exosome release in different cellular backgrounds. The present study presents diverse biological aspects of exosomes, and highlights their diagnostic and therapeutic potentials.

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Extracellular Vesicles: An Emerging Mechanism Governing the Secretion and Biological Roles of Tenascin-C

Frontiers in Immunology ◽

10.3389/fimmu.2021.671485 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lucas Albacete-Albacete ◽

Miguel Sánchez-Álvarez ◽

Miguel Angel del Pozo

Keyword(s):

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Cell Communication ◽

Cell Types ◽

Target Cells ◽

Tenascin C ◽

Signalling Molecules ◽

Inflammatory Processes ◽

Bona Fide ◽

Cellular Components

ECM composition and architecture are tightly regulated for tissue homeostasis. Different disorders have been associated to alterations in the levels of proteins such as collagens, fibronectin (FN) or tenascin-C (TnC). TnC emerges as a key regulator of multiple inflammatory processes, both during physiological tissue repair as well as pathological conditions ranging from tumor progression to cardiovascular disease. Importantly, our current understanding as to how TnC and other non-collagen ECM components are secreted has remained elusive. Extracellular vesicles (EVs) are small membrane-bound particles released to the extracellular space by most cell types, playing a key role in cell-cell communication. A broad range of cellular components can be transported by EVs (e.g. nucleic acids, lipids, signalling molecules and proteins). These cargoes can be transferred to target cells, potentially modulating their function. Recently, several extracellular matrix (ECM) proteins have been characterized as bona fide EV cargoes, exosomal secretion being particularly critical for TnC. EV-dependent ECM secretion might underpin diseases where ECM integrity is altered, establishing novel concepts in the field such as ECM nucleation over long distances, and highlighting novel opportunities for diagnostics and therapeutic intervention. Here, we review recent findings and standing questions on the molecular mechanisms governing EV–dependent ECM secretion and its potential relevance for disease, with a focus on TnC.

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Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

Blood ◽

10.1182/blood.v60.3.583.583 ◽

1982 ◽

Vol 60 (3) ◽

pp. 583-594 ◽

Cited By ~ 26

Author(s):

N Dainiak ◽

CM Cohen

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Mononuclear Cells ◽

Surface Membrane ◽

Freeze Fracture ◽

Membrane Vesicles ◽

Cell Types ◽

Target Cells ◽

Freeze Fracture Electron Microscopy

Abstract In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.

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The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

Viruses ◽

10.3390/v13112272 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2272

Author(s):

Hussein Kaddour ◽

Malik Tranquille ◽

Chioma M. Okeoma

Keyword(s):

Size Exclusion Chromatography ◽

Extracellular Vesicles ◽

Cell Types ◽

Size Exclusion ◽

Target Cells ◽

Purification And Characterization ◽

Extracellular Milieu ◽

Exclusion Chromatography ◽

Single Vesicle

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.

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Heme-Oxygenase-1 Attenuates Oxidative Functions of Antigen Presenting Cells and Promotes Regulatory T Cell Differentiation during Fasciola hepatica Infection

Antioxidants ◽

10.3390/antiox10121938 ◽

2021 ◽

Vol 10 (12) ◽

pp. 1938

Author(s):

Monique Costa ◽

Valeria da Costa ◽

Sofía Frigerio ◽

María Florencia Festari ◽

Mercedes Landeira ◽

...

Keyword(s):

