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Sutton ER, Beauvais A, Yaworski R, De Repentigny Y, Reilly A, Alves de Almeida MM, Deguise MO, Poulin KL, Parks RJ, Schneider BL, Kothary R. Liver SMN restoration rescues the Smn 2B/- mouse model of spinal muscular atrophy. EBioMedicine 2024; 110:105444. [PMID: 39515026 PMCID: PMC11583733 DOI: 10.1016/j.ebiom.2024.105444] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2024] [Revised: 10/21/2024] [Accepted: 10/23/2024] [Indexed: 11/16/2024] Open
Abstract
BACKGROUND The liver is a key metabolic organ, acting as a hub to metabolically connect various tissues. Spinal muscular atrophy (SMA) is a neuromuscular disorder whereby patients have an increased susceptibility to developing dyslipidaemia and liver steatosis. It remains unknown whether fatty liver is due to an intrinsic or extrinsic impact of survival motor neuron (SMN) protein depletion. METHODS Using an adeno-associated viral vector with a liver specific promoter (albumin), we restored SMN protein levels in the liver alone in Smn2B/- mice, a model of SMA. Experiments assessed central and peripheral impacts using immunoblot, immunohistochemistry, and electron microscopy techniques. FINDINGS We demonstrate that AAV9-albumin-SMN successfully expresses SMN protein in the liver with no detectable expression in the spinal cord or muscle in Smn2B/- mice. Liver intrinsic rescue of SMN protein was sufficient to increase survival of Smn2B/- mice. Fatty liver was ameliorated while key markers of liver function were also restored to normal levels. Certain peripheral pathologies were rescued including muscle size and pancreatic cell imbalance. Only a partial CNS recovery was seen using a liver therapeutic strategy alone. INTERPRETATION The fatty liver phenotype is a direct impact of liver intrinsic SMN protein loss. Correction of SMN protein levels in liver is enough to restore some aspects of disease in SMA. We conclude that the liver is an important contributor to whole-body pathology in Smn2B/- mice. FUNDING This work was funded by Muscular Dystrophy Association (USA) [grant number 963652 to R.K.]; the Canadian Institutes of Health Research [grant number PJT-186300 to R.K.].
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Affiliation(s)
- Emma R Sutton
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada
| | - Ariane Beauvais
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada
| | - Rebecca Yaworski
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada
| | - Yves De Repentigny
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada
| | - Aoife Reilly
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada
| | | | - Marc-Olivier Deguise
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada
| | - Kathy L Poulin
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada
| | - Robin J Parks
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada; Department of Medicine, University of Ottawa, Ottawa, Canada
| | - Bernard L Schneider
- Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland; Bertarelli Platform for Gene Therapy, Polytechnique Fédérale de Lausanne (EPFL), Geneva, Switzerland
| | - Rashmi Kothary
- Regenerative Medicine Program, The Ottawa Hospital Research Institute, Ottawa, Canada; Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada; Department of Medicine, University of Ottawa, Ottawa, Canada.
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2
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Fernandes CJC, Silva RA, Ferreira MR, Fuhler GM, Peppelenbosch MP, van der Eerden BC, Zambuzzi WF. Vascular smooth muscle cell-derived exosomes promote osteoblast-to-osteocyte transition via β-catenin signaling. Exp Cell Res 2024; 442:114211. [PMID: 39147261 DOI: 10.1016/j.yexcr.2024.114211] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2024] [Revised: 08/01/2024] [Accepted: 08/13/2024] [Indexed: 08/17/2024]
Abstract
Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via β-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/β-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.
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Affiliation(s)
- Célio J C Fernandes
- Bioassays and Cell Dynamics Lab, Dept. of Chemistry and Biochemistry, Bioscience Institute, UNESP, Botucatu, 18603-100, Sao Paulo, Brazil
| | - Rodrigo A Silva
- School of Dentistry, University of Taubaté, 12020-340, Taubaté, São Paulo, Brazil
| | - Marcel R Ferreira
- Bioassays and Cell Dynamics Lab, Dept. of Chemistry and Biochemistry, Bioscience Institute, UNESP, Botucatu, 18603-100, Sao Paulo, Brazil
| | - Gwenny M Fuhler
- Department of Gastroenterology and Hepatology, Erasmus MC, Erasmus University Medical Center, Dr Molewaterplein 40, 3015 GD, Rotterdam, the Netherlands
| | - Maikel P Peppelenbosch
- Department of Gastroenterology and Hepatology, Erasmus MC, Erasmus University Medical Center, Dr Molewaterplein 40, 3015 GD, Rotterdam, the Netherlands
| | - Bram Cj van der Eerden
- Department of Internal Medicine, Erasmus MC, Erasmus University Medical Center, Dr Molewaterplein 40, 3015 GD, Rotterdam, the Netherlands
| | - Willian F Zambuzzi
- Bioassays and Cell Dynamics Lab, Dept. of Chemistry and Biochemistry, Bioscience Institute, UNESP, Botucatu, 18603-100, Sao Paulo, Brazil.
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3
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Nair S, Nova-Lamperti E, Labarca G, Kulasinghe A, Short KR, Carrión F, Salomon C. Genomic communication via circulating extracellular vesicles and long-term health consequences of COVID-19. J Transl Med 2023; 21:709. [PMID: 37817137 PMCID: PMC10563316 DOI: 10.1186/s12967-023-04552-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2023] [Accepted: 09/22/2023] [Indexed: 10/12/2023] Open
Abstract
COVID-19 continues to affect an unprecedented number of people with the emergence of new variants posing a serious challenge to global health. There is an expansion of knowledge in understanding the pathogenesis of Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the impact of the acute disease on multiple organs. In addition, growing evidence reports that the impact of COVID-19 on different organs persists long after the recovery phase of the disease, leading to long-term consequences of COVID-19. These long-term consequences involve pulmonary as well as extra-pulmonary sequelae of the disease. Noteably, recent research has shown a potential association between COVID-19 and change in the molecular cargo of extracellular vesicles (EVs). EVs are vesicles released by cells and play an important role in cell communication by transfer of bioactive molecules between cells. Emerging evidence shows a strong link between EVs and their molecular cargo, and regulation of metabolism in health and disease. This review focuses on current knowledge about EVs and their potential role in COVID-19 pathogenesis, their current and future implications as tools for biomarker and therapeutic development and their possible effects on long-term impact of COVID-19.
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Affiliation(s)
- Soumyalekshmi Nair
- Translational Extracellular Vesicles in Obstetrics and Gynae-Oncology Group, UQ Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, Qld, 4072, Australia
| | - Estefania Nova-Lamperti
- Molecular and Translational Immunology Laboratory, Clinical Biochemistry and Immunology Department, Pharmacy Faculty, Universidad de Concepción, Concepción, Chile
| | - Gonzalo Labarca
- Molecular and Translational Immunology Laboratory, Clinical Biochemistry and Immunology Department, Pharmacy Faculty, Universidad de Concepción, Concepción, Chile
| | - Arutha Kulasinghe
- Frazer Institute, Faculty of Medicine, The University of Queensland, Brisbane, Qld, 4102, Australia
| | - Kirsty R Short
- School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
| | - Flavio Carrión
- Departamento de Investigación, Postgrado y Educación Continua (DIPEC), Facultad de Ciencias de la Salud, Universidad del Alba, Santiago, Chile.
| | - Carlos Salomon
- Translational Extracellular Vesicles in Obstetrics and Gynae-Oncology Group, UQ Centre for Clinical Research, Royal Brisbane and Women's Hospital, Faculty of Medicine, The University of Queensland, Brisbane, Qld, 4072, Australia.
- Departamento de Investigación, Postgrado y Educación Continua (DIPEC), Facultad de Ciencias de la Salud, Universidad del Alba, Santiago, Chile.