Heme Oxygenase ◽

Molecular Mechanisms ◽

Fasciola Hepatica ◽

Antioxidant Properties ◽

Antigen Presenting Cells ◽

Parasite Infection ◽

Economic Losses ◽

Heme Oxygenase 1 ◽

Host Immune System ◽

Antigen Presenting

Fasciola hepatica is a fluke that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. The parasite regulates the host immune system by inducing a strong Th2 and regulatory T (Treg) cell immune response through mechanisms that might involve the expression or activity of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that also has immunoregulatory and antioxidant properties. In this paper, we show that F. hepatica-infected mice upregulate HO-1 on peritoneal antigen-presenting cells (APC), which produce decreased levels of both reactive oxygen and nitrogen species (ROS/RNS). The presence of these cells was associated with increased levels of regulatory T cells (Tregs). Blocking the IL-10 receptor (IL-10R) during parasite infection demonstrated that the presence of splenic Tregs and peritoneal APC expressing HO-1 were both dependent on IL-10 activity. Furthermore, IL-10R neutralization as well as pharmacological treatment with the HO-1 inhibitor SnPP protected mice from parasite infection and allowed peritoneal APC to produce significantly higher ROS/RNS levels than those detected in cells from infected control mice. Finally, parasite infection carried out in gp91phox knockout mice with inactive NADPH oxidase was associated with decreased levels of peritoneal HO-1+ cells and splenic Tregs, and partially protected mice from the hepatic damage induced by the parasite, revealing the complexity of the molecular mechanisms involving ROS production that participate in the complex pathology induced by this helminth. Altogether, these results contribute to the elucidation of the immunoregulatory and antioxidant role of HO-1 induced by F. hepatica in the host, providing alternative checkpoints that might control fasciolosis.

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Molecular Background of Leak K+ Currents: Two-Pore Domain Potassium Channels

Physiological Reviews ◽

10.1152/physrev.00029.2009 ◽

2010 ◽

Vol 90 (2) ◽

pp. 559-605 ◽

Cited By ~ 491

Author(s):

Péter Enyedi ◽

Gábor Czirják

Keyword(s):

Intracellular Signaling ◽

Molecular Mechanisms ◽

Cell Types ◽

Detailed Examination ◽

Resting Membrane Potential ◽

Membrane Stretch ◽

Physiological Processes ◽

Pore Domain ◽

K Currents

Two-pore domain K+ (K2P) channels give rise to leak (also called background) K+ currents. The well-known role of background K+ currents is to stabilize the negative resting membrane potential and counterbalance depolarization. However, it has become apparent in the past decade (during the detailed examination of the cloned and corresponding native K2P channel types) that this primary hyperpolarizing action is not performed passively. The K2P channels are regulated by a wide variety of voltage-independent factors. Basic physicochemical parameters (e.g., pH, temperature, membrane stretch) and also several intracellular signaling pathways substantially and specifically modulate the different members of the six K2P channel subfamilies (TWIK, TREK, TASK, TALK, THIK, and TRESK). The deep implication in diverse physiological processes, the circumscribed expression pattern of the different channels, and the interesting pharmacological profile brought the K2P channel family into the spotlight. In this review, we focus on the physiological roles of K2P channels in the most extensively investigated cell types, with special emphasis on the molecular mechanisms of channel regulation.

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Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments

Cells ◽

10.3390/cells9051180 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1180 ◽

Cited By ~ 1

Author(s):

Enrico Ragni ◽

Silvia Palombella ◽

Silvia Lopa ◽

Giuseppe Talò ◽

Carlotta Perucca Orfei ◽

...

Keyword(s):

Confocal Microscopy ◽

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Articular Chondrocytes ◽

Basic Research ◽

Cell Types ◽

Target Cells ◽

Internal Cell ◽

2D System ◽

2D And 3D

Extracellular vesicles (EVs) showed therapeutic properties in several applications, many in regenerative medicine. A clear example is in the treatment of osteoarthritis (OA), where adipose-derived mesenchymal stem cells (ASCs)-EVs were able to promote regeneration and reduce inflammation in both synovia and cartilage. A still obscure issue is the effective ability of EVs to be internalized by target cells, rather than simply bound to the extracellular matrix (ECM) or plasma membrane, since the current detection or imaging technologies cannot fully decipher it due to technical limitations. In the present study, human articular chondrocytes (ACHs) and fibroblast-like synoviocytes (FLSs) isolated from the same OA patients were cocultured in 2D as well as in 3D conditions with fluorescently labeled ASC-EVs, and analyzed by flow cytometry or confocal microscopy, respectively. In contrast with conventional 2D, in 3D cultures, confocal microscopy allowed a clear detection of the tridimensional morphology of the cells and thus an accurate discrimination of EV interaction with the external and/or internal cell environment. In both 2D and 3D conditions, FLSs were more efficient in interacting with ASC-EVs and 3D imaging demonstrated a faster uptake process. The removal of the hyaluronic acid component from the ECM of both cell types reduced their interaction with ASC-EVs only in the 2D system, showing that 2D and 3D conditions can yield different outcomes when investigating events where ECM plays a key role. These results indicate that studying EVs binding and uptake both in 2D and 3D guarantees a more precise and complementary characterization of the molecular mechanisms involved in the process. The implementation of this strategy can become a valuable tool not only for basic research, but also for release assays and potency prediction for clinical EV batches.