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4
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Cha M, Jeong SH, Bae S, Park JH, Baeg Y, Han DW, Kim SS, Shin J, Park JE, Oh SW, Gho YS, Shon MJ. Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method. Anal Chem 2023; 95:5843-5849. [PMID: 36990442 PMCID: PMC10100391 DOI: 10.1021/acs.analchem.2c05166] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/31/2023]
Abstract
Fluorescent labeling allows for imaging and tracking of vesicles down to single-particle level. Among several options to introduce fluorescence, staining of lipid membranes with lipophilic dyes provides a straightforward approach without interfering with vesicle content. However, incorporating lipophilic molecules into vesicle membranes in an aqueous solution is generally not efficient because of their low water solubility. Here, we describe a simple, fast (<30 min), and highly effective procedure for fluorescent labeling of vesicles including natural extracellular vesicles. By adjusting the ionic strength of the staining buffer with NaCl, the aggregation status of DiI, a representative lipophilic tracer, can be controlled reversibly. Using cell-derived vesicles as a model system, we show that dispersion of DiI under low-salt condition improved its incorporation into vesicles by a factor of 290. In addition, increasing NaCl concentration after labeling induced free dye molecules to form aggregates, which can be filtered and thus effectively removed without ultracentrifugation. We consistently observed 6- to 85-fold increases in the labeled vesicle count across different types of dyes and vesicles. The method is expected to reduce the concern about off-target labeling resulting from the use of high concentrations of dyes.
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Affiliation(s)
- Minkwon Cha
- Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
- POSTECH Biotech Center, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic Korea
| | - Sang Hyeok Jeong
- Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Seoyoon Bae
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Jun Hyuk Park
- Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Yoonjin Baeg
- Biodrone Research Institute, MDimune Inc., Seoul 04790, Republic of Korea
| | - Dong Woo Han
- Biodrone Research Institute, MDimune Inc., Seoul 04790, Republic of Korea
| | - Sang Soo Kim
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Jaehyeon Shin
- Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Jeong Eun Park
- Biodrone Research Institute, MDimune Inc., Seoul 04790, Republic of Korea
| | - Seung Wook Oh
- Biodrone Research Institute, MDimune Inc., Seoul 04790, Republic of Korea
| | - Yong Song Gho
- Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
| | - Min Ju Shon
- Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
- School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea
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5
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Xia H, Yu Z, Zhang L, Liu S, Zhao Y, Huang J, Fu D, Xie Q, Liu H, Zhang Z, Zhao Y, Wu M, Zhang W, Pang D, Chen G. Real-Time Dissection of the Transportation and miRNA-Release Dynamics of Small Extracellular Vesicles. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2205566. [PMID: 36599707 PMCID: PMC9982592 DOI: 10.1002/advs.202205566] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Revised: 11/28/2022] [Indexed: 06/17/2023]
Abstract
Extracellular vesicles (EVs) are cell-derived membrane-enclosed structures that deliver biomolecules for intercellular communication. Developing visualization methods to elucidate the spatiotemporal dynamics of EVs' behaviors will facilitate their understanding and translation. With a quantum dot (QD) labeling strategy, a single particle tracking (SPT) platform is proposed here for dissecting the dynamic behaviors of EVs. The interplays between tumor cell-derived small EVs (T-sEVs) and endothelial cells (ECs) are specifically investigated based on this platform. It is revealed that, following a clathrin-mediated endocytosis by ECs, T-sEVs are transported to the perinuclear region in a typical three-stage pattern. Importantly, T-sEVs frequently interact with and finally enter lysosomes, followed by quick release of their carried miRNAs. This study, for the first time, reports the entire process and detailed dynamics of T-sEV transportation and cargo-release in ECs, leading to better understanding of their proangiogenic functions. Additionally, the QD-based SPT technique will help uncover more secrets of sEV-mediated cell-cell communication.
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Affiliation(s)
- Hou‐Fu Xia
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
- Department of Oral and Maxillofacial SurgerySchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Zi‐Li Yu
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
- Department of Oral and Maxillofacial SurgerySchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Li‐Juan Zhang
- College of Chemistry and Molecular SciencesWuhan UniversityWuhan430072P. R. China
| | - Shu‐Lin Liu
- State Key Laboratory of Medicinal Chemical BiologyTianjin Key Laboratory of Biosensing and Molecular RecognitionResearch Center for Analytical Sciencesand College of ChemistryNankai UniversityTianjin300071P. R. China
| | - Yi Zhao
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
- Department of ProsthodonticsSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Jue Huang
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Dan‐Dan Fu
- College of Chemistry and Molecular SciencesWuhan UniversityWuhan430072P. R. China
| | - Qi‐Hui Xie
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Hai‐Ming Liu
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Zhi‐Ling Zhang
- College of Chemistry and Molecular SciencesWuhan UniversityWuhan430072P. R. China
| | - Yi‐Fang Zhao
- Department of Oral and Maxillofacial SurgerySchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Min Wu
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Wei Zhang
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
- Department of Oral and Maxillofacial SurgerySchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
| | - Dai‐Wen Pang
- State Key Laboratory of Medicinal Chemical BiologyTianjin Key Laboratory of Biosensing and Molecular RecognitionResearch Center for Analytical Sciencesand College of ChemistryNankai UniversityTianjin300071P. R. China
| | - Gang Chen
- The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei‐MOST) and Key Laboratory of Oral Biomedicine Ministry of EducationSchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
- Department of Oral and Maxillofacial SurgerySchool and Hospital of StomatologyWuhan UniversityWuhan430079P. R. China
- TaiKang Center for Life and Medical SciencesWuhan UniversityWuhan430071P. R. China
- Frontier Science Center for Immunology and MetabolismWuhan UniversityWuhan430071P. R. China
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6
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Repiska G, Crescitelli R, Lunavat TR, Soekmadji C, Cho WC. Editorial: The role of extracellular vesicles in diseases: Shedding light on their role in cell-to-cell communication. Front Genet 2023; 14:1123822. [PMID: 36760996 PMCID: PMC9904441 DOI: 10.3389/fgene.2023.1123822] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2022] [Accepted: 01/16/2023] [Indexed: 01/26/2023] Open
Affiliation(s)
- Gabriela Repiska
- Institute of Physiology, Faculty of Medicine, Comenius University in Bratislava, Bratislava, Slovakia
| | - Rossella Crescitelli
- Sahlgrenska Center for Cancer Research and Wallenberg Centre for Molecular and Translational Medicine, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
| | - Taral R. Lunavat
- Department of Neurology, Molecular Neurogenetics Unit-West, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States
- Department of Biomedicine, University of Bergen, Bergen, Norway
| | - Carolina Soekmadji
- School of Biomedical Sciences, Faculty of Medicine, University of Queensland, Brisbane, QLD, Australia
| | - William C. Cho
- Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong SAR, China
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7
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Toribio V, Yáñez-Mó M. Two Complementary Strategies to Quantitate Extracellular Vesicle Uptake Using Bioluminescence and Non-Lipidic Dyes. Methods Mol Biol 2023; 2668:133-144. [PMID: 37140795 DOI: 10.1007/978-1-0716-3203-1_11] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/05/2023]
Abstract
The study of the molecular mechanisms controlling extracellular vesicle uptake by a target cell is an aspect of great interest within the EV community due to EV relevance in intercellular communication for tissue homeostasis or different disease progressions such as cancer or Alzheimer's. Since the EV field is relatively young, standardization of techniques for even basic aspects such as their isolation and characterization is still under development and debate. So it is for the study of EV uptake, where the currently most used strategies have critical limitations. Newly designed techniques should try to discern the uptake events from the surface EV binding or to improve the sensitivity and fidelity of the assays. Here, we describe two different complementary methods to measure and quantify EV uptake that we believe, help to overcome certain limitations of the currently used techniques. One is based on a mEGFP-Tspn-Rluc construct, to sort these two reporters into EVs. The use of bioluminescence signal to measure EV uptake allows for a better sensitivity, discerns EV binding from uptake, and allows kinetics measurement in alive cells, being compatible with a high-throughput screen format. The second one is a flow cytometry assay based in EV staining with a maleimide conjugated with a fluorophore, a chemical compound that covalently binds to proteins within sulfhydryl residues, being a good alternative to lipidic dyes and compatible with flow cytometry sorting of cell populations that have captured the labeled EVs.
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Affiliation(s)
- Víctor Toribio
- Departamento de Biología Molecular, Instituto Universitario de Biología Molecular (IUBM), Universidad Autónoma de Madrid (UAM), Madrid, Spain
- Centro de Biología Molecular Severo Ochoa (CBMSO), Instituto de Investigación Sanitaria La Princesa (IIS-IP), Madrid, Spain
| | - María Yáñez-Mó
- Departamento de Biología Molecular, Instituto Universitario de Biología Molecular (IUBM), Universidad Autónoma de Madrid (UAM), Madrid, Spain.
- Centro de Biología Molecular Severo Ochoa (CBMSO), Instituto de Investigación Sanitaria La Princesa (IIS-IP), Madrid, Spain.
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8
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Gomes P, Tzouanou F, Skolariki K, Vamvaka-Iakovou A, Noguera-Ortiz C, Tsirtsaki K, Waites CL, Vlamos P, Sousa N, Costa-Silva B, Kapogiannis D, Sotiropoulos I. Extracellular vesicles and Alzheimer's disease in the novel era of Precision Medicine: implications for disease progression, diagnosis and treatment. Exp Neurol 2022; 358:114183. [PMID: 35952764 PMCID: PMC9985072 DOI: 10.1016/j.expneurol.2022.114183] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Revised: 06/17/2022] [Accepted: 07/21/2022] [Indexed: 12/13/2022]
Abstract
Extracellular vesicles (EVs), secreted membranous nano-sized particles, are critical intercellular messengers participating in nervous system homeostasis, while recent evidence implicates EVs in Alzheimer's disease (AD) pathogenesis. Specifically, small EVs have been shown to spread toxic proteins, induce neuronal loss, and contribute to neuroinflammation and AD progression. On the other hand, EVs can reduce amyloid-beta deposition and transfer neuroprotective substances between cells, mitigating disease mechanisms. In addition to their roles in AD pathogenesis, EVs also exhibit great potential for the diagnosis and treatment of other brain disorders, representing an advantageous tool for Precision Medicine. Herein, we summarize the contribution of small EVs to AD-related mechanisms and disease progression, as well as their potential as diagnostic and therapeutic agents for AD.
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Affiliation(s)
- Patrícia Gomes
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Foteini Tzouanou
- Institute of Biosciences & Applications NCSR "Demokritos", Athens, Greece
| | | | - Anastasia Vamvaka-Iakovou
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal; Institute of Biosciences & Applications NCSR "Demokritos", Athens, Greece
| | - Carlos Noguera-Ortiz
- Laboratory of Clinical Investigation, Intramural Research Program, National Institute on Aging, NIH, Baltimore, MD, USA
| | - Katerina Tsirtsaki
- Institute of Biosciences & Applications NCSR "Demokritos", Athens, Greece
| | - Clarissa L Waites
- Department of Pathology and Cell Biology, Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University Medical Center, New York, NY, USA
| | | | - Nuno Sousa
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal
| | - Bruno Costa-Silva
- Systems Oncology Group, Champalimaud Research, Champalimaud Centre for the Unknown, Av. Brasília, 1400-038 Lisbon, Portugal
| | - Dimitrios Kapogiannis
- Laboratory of Clinical Investigation, Intramural Research Program, National Institute on Aging, NIH, Baltimore, MD, USA
| | - Ioannis Sotiropoulos
- Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; ICVS/3B's - PT Government Associate Laboratory, Braga/Guimarães, Portugal; Institute of Biosciences & Applications NCSR "Demokritos", Athens, Greece.
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9
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Kimiz-Gebologlu I, Oncel SS. Exosomes: Large-scale production, isolation, drug loading efficiency, and biodistribution and uptake. J Control Release 2022; 347:533-543. [PMID: 35597405 DOI: 10.1016/j.jconrel.2022.05.027] [Citation(s) in RCA: 299] [Impact Index Per Article: 99.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2022] [Revised: 05/13/2022] [Accepted: 05/14/2022] [Indexed: 12/24/2022]
Abstract
Exosomes are nanovesicles with different contents that play a role in various biological and pathological processes. It offers significant advantages over other delivery systems such as liposomes and polymeric nanoparticles. Although exosomes are expected to be effective therapeutic agents, their optimal use remains a challenge. The development of methods for large-scale production, isolation, and drug loading is necessary to improve their efficiency and therapeutic potential. In this review, after mentioning general properties and biological functions of the exosomes, details of their potential for use in the drug delivery system are presented. For this purpose, methodologies for the large-scale production of exosomes, exosome isolation, exosomal cargo loading, and exosome uptake by the recipient cell are reviewed. The current challenges and potential directions of this new area of drug delivery that has become popular recently are also investigated.
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Affiliation(s)
| | - Suphi S Oncel
- Department of Bioengineering, Faculty of Engineering, Ege University, Izmir, Turkey..
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10
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Newman LA, Useckaite Z, Johnson J, Sorich MJ, Hopkins AM, Rowland A. Selective Isolation of Liver-Derived Extracellular Vesicles Redefines Performance of miRNA Biomarkers for Non-Alcoholic Fatty Liver Disease. Biomedicines 2022; 10:biomedicines10010195. [PMID: 35052873 PMCID: PMC8773667 DOI: 10.3390/biomedicines10010195] [Citation(s) in RCA: 39] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Revised: 01/10/2022] [Accepted: 01/12/2022] [Indexed: 02/04/2023] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH.
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Affiliation(s)
- Lauren A. Newman
- College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia; (L.A.N.); (Z.U.); (M.J.S.); (A.M.H.)
| | - Zivile Useckaite
- College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia; (L.A.N.); (Z.U.); (M.J.S.); (A.M.H.)
| | - Jillian Johnson
- Early Clinical Development, Pfizer Global Research and Development, Groton, CT 06340, USA;
| | - Michael J. Sorich
- College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia; (L.A.N.); (Z.U.); (M.J.S.); (A.M.H.)
| | - Ashley M. Hopkins
- College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia; (L.A.N.); (Z.U.); (M.J.S.); (A.M.H.)
| | - Andrew Rowland
- College of Medicine and Public Health, Flinders University, Adelaide, SA 5042, Australia; (L.A.N.); (Z.U.); (M.J.S.); (A.M.H.)
- Correspondence: ; Tel.: +61-882-047-546
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11
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Gómez O, Perini-Villanueva G, Yuste A, Rodríguez-Navarro JA, Poch E, Bejarano E. Autophagy and Glycative Stress: A Bittersweet Relationship in Neurodegeneration. Front Cell Dev Biol 2022; 9:790479. [PMID: 35004686 PMCID: PMC8733682 DOI: 10.3389/fcell.2021.790479] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Accepted: 12/06/2021] [Indexed: 12/27/2022] Open
Abstract
Autophagy is a fine-tuned proteolytic pathway that moves dysfunctional/aged cellular components into the lysosomal compartment for degradation. Over the last 3 decades, global research has provided evidence for the protective role of autophagy in different brain cell components. Autophagic capacities decline with age, which contributes to the accumulation of obsolete/damaged organelles and proteins and, ultimately, leads to cellular aging in brain tissues. It is thus well-accepted that autophagy plays an essential role in brain homeostasis, and malfunction of this catabolic system is associated with major neurodegenerative disorders. Autophagy function can be modulated by different types of stress, including glycative stress. Glycative stress is defined as a cellular status with abnormal and accelerated accumulation of advanced glycation end products (AGEs). It occurs in hyperglycemic states, both through the consumption of high-sugar diets or under metabolic conditions such as diabetes. In recent years, glycative stress has gained attention for its adverse impact on brain pathology. This is because glycative stress stimulates insoluble, proteinaceous aggregation that is linked to the malfunction of different neuropathological proteins. Despite the emergence of new literature suggesting that autophagy plays a major role in fighting glycation-derived damage by removing cytosolic AGEs, excessive glycative stress might also negatively impact autophagic function. In this mini-review, we provide insight on the status of present knowledge regarding the role of autophagy in brain physiology and pathophysiology, with an emphasis on the cytoprotective role of autophagic function to ameliorate the adverse effects of glycation-derived damage in neurons, glia, and neuron-glia interactions.
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Affiliation(s)
- Olga Gómez
- School of Health Sciences and Veterinary School, Universidad CEU Cardenal Herrera, CEU Universities, Valencia, Spain
| | - Giuliana Perini-Villanueva
- Laboratory for Nutrition and Vision Research, USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA, United States
| | - Andrea Yuste
- School of Health Sciences and Veterinary School, Universidad CEU Cardenal Herrera, CEU Universities, Valencia, Spain
| | | | - Enric Poch
- School of Health Sciences and Veterinary School, Universidad CEU Cardenal Herrera, CEU Universities, Valencia, Spain
| | - Eloy Bejarano
- School of Health Sciences and Veterinary School, Universidad CEU Cardenal Herrera, CEU Universities, Valencia, Spain
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12
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Dantas-Pereira L, Menna-Barreto R, Lannes-Vieira J. Extracellular Vesicles: Potential Role in Remote Signaling and Inflammation in Trypanosoma cruzi-Triggered Disease. Front Cell Dev Biol 2022; 9:798054. [PMID: 34988085 PMCID: PMC8721122 DOI: 10.3389/fcell.2021.798054] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2021] [Accepted: 11/26/2021] [Indexed: 12/12/2022] Open
Abstract
Extracellular vesicles (EVs) act as cell communicators and immune response modulators and may be employed as disease biomarkers and drug delivery systems. In infectious diseases, EVs can be released by the pathogen itself or by the host cells (infected or uninfected), potentially impacting the outcome of the immune response and pathological processes. Chagas disease (CD) is caused by infection by the protozoan Trypanosoma cruzi and is the main cause of heart failure in endemic areas. This illness attracted worldwide attention due to the presence of symptomatic seropositive subjects in North America, Asia, Oceania, and Europe. In the acute phase of infection, nonspecific signs, and symptoms contribute to miss diagnosis and early etiological treatment. In this phase, the immune response is crucial for parasite control; however, parasite persistence, dysregulated immune response, and intrinsic tissue factors may contribute to the pathogenesis of chronic CD. Most seropositive subjects remain in the indeterminate chronic form, and from 30 to 40% of the subjects develop cardiac, digestive, or cardio-digestive manifestations. Identification of EVs containing T. cruzi antigens suggests that these vesicles may target host cells and regulate cellular processes and the immune response by molecular mechanisms that remain to be determined. Parasite-released EVs modulate the host-parasite interplay, stimulate intracellular parasite differentiation and survival, and promote a regulatory cytokine profile in experimental models of CD. EVs derived from the parasite-cell interaction inhibit complement-mediated parasite lysis, allowing evasion. EVs released by T. cruzi-infected cells also regulate surrounding cells, maintaining a proinflammatory profile. After a brief review of the basic features of EVs, the present study focuses on potential participation of T. cruzi-secreted EVs in cell infection and persistence of low-grade parasite load in the chronic phase of infection. We also discuss the role of EVs in shaping the host immune response and in pathogenesis and progression of CD.
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Affiliation(s)
- Luíza Dantas-Pereira
- Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil.,Laboratório de Biologia das Interações, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
| | - Rubem Menna-Barreto
- Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
| | - Joseli Lannes-Vieira
- Laboratório de Biologia das Interações, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
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13
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Ashcroft J, Leighton P, Elliott TR, Hosgood SA, Nicholson ML, Kosmoliaptsis V. Extracellular vesicles in kidney transplantation: a state-of-the-art review. Kidney Int 2021; 101:485-497. [PMID: 34838864 DOI: 10.1016/j.kint.2021.10.038] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2021] [Revised: 10/11/2021] [Accepted: 10/26/2021] [Indexed: 12/16/2022]
Abstract
Kidney transplantation is the optimal treatment for patients with kidney failure; however, early detection and timely treatment of graft injury remain a challenge. Precise and noninvasive techniques of graft assessment and innovative therapeutics are required to improve kidney transplantation outcomes. Extracellular vesicles (EVs) are lipid bilayer-delimited particles with unique biosignatures and immunomodulatory potential, functioning as intermediaries of cell signalling. Promising evidence exists for the potential of EVs to develop precision diagnostics of graft dysfunction, and prognostic biomarkers for clinician decision making. The inherent targeting characteristics of EVs and their low immunogenic and toxicity profiles combined with their potential as vehicles for drug delivery make them ideal targets for development of therapeutics to improve kidney transplant outcomes. In this review, we summarize the current evidence for EVs in kidney transplantation, discuss common methodological principles of EV isolation and characterization, explore upcoming innovative approaches in EV research, and discuss challenges and opportunities to enable translation of research findings into clinical practice.
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Affiliation(s)
- James Ashcroft
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Cambridge, UK
| | - Philippa Leighton
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Cambridge, UK
| | - Tegwen R Elliott
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Cambridge, UK
| | - Sarah A Hosgood
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Cambridge, UK; NIHR Blood and Transplant Research Unit in Organ Donation and Transplantation, University of Cambridge, Cambridge, UK
| | - Michael L Nicholson
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Cambridge, UK; NIHR Blood and Transplant Research Unit in Organ Donation and Transplantation, University of Cambridge, Cambridge, UK
| | - Vasilis Kosmoliaptsis
- Department of Surgery, University of Cambridge and NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Cambridge, UK; NIHR Blood and Transplant Research Unit in Organ Donation and Transplantation, University of Cambridge, Cambridge, UK.
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14
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Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human plasma by LC-MS. Pract Lab Med 2021; 26:e00241. [PMID: 34258353 PMCID: PMC8254000 DOI: 10.1016/j.plabm.2021.e00241] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2020] [Revised: 04/29/2021] [Accepted: 06/17/2021] [Indexed: 12/13/2022] Open
Abstract
Objectives Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30–140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids. Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins. Design and methods We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes. We compared the proteins detected by UC alone and UC coupled with SEC. Results In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone. Conclusions We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.
Isolating exosomes with the least possible contaminants is very important for their clinical application. Isolating exosomes from human plasma faces considerable challenges. There is no ideal single isolation technique until this date. Exosomes can be successfully isolated from plasma using a combination of Ultracentrifugation and SEC.
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Key Words
- AGO2, Argonaute protein
- CSF, cerebrospinal fluid
- EVs, extracellular vesicles
- Exosomes
- FDR, false discovery rate
- Human plasma
- ILVs, intraluminal vesicles
- MVBs, multivesicular bodies
- Mass spectrometry
- SEC, size exclusion chromatography
- Size-exclusion chromatography
- UC, ultracentrifugation
- Ultracentrifugation
- cfNAs, Cell-free nucleic acids
- ctDNA, circulating tumor DNA
- miRNA, microRNA
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15
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Zheng X, Hermann DM, Bähr M, Doeppner TR. The role of small extracellular vesicles in cerebral and myocardial ischemia-Molecular signals, treatment targets, and future clinical translation. Stem Cells 2021; 39:403-413. [PMID: 33432732 DOI: 10.1002/stem.3329] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2020] [Revised: 12/09/2020] [Accepted: 12/13/2020] [Indexed: 12/17/2022]
Abstract
The heart and the brain mutually interact with each other, forming a functional axis that is disturbed under conditions of ischemia. Stem cell-derived extracellular vesicles (EVs) show great potential for the treatment of ischemic stroke and myocardial infarction. Due to heart-brain interactions, therapeutic actions of EVs in the brain and the heart cannot be regarded in an isolated way. Effects in each of the two organs reciprocally influence the outcome of the other. Stem cell-derived EVs modulate a large number of signaling pathways in both tissues. Upon ischemia, EVs prevent delayed injury, promote angiogenesis, enhance parenchymal remodeling, and enable functional tissue recovery. The therapeutic effects greatly depend on EV cargos, among which are noncoding RNAs like microRNAs (miRNAs) and proteins, which modulate cell signaling in a differential way that not always corresponds to each other in the two tissues. Interestingly, the same miRNA or protein localized in EVs can modulate different signaling pathways in the ischemic heart and brain, which may have diverse consequences for disease outcomes. Paying careful attention to unveiling these underlying mechanisms may provide new insights into tissue remodeling processes and identify targets for ischemic stroke and myocardial infarction therapies. Some of these mechanisms are discussed in this concise review, and consequences for the clinical translation of EVs are presented.
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Affiliation(s)
- Xuan Zheng
- Department of Neurology, University Medical Center Goettingen, Goettingen, Germany
| | - Dirk M Hermann
- Department of Neurology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
| | - Mathias Bähr
- Department of Neurology, University Medical Center Goettingen, Goettingen, Germany
| | - Thorsten R Doeppner
- Department of Neurology, University Medical Center Goettingen, Goettingen, Germany
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16
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Najar M, Martel-Pelletier J, Pelletier JP, Fahmi H. Novel insights for improving the therapeutic safety and efficiency of mesenchymal stromal cells. World J Stem Cells 2020; 12:1474-1491. [PMID: 33505596 PMCID: PMC7789128 DOI: 10.4252/wjsc.v12.i12.1474] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Revised: 08/13/2020] [Accepted: 09/25/2020] [Indexed: 02/06/2023] Open
Abstract
Mesenchymal stromal cells (MSCs) have attracted great interest in the field of regenerative medicine. They can home to damaged tissue, where they can exert pro-regenerative and anti-inflammatory properties. These therapeutic effects involve the secretion of growth factors, cytokines, and chemokines. Moreover, the functions of MSCs could be mediated by extracellular vesicles (EVs) that shuttle various signaling messengers. Although preclinical studies and clinical trials have demonstrated promising therapeutic results, the efficiency and the safety of MSCs need to be improved. After transplantation, MSCs face harsh environmental conditions, which likely dampen their therapeutic efficacy. A possible strategy aiming to improve the survival and therapeutic functions of MSCs needs to be developed. The preconditioning of MSCs ex vivo would strength their capacities by preparing them to survive and to better function in this hostile environment. In this review, we will discuss several preconditioning approaches that may improve the therapeutic capacity of MSCs. As stated above, EVs can recapitulate the beneficial effects of MSCs and may help avoid many risks associated with cell transplantation. As a result, this novel type of cell-free therapy may be safer and more efficient than the whole cell product. We will, therefore, also discuss current knowledge regarding the therapeutic properties of MSC-derived EVs.
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Affiliation(s)
- Mehdi Najar
- Department of Medicine, University of Montreal, Osteoarthritis Research Unit, University of Montreal Hospital Research Center (CRCHUM), Montreal, QC H2X 0A9, Canada.
| | - Johanne Martel-Pelletier
- Department of Medicine, University of Montreal, Osteoarthritis Research Unit, University of Montreal Hospital Research Center (CRCHUM), Montreal, QC H2X 0A9, Canada
| | - Jean Pierre Pelletier
- Department of Medicine, University of Montreal, Osteoarthritis Research Unit, University of Montreal Hospital Research Center (CRCHUM), Montreal, QC H2X 0A9, Canada
| | - Hassan Fahmi
- Department of Medicine, University of Montreal, Osteoarthritis Research Unit, University of Montreal Hospital Research Center (CRCHUM), Montreal, QC H2X 0A9, Canada
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17
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Willis CM, Nicaise AM, Hamel R, Pappa V, Peruzzotti-Jametti L, Pluchino S. Harnessing the Neural Stem Cell Secretome for Regenerative Neuroimmunology. Front Cell Neurosci 2020; 14:590960. [PMID: 33250716 PMCID: PMC7674923 DOI: 10.3389/fncel.2020.590960] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 10/06/2020] [Indexed: 12/15/2022] Open
Abstract
Increasing evidence foresees the secretome of neural stem cells (NSCs) to confer superimposable beneficial properties as exogenous NSC transplants in experimental treatments of traumas and diseases of the central nervous system (CNS). Naturally produced secretome biologics include membrane-free signaling molecules and extracellular membrane vesicles (EVs) capable of regulating broad functional responses. The development of high-throughput screening pipelines for the identification and validation of NSC secretome targets is still in early development. Encouraging results from pre-clinical animal models of disease have highlighted secretome-based (acellular) therapeutics as providing significant improvements in biochemical and behavioral measurements. Most of these responses are being hypothesized to be the result of modulating and promoting the restoration of key inflammatory and regenerative programs in the CNS. Here, we will review the most recent findings regarding the identification of NSC-secreted factors capable of modulating the immune response to promote the regeneration of the CNS in animal models of CNS trauma and inflammatory disease and discuss the increased interest to refine the pro-regenerative features of the NSC secretome into a clinically available therapy in the emerging field of Regenerative Neuroimmunology.
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Affiliation(s)
- Cory M. Willis
- Department of Clinical Neurosciences and NIHR Biomedical Research Centre, University of Cambridge, Cambridge, United Kingdom
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18
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Vilanova-Perez T, Jones C, Balint S, Dragovic R, L Dustin M, Yeste M, Coward K. Exosomes derived from HEK293T cells interact in an efficient and noninvasive manner with mammalian sperm in vitro. Nanomedicine (Lond) 2020; 15:1965-1980. [PMID: 32794431 DOI: 10.2217/nnm-2020-0056] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Aim: To investigate exosomes as a noninvasive delivery tool for mammalian sperm. Materials & Methods: Exosomes were isolated from HEK293T cells and co-incubated with boar sperm in vitro. Results: Internalized exosomes were detected within 10 min of co-incubation. Computer-assisted sperm analysis and flow cytometry demonstrated that even after 5-h of exposure to exosomes, there were no significant deleterious effects with regard to sperm motility, viability, membrane integrity and mitochondrial membrane potential (p > 0.05), thus indicating that exosomes did not interfere with basic sperm function. Conclusion: HEK293T-derived exosomes interacted with boar sperm without affecting sperm function. Exosomes represent a versatile and promising research tool for studying sperm biology and provide new options for the diagnosis and treatment of male infertility.
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Affiliation(s)
- Teresa Vilanova-Perez
- Nuffield Department of Women's & Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, OX3 9DU, Oxford, UK
| | - Celine Jones
- Nuffield Department of Women's & Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, OX3 9DU, Oxford, UK
| | - Stefan Balint
- Nuffield Department of Orthopaedics, The Kennedy Institute of Rheumatology, Rheumatology & Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, OX3 7FY, Oxford, UK
| | - Rebecca Dragovic
- Nuffield Department of Women's & Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, OX3 9DU, Oxford, UK
| | - Michael L Dustin
- Nuffield Department of Orthopaedics, The Kennedy Institute of Rheumatology, Rheumatology & Musculoskeletal Sciences, University of Oxford, Roosevelt Drive, Headington, OX3 7FY, Oxford, UK
| | - Marc Yeste
- Department of Biology, Biotechnology of Animal & Human Reproduction (TechnoSperm), Unit of Cell Biology, Institute of Food & Agricultural Technology, University of Girona, E-17003, Girona, Spain
| | - Kevin Coward
- Nuffield Department of Women's & Reproductive Health, University of Oxford, Level 3, Women's Centre, John Radcliffe Hospital, OX3 9DU, Oxford, UK
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19
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Naseri M, Bozorgmehr M, Zöller M, Ranaei Pirmardan E, Madjd Z. Tumor-derived exosomes: the next generation of promising cell-free vaccines in cancer immunotherapy. Oncoimmunology 2020; 9:1779991. [PMID: 32934883 PMCID: PMC7466856 DOI: 10.1080/2162402x.2020.1779991] [Citation(s) in RCA: 105] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Identification of immunogenic tumor antigens that are efficiently processed and delivered by dendritic cells to prime the immune system and to induce an appropriate immune response is a research hotspot in the field of cancer vaccine development. High biosafety is an additional demand. Tumor-derived exosomes (TEXs) are nanosized lipid bilayer encapsulated vesicles that shuttle bioactive information to the tumor microenvironment facilitating tumor progression. However, accumulating evidence points toward the capacity of TEXs to efficiently stimulate immune responses against tumors provided they are appropriately administered. After briefly describing the function of exosomes in cancer biology and their communication with immune cells, we summarize in this review in vitro and preclinical studies eliciting the potency of TEXs in inducing effective anti-tumor responses and recently modified strategies further improving TEX-vaccination efficacy. We interpret the available data as TEXs becoming a lead in cancer vaccination based on tumor antigen-selective high immunogenicity.
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Affiliation(s)
- Marzieh Naseri
- Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.,Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Mahmood Bozorgmehr
- Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran
| | - Margot Zöller
- Section Pancreas Research, University Hospital of Surgery, Heidelberg, Germany
| | - Ehsan Ranaei Pirmardan
- Molecular Biomarkers Nano-imaging Laboratory, Brigham & Women's Hospital, Department of Radiology, Harvard Medical School, Boston, MA, USA
| | - Zahra Madjd
- Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.,Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran
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20
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Innovative Visualization and Quantification of Extracellular Vesicles Interaction with and Incorporation in Target Cells in 3D Microenvironments. Cells 2020; 9:cells9051180. [PMID: 32397409 PMCID: PMC7291305 DOI: 10.3390/cells9051180] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2020] [Revised: 04/30/2020] [Accepted: 05/06/2020] [Indexed: 02/07/2023] Open
Abstract
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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21
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Luo W, Dai Y, Chen Z, Yue X, Andrade-Powell KC, Chang J. Spatial and temporal tracking of cardiac exosomes in mouse using a nano-luciferase-CD63 fusion protein. Commun Biol 2020; 3:114. [PMID: 32157172 PMCID: PMC7064570 DOI: 10.1038/s42003-020-0830-7] [Citation(s) in RCA: 49] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2019] [Accepted: 02/12/2020] [Indexed: 12/15/2022] Open
Abstract
Exosomes are secreted extracellular vesicles with lipid bilayer membranes. They are emerging as a new category of messengers that facilitate cross-talk between cells, tissues, and organs. Thus, a critical demand arises for the development of a sensitive and non-invasive tracking system for endogenous exosomes. We have generated a genetic mouse model that meets this goal. The Nano-luciferase (NanoLuc) reporter was fused with the exosome surface marker CD63 for exosome labeling. The cardiomyocyte-specific αMHC promoter followed by the loxP-STOP-loxP cassette was engineered for temporal and spatial labeling of exosomes originated from cardiomyocytes. The transgenic mouse was bred with a tamoxifen-inducible Cre mouse (Rosa26Cre-ERT2) to achieve inducible expression of CD63NanoLuc reporter. The specific labeling and tissue distribution of endogenous exosomes released from cardiomyocytes were demonstrated by luciferase assay and non-invasive bioluminescent live imaging. This endogenous exosome tracking mouse provides a useful tool for a range of research applications. Using nano-luciferase-CD63, Weijia Luo et al. develop transgenic mice where cardiac exosomes can be tracked with non-invasive bioluminescent live imaging in a tamoxifen-inducible manner. These mice serve as a valuable tool that allow researchers to track cardiac exosomes in a defined time window.
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Affiliation(s)
- Weijia Luo
- Center for Genomic and Precision Medicine, Texas A&M University, Institute of Biosciences and Technology, Houston, TX, 77030, USA
| | - Yuan Dai
- Center for Genomic and Precision Medicine, Texas A&M University, Institute of Biosciences and Technology, Houston, TX, 77030, USA
| | - Zhishi Chen
- Center for Genomic and Precision Medicine, Texas A&M University, Institute of Biosciences and Technology, Houston, TX, 77030, USA
| | - Xiaojing Yue
- Department of Obstetrics and Gynecology, Nanfang Hospital, Southern Medical University, 510515, Guangzhou, China
| | - Kelsey C Andrade-Powell
- Center for Genomic and Precision Medicine, Texas A&M University, Institute of Biosciences and Technology, Houston, TX, 77030, USA
| | - Jiang Chang
- Center for Genomic and Precision Medicine, Texas A&M University, Institute of Biosciences and Technology, Houston, TX, 77030, USA.
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22
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Schira-Heinen J, Grube L, Waldera-Lupa DM, Baberg F, Langini M, Etemad-Parishanzadeh O, Poschmann G, Stühler K. Pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2019; 1867:140237. [DOI: 10.1016/j.bbapap.2019.06.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Revised: 06/04/2019] [Accepted: 06/06/2019] [Indexed: 02/07/2023]
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Abstract
The outstanding potential of Extracellular Vesicles (EVs) in medicine, deserves a detailed study of the molecular aspects regulating their incorporation into target cells. However, because EV size lies below the limit of resolution of optical techniques, quantification together with discrimination between EV binding to the target cell and uptake is usually not completely achieved with current techniques. Human tetraspanins CD9 and CD63 were fused to a dual EGFP-Renilla-split tag. Subcellular localization and incorporation of these fusion proteins into EVs was assessed by western-blot and fluorescence microscopy. EV binding and uptake was measured using either a classical Renilla substrate or a cytopermeable one. Incubation of target cells expressing DSP2 with EVs containing the complementary DSP1 portion could not recover fluorescence or luciferase activity. However, using EVs carrying the fully reconstituted Dual-EGFP-Renilla protein and the cytopermeable Renilla luciferase substrate, we could distinguish EV binding from uptake. We provide proof of concept of the system by analysing the effect of different chemical inhibitors, demonstrating that this method is highly sensitive and quantitative, allowing a dynamic follow-up in a high-throughput scheme to unravel the molecular mechanisms of EV uptake in different biological systems.
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24
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Ibáñez F, Montesinos J, Ureña-Peralta JR, Guerri C, Pascual M. TLR4 participates in the transmission of ethanol-induced neuroinflammation via astrocyte-derived extracellular vesicles. J Neuroinflammation 2019; 16:136. [PMID: 31272469 PMCID: PMC6610989 DOI: 10.1186/s12974-019-1529-x] [Citation(s) in RCA: 91] [Impact Index Per Article: 15.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2019] [Accepted: 06/24/2019] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Current evidence indicates that extracellular vesicles (EVs) participate in intercellular signaling, and in the regulation and amplification of neuroinflammation. We have previously shown that ethanol activates glial cells through Toll-like receptor 4 (TLR4) by triggering neuroinflammation. Here, we evaluate if ethanol and the TLR4 response change the release and inflammatory content of astrocyte-derived EVs, and whether these vesicles are capable of communicating with neurons by spreading neuroinflammation. METHODS Cortical neurons and astrocytes in culture were used. EVs were isolated from the extracellular medium of the primary culture of the WT and TLR4-KO astrocytes treated with or without ethanol (40 mM) for 24 h. Flow cytometry, nanoparticle tracking analysis technology, combined with exosomal molecular markers (tetraspanins) along with electron microscopy, were used to characterize and quantify EVs. The content of EVs in inflammatory proteins, mRNA, and miRNAs was analyzed by Western blot and RT-PCR in both astrocyte-derived EVs and the neurons incubated or not with these EVs. Functional analyses of miRNAs were also performed. RESULTS We show that ethanol increases the number of secreted nanovesicles and their content by raising the levels of both inflammatory-related proteins (TLR4, NFκB-p65, IL-1R, caspase-1, NLRP3) and by changing miRNAs (mir-146a, mir-182, and mir-200b) in the EVs from the WT-astrocytes compared with those from the untreated WT cells. No changes were observed in either the number of isolated EVs or their content between the untreated and ethanol-treated TLR4-KO astrocytes. We also show that astrocyte-derived EVs could be internalized by naïve cortical neurons to increase the neuronal levels of inflammatory protein (COX-2) and miRNAs (e.g., mir-146a) and to compromise their survival. The functional analysis of miRNAs revealed the regulatory role of the expressed miRNAs in some genes involved in several inflammatory pathways. CONCLUSIONS These results suggest that astrocyte-derived EVs could act as cellular transmitters of inflammation signaling by spreading and amplifying the neuroinflammatory response induced by ethanol through TLR4 activation.
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Affiliation(s)
- Francesc Ibáñez
- Department of Molecular and Cellular Pathology of Alcohol, Centro de Investigación Príncipe Felipe, C/Eduardo Primo Yúfera 3, 46012, Valencia, Spain
| | - Jorge Montesinos
- Department of Molecular and Cellular Pathology of Alcohol, Centro de Investigación Príncipe Felipe, C/Eduardo Primo Yúfera 3, 46012, Valencia, Spain.,Department of Neurology, Columbia University Medical Center, New York, USA
| | - Juan R Ureña-Peralta
- Department of Molecular and Cellular Pathology of Alcohol, Centro de Investigación Príncipe Felipe, C/Eduardo Primo Yúfera 3, 46012, Valencia, Spain
| | - Consuelo Guerri
- Department of Molecular and Cellular Pathology of Alcohol, Centro de Investigación Príncipe Felipe, C/Eduardo Primo Yúfera 3, 46012, Valencia, Spain.
| | - María Pascual
- Department of Molecular and Cellular Pathology of Alcohol, Centro de Investigación Príncipe Felipe, C/Eduardo Primo Yúfera 3, 46012, Valencia, Spain. .,Department of Physiology, School of Medicine and Dentistry, University of Valencia, 46012, Valencia, Spain.
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25
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Ressel S, Rosca A, Gordon K, Buck AH. Extracellular RNA in viral-host interactions: Thinking outside the cell. WILEY INTERDISCIPLINARY REVIEWS. RNA 2019; 10:e1535. [PMID: 30963709 PMCID: PMC6617787 DOI: 10.1002/wrna.1535] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/05/2019] [Revised: 03/13/2019] [Accepted: 03/14/2019] [Indexed: 12/15/2022]
Abstract
Small RNAs and their associated RNA interference (RNAi) pathways underpin diverse mechanisms of gene regulation and genome defense across all three kingdoms of life and are integral to virus-host interactions. In plants, fungi and many animals, an ancestral RNAi pathway exists as a host defense mechanism whereby viral double-stranded RNA is processed to small RNAs that enable recognition and degradation of the virus. While this antiviral RNAi pathway is not generally thought to be present in mammals, other RNAi mechanisms can influence infection through both viral- and host-derived small RNAs. Furthermore, a burgeoning body of data suggests that small RNAs in mammals can function in a non-cell autonomous manner to play various roles in cell-to-cell communication and disease through their transport in extracellular vesicles. While vesicular small RNAs have not been proposed as an antiviral defense pathway per se, there is increasing evidence that the export of host- or viral-derived RNAs from infected cells can influence various aspects of the infection process. This review discusses the current knowledge of extracellular RNA functions in viral infection and the technical challenges surrounding this field of research. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action.
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Affiliation(s)
- Sarah Ressel
- Institute of Immunology and Infection Research, School of Biological SciencesUniversity of EdinburghEdinburghUK
| | - Adelina Rosca
- Department of VirologyCarol Davila University of Medicine and PharmacyBucharestRomania
| | - Katrina Gordon
- Institute of Immunology and Infection Research, School of Biological SciencesUniversity of EdinburghEdinburghUK
| | - Amy H. Buck
- Institute of Immunology and Infection Research, School of Biological SciencesUniversity of EdinburghEdinburghUK
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26
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Delivery of microRNAs by Extracellular Vesicles in Viral Infections: Could the News be Packaged? Cells 2019; 8:cells8060611. [PMID: 31216738 PMCID: PMC6627707 DOI: 10.3390/cells8060611] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Revised: 06/04/2019] [Accepted: 06/13/2019] [Indexed: 12/18/2022] Open
Abstract
Extracellular vesicles (EVs) are released by various cells and recently have attracted attention because they constitute a refined system of cell-cell communication. EVs deliver a diverse array of biomolecules including messenger RNAs (mRNAs), microRNAs (miRNAs), proteins and lipids, and they can be used as potential biomarkers in normal and pathological conditions. The cargo of EVs is a snapshot of the donor cell profile; thus, in viral infections, EVs produced by infected cells could be a central player in disease pathogenesis. In this context, miRNAs incorporated into EVs can affect the immune recognition of viruses and promote or restrict their replication in target cells. In this review, we provide an updated overview of the roles played by EV-delivered miRNAs in viral infections and discuss the potential consequences for the host response. The full understanding of the functions of EVs and miRNAs can turn into useful biomarkers for infection detection and monitoring and/or uncover potential therapeutic targets.
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27
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Fafián-Labora J, Carpintero-Fernández P, Jordan SJD, Shikh-Bahaei T, Abdullah SM, Mahenthiran M, Rodríguez-Navarro JA, Niklison-Chirou MV, O'Loghlen A. FASN activity is important for the initial stages of the induction of senescence. Cell Death Dis 2019; 10:318. [PMID: 30962418 PMCID: PMC6453932 DOI: 10.1038/s41419-019-1550-0] [Citation(s) in RCA: 60] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Revised: 03/18/2019] [Accepted: 03/21/2019] [Indexed: 02/08/2023]
Abstract
Senescent cells accumulate in several tissues during ageing and contribute to several pathological processes such as ageing and cancer. Senescence induction is a complex process not well defined yet and is characterized by a series of molecular changes acquired after an initial growth arrest. We found that fatty acid synthase (FASN) levels increase during the induction of senescence in mouse hepatic stellate cells and human primary fibroblasts. Importantly, we also observed a significant increase in FASN levels during ageing in mouse liver tissues. To probe the central role of FASN in senescence induction, we used a small-molecule inhibitor of FASN activity, C75. We found that C75 treatment prevented the induction of senescence in mouse and human senescent cells. Importantly, C75 also reduced the expression of the signature SASP factors interleukin 1α (IL-1α), IL-1β and IL-6, and suppressed the secretion of small extracellular vesicles. These findings were confirmed using a shRNA targeting FASN. In addition, we find that FASN inhibition induces metabolic changes in senescent cells. Our work underscores the importance of C75 as a pharmacological inhibitor for reducing the impact of senescent cell accumulation.
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Affiliation(s)
- Juan Fafián-Labora
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK
| | - Paula Carpintero-Fernández
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK
| | - Samuel James Davison Jordan
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK
| | - Tamanna Shikh-Bahaei
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK
| | - Sana Mohammad Abdullah
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK
| | - Midusa Mahenthiran
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK.,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK
| | - José Antonio Rodríguez-Navarro
- Instituto Ramón y Cajal de Investigaciones Sanitarias, Neurobiología-Investigación. Hospital Ramón y Cajal, 28034, Madrid, Spain
| | - Maria Victoria Niklison-Chirou
- Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK.
| | - Ana O'Loghlen
- Epigenetics and Cellular Senescence Group, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London, E1 2AT, UK. .,Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK.
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28
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Gámez-Valero A, Beyer K, Borràs FE. Extracellular vesicles, new actors in the search for biomarkers of dementias. Neurobiol Aging 2019; 74:15-20. [DOI: 10.1016/j.neurobiolaging.2018.10.006] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2018] [Revised: 09/14/2018] [Accepted: 10/04/2018] [Indexed: 02/07/2023]
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29
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Barrachina MN, Calderón-Cruz B, Fernandez-Rocca L, García Á. Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives. Proteomics 2019; 19:e1800247. [PMID: 30467982 DOI: 10.1002/pmic.201800247] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2018] [Revised: 11/09/2018] [Indexed: 02/06/2023]
Abstract
Extracellular vesicles (EVs) are a heterogeneous population of vesicles composed of a lipid bilayer that carry a large repertoire of molecules including proteins, lipids, and nucleic acids. In this review, some guidelines for plasma-derived EVs isolation, characterization, and proteomic analysis, and the application of the above to cardiovascular disease (CVD) studies are provided. For EVs analysis, blood samples should be collected using a 21-gauge needle, preferably in citrate tubes, and plasma stored for up to 1 year at -80°, using a single freeze-thaw cycle. For proteomic applications, differential centrifugation (including ultracentrifugation steps) is a good option for EVs isolation. EVs characterization is done by transmission electron microscopy, particle enumeration techniques (nanoparticle-tracking analysis, dynamic light scattering), and flow cytometry. Regarding the proteomics strategy, a label-free and gel-free quantitative method is a good choice due to its accuracy and because it minimizes the amount of sample required for clinical applications. Besides the above, main EVs proteomic findings in cardiovascular-related diseases are presented and analyzed in this review, paying especial attention to overlapping results between studies. The latter might offer new insights into the clinical relevance and potential of novel EVs biomarkers identified to date in the context of CVD.
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Affiliation(s)
- Maria N Barrachina
- Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade Santiago de Compostela, Santiago de Compostela, 15782, Spain.,Instituto de Investigación, Sanitaria de Santiago (IDIS), Santiago de Compostela, 15706, Spain
| | - Beatriz Calderón-Cruz
- Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade Santiago de Compostela, Santiago de Compostela, 15782, Spain.,Instituto de Investigación, Sanitaria de Santiago (IDIS), Santiago de Compostela, 15706, Spain
| | - Lucía Fernandez-Rocca
- Clinical Analysis Laboratory, Maciel Hospital, Faculty of Chemistry, University of the Republic, Montevideo, 11000, Uruguay
| | - Ángel García
- Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade Santiago de Compostela, Santiago de Compostela, 15782, Spain.,Instituto de Investigación, Sanitaria de Santiago (IDIS), Santiago de Compostela, 15706, Spain
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30
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A new level of complexity in parasite-host interaction: The role of extracellular vesicles. ADVANCES IN PARASITOLOGY 2019; 104:39-112. [PMID: 31030771 DOI: 10.1016/bs.apar.2019.02.003] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Humans and animals have co-existed with parasites in a battle of constant adaptation to one another. It is becoming increasingly clear that extracellular vesicles (EVs) play important roles in this co-existence and pathology. This chapter reviews the current research on EVs released by protozoa, nematodes, trematodes, and cestodes with a special focus on EVs in parasite life cycles. The environmental changes experienced by the parasite during its life cycle is associated with distinct changes in EV release and content. The function of these EV seems to have a significant influence on parasite pathology and survival in the host by concomitantly modulating host immune responses and triggering parasite differentiation. The role of EVs in communication between the parasites and the host adds a new level of complexity in our understanding of parasite biology, which may be a key to further understand the complexity behind host-parasite interactions and communication. This increased understanding can, in turn, open up new avenues for vaccine, diagnostic, and therapeutic development for a wide variety of diseases such as parasite infection, cancers, and immunological disorders.
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31
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Mobarrez F, Svenungsson E, Pisetsky DS. Microparticles as autoantigens in systemic lupus erythematosus. Eur J Clin Invest 2018; 48:e13010. [PMID: 30062774 DOI: 10.1111/eci.13010] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Accepted: 07/29/2018] [Indexed: 12/17/2022]
Abstract
Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antibodies to components of the cell nucleus (antinuclear antibodies or ANAs) and the formation of immune complexes with nuclear antigens. These complexes can drive pathogenesis by depositing in the tissue to incite inflammation or induce cytokine production by cells of the innate immune system. While ANAs can bind to purified nuclear molecules, nuclear autoantigens in vivo most likely exist attached to other molecules or embedded in larger structures. Among these structures, microparticles (MPs) are membrane bound vesicles that are released from dead and dying cells by a blebbing process; MPs can also be released during activation of platelets. The presence of MPs in the blood or tissue culture media can be assayed by flow cytometry on the basis of light scattering as well as binding of marker antibodies to identify the cell of origin. As shown by biochemical analyses, MPs contain an ensemble of intracellular components including nuclear, cytoplasmic and membrane molecules. Because of the display of these molecules on the particle surface or in an otherwise accessible form, ANAs, including anti-DNA, can bind to particles. Levels of MPs are increased in the blood of patients with SLE, with flow cytometry demonstrating the presence of IgG-containing particles. In addition to forming immune complexes, MPs can directly stimulate immune responses. Together, these findings suggest an important role of particles in the pathogenesis of SLE and their utility as biomarkers.
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Affiliation(s)
- Fariborz Mobarrez
- Department of Medicine, Unit of Rheumatology, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden
| | - Elisabet Svenungsson
- Department of Medicine, Unit of Rheumatology, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden
| | - David S Pisetsky
- Department of Medicine, Duke University Medical Center, Durham, North Carolina.,Medical Research Service, Durham VA Hospital, Durham, North Carolina
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32
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Ilgın C, Topuzoğlu A. Extracellular Vesicles in Psychiatry Research in the Context of RDoC Criteria. Psychiatry Investig 2018; 15:1011-1018. [PMID: 30380817 PMCID: PMC6259002 DOI: 10.30773/pi.2018.09.17] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/27/2018] [Revised: 09/06/2018] [Accepted: 09/17/2018] [Indexed: 12/18/2022] Open
Abstract
The analysis of extracellular vesicles has been accelerated because of the technological advancements in omics methods in recent decades. Extracellular vesicles provide multifaceted information regarding the functional status of the cells. This information would be critical in case of central nervous system cells, which are confined in a relatively sealed biological compartment. This obstacle is more dramatic in psychiatric disorders since their diagnosis primarily depend on the symptoms and signs of the patients. In this paper, we reviewed this rapidly advancing field by discussing definition of extracellular vesicles, their biogenesis and potential use as clinical biomarkers. Then we focused on their potential use in psychiatric disorders in the context of diagnosis and treatment of these disorders. Finally, we tried to combine the RDoC (Research Domain Criteria) with the use of extracellular vesicles in psychiatry research and practice. This review may offer new insights in both basic and translational research focusing on psychiatric disorders.
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Affiliation(s)
- Can Ilgın
- Department of Public Health, Marmara University School of Medicine, Istanbul, Turkey
| | - Ahmet Topuzoğlu
- Department of Public Health, Marmara University School of Medicine, Istanbul, Turkey
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33
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Tengattini S. Chromatographic Approaches for Purification and Analytical Characterization of Extracellular Vesicles: Recent Advancements. Chromatographia 2018. [DOI: 10.1007/s10337-018-3637-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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34
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Hikita T, Miyata M, Watanabe R, Oneyama C. Sensitive and rapid quantification of exosomes by fusing luciferase to exosome marker proteins. Sci Rep 2018; 8:14035. [PMID: 30232365 PMCID: PMC6145919 DOI: 10.1038/s41598-018-32535-7] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2018] [Accepted: 09/03/2018] [Indexed: 12/18/2022] Open
Abstract
Exosomes have emerged as important mediators of intercellular communication. Although their modes of action have been elucidated, the molecular mechanisms underlying their secretion, sorting of molecules, uptake into recipient cells, and biological distribution in vivo remain elusive. Here, we present a novel system for quantifying secreted exosomes by introducing ectopic or CRISPR/Cas9-mediated knock-in of luciferase-fusion exosome markers such as CD63. This luciferase-based method makes it possible to measure exosomes secreted into the culture medium with high linearity and wide dynamic range in a high-throughput manner. We demonstrate that data obtained by luminescent quantification are well correlated with data obtained by conventional nanoparticle tracking analysis under multiple conditions. In addition, our system is capable of evaluating the recipient cells or tissues that take up exosomes, as well as visualizing exosomes in vivo. The proposed system represents a powerful tool for understanding the molecular mechanisms underlying exosome production, uptake, and long-term distribution.
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Affiliation(s)
- Tomoya Hikita
- Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan
| | - Mamiko Miyata
- Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan
| | - Risayo Watanabe
- Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan
| | - Chitose Oneyama
- Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Japan. .,JST, PRESTO, Nagoya, Japan.
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35
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Pick H, Alves AC, Vogel H. Single-Vesicle Assays Using Liposomes and Cell-Derived Vesicles: From Modeling Complex Membrane Processes to Synthetic Biology and Biomedical Applications. Chem Rev 2018; 118:8598-8654. [PMID: 30153012 DOI: 10.1021/acs.chemrev.7b00777] [Citation(s) in RCA: 109] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
The plasma membrane is of central importance for defining the closed volume of cells in contradistinction to the extracellular environment. The plasma membrane not only serves as a boundary, but it also mediates the exchange of physical and chemical information between the cell and its environment in order to maintain intra- and intercellular functions. Artificial lipid- and cell-derived membrane vesicles have been used as closed-volume containers, representing the simplest cell model systems to study transmembrane processes and intracellular biochemistry. Classical examples are studies of membrane translocation processes in plasma membrane vesicles and proteoliposomes mediated by transport proteins and ion channels. Liposomes and native membrane vesicles are widely used as model membranes for investigating the binding and bilayer insertion of proteins, the structure and function of membrane proteins, the intramembrane composition and distribution of lipids and proteins, and the intermembrane interactions during exo- and endocytosis. In addition, natural cell-released microvesicles have gained importance for early detection of diseases and for their use as nanoreactors and minimal protocells. Yet, in most studies, ensembles of vesicles have been employed. More recently, new micro- and nanotechnological tools as well as novel developments in both optical and electron microscopy