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Durable anticancer immunity from intratumoral administration of IL-23, IL-36γ, and OX40L mRNAs

Science Translational Medicine ◽

10.1126/scitranslmed.aat9143 ◽

2019 ◽

Vol 11 (477) ◽

pp. eaat9143 ◽

Cited By ~ 27

Author(s):

Susannah L. Hewitt ◽

Ailin Bai ◽

Dyane Bailey ◽

Kana Ichikawa ◽

John Zielinski ◽

...

Keyword(s):

T Cell ◽

Immune Cell ◽

Inflammatory Diseases ◽

Interleukin 1 ◽

Complete Response ◽

Cell Types ◽

Immunomodulatory Activity ◽

Messenger Rnas ◽

Tumor Microenvironments ◽

Microbial Invasion

Many solid cancers contain dysfunctional immune microenvironments. Immune system modulators that initiate responses to foreign pathogens could be promising candidates for reigniting productive responses toward tumors. Interleukin-1 (IL-1) and IL-12 cytokine family members cooperate at barrier tissues after microbial invasion, in human inflammatory diseases, and in antitumoral immunity. IL-36γ, in classic alarmin fashion, acts in damaged tissues, whereas IL-23 centrally coordinates immune responses to danger signals. In this study, direct intratumoral delivery of messenger RNAs (mRNAs) encoding these cytokines produced robust anticancer responses in a broad range of tumor microenvironments. The addition of mRNA encoding the T cell costimulator OX40L increased complete response rates in treated and untreated distal tumors compared to the cytokine mRNAs alone. Mice exhibiting complete responses were subsequently protected from tumor rechallenge. Treatments with these mRNA mixtures induced downstream cytokine and chemokine expression, and also activated multiple dendritic cell (DC) and T cell types. Consistent with this, efficacy was dependent on Batf3-dependent cross-presenting DCs and cytotoxic CD8+T cells. IL-23/IL-36γ/OX40L triplet mRNA mixture triggered substantial immune cell recruitment into tumors, enabling effective tumor destruction irrespective of previous tumoral immune infiltrates. Last, combining triplet mRNA with checkpoint blockade led to efficacy in models otherwise resistant to systemic immune checkpoint inhibition. Human cell studies showed similar cytokine responses to the individual components of this mRNA mixture, suggesting translatability of immunomodulatory activity to human patients.

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Exosomes are the Driving Force in Preparing the Soil for the Metastatic Seeds: Lessons from the Prostate Cancer

Cells ◽

10.3390/cells9030564 ◽

2020 ◽

Vol 9 (3) ◽

pp. 564 ◽

Cited By ~ 4

Author(s):

Saber H. Saber ◽

Hamdy E. A. Ali ◽

Rofaida Gaballa ◽

Mohamed Gaballah ◽

Hamed I. Ali ◽

...

Keyword(s):

Prostate Cancer ◽

Molecular Mechanisms ◽

Cancer Metastasis ◽

Membrane Vesicles ◽

Cell Types ◽

Bioactive Molecules ◽

Therapeutic Interventions ◽

Target Cells ◽

Molecular Signatures ◽

Cellular Processes

Exosomes are nano-membrane vesicles that various cell types secrete during physiological and pathophysiological conditions. By shuttling bioactive molecules such as nucleic acids, proteins, and lipids to target cells, exosomes serve as key regulators for multiple cellular processes, including cancer metastasis. Recently, microvesicles have emerged as a challenge in the treatment of prostate cancer (PCa), encountered either when the number of vesicles increases or when the vesicles move into circulation, potentially with an ability to induce drug resistance, angiogenesis, and metastasis. Notably, the exosomal cargo can induce the desmoplastic response of PCa-associated cells in a tumor microenvironment (TME) to promote PCa metastasis. However, the crosstalk between PCa-derived exosomes and the TME remains only partially understood. In this review, we provide new insights into the metabolic and molecular signatures of PCa-associated exosomes in reprogramming the TME, and the subsequent promotion of aggressive phenotypes of PCa cells. Elucidating the molecular mechanisms of TME reprogramming by exosomes draws more practical and universal conclusions for the development of new therapeutic interventions when considering TME in the treatment of PCa patients.

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Current methods for the isolation of extracellular vesicles

Biological Chemistry ◽

10.1515/hsz-2013-0141 ◽

2013 ◽

Vol 394 (10) ◽

pp. 1253-1262 ◽

Cited By ~ 279

Author(s):

Fatemeh Momen-Heravi ◽

Leonora Balaj ◽

Sara Alian ◽

Pierre-Yves Mantel ◽

Allison E. Halleck ◽

...

Keyword(s):

Extracellular Vesicles ◽

Molecular Mechanisms ◽

Magnetic Beads ◽

Cell Types ◽

Target Cells ◽

Cell Of Origin ◽

Novel Technologies ◽

Advantages And Disadvantages ◽

Isolation Methods ◽

Technical Advances

Abstract Extracellular vesicles (EVs), including microvesicles and exosomes, are nano- to micron-sized vesicles, which may deliver bioactive cargos that include lipids, growth factors and their receptors, proteases, signaling molecules, as well as mRNA and non-coding RNA, released from the cell of origin, to target cells. EVs are released by all cell types and likely induced by mechanisms involved in oncogenic transformation, environmental stimulation, cellular activation, oxidative stress, or death. Ongoing studies investigate the molecular mechanisms and mediators of EVs-based intercellular communication at physiological and oncogenic conditions with the hope of using this information as a possible source for explaining physiological processes in addition to using them as therapeutic targets and disease biomarkers in a variety of diseases. A major limitation in this evolving discipline is the hardship and the lack of standardization for already challenging techniques to isolate EVs. Technical advances have been accomplished in the field of isolation with improving knowledge and emerging novel technologies, including ultracentrifugation, microfluidics, magnetic beads and filtration-based isolation methods. In this review, we will discuss the latest advances in methods of isolation methods and production of clinical grade EVs as well as their advantages and disadvantages, and the justification for their support and the challenges that they encounter.

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NK cells promote transplant tolerance by killing donor antigen-presenting cells

Journal of Experimental Medicine ◽

10.1084/jem.20060603 ◽

2006 ◽

Vol 203 (8) ◽

pp. 1851-1858 ◽

Cited By ~ 203

Author(s):

Guang Yu ◽

Xuemin Xu ◽

Minh Diem Vu ◽

Elizabeth D. Kilpatrick ◽

Xian Chang Li

Keyword(s):

T Cells ◽

Nk Cells ◽

Tolerance Induction ◽

Antigen Presenting Cells ◽

Cell Types ◽

Target Cells ◽

Skin Allograft ◽

Transplant Tolerance ◽

Skin Allografts ◽

Antigen Presenting

Natural killer (NK) cells are programmed to kill target cells without prior antigen priming. Because of their potent cytolytic activities, NK cells are one of the key cell types involved in dismantling allografts. However, in certain transplant models, NK cells also express potent immunoregulatory properties that promote tolerance induction. The precise mechanism for such striking dichotomy remains unknown. In the present study, we showed in a skin transplant model that the skin allografts contain a subset of antigen-presenting cells (APCs) that can home to the recipient mice. We also showed that such graft-derived APCs are usually destroyed by the host NK cells. But in the absence of NK cells, donor APCs can survive and then migrate to the host lymphoid and extralymphoid sites where they directly stimulate the activation of alloreactive T cells. T cells activated in the absence of NK cells are more resistant to costimulatory blockade treatment, and under such conditions stable skin allograft survival is difficult to achieve. Our study identified a novel role for NK cells in regulating T cell priming in transplant models, and may have important clinical implications in tolerance induction.

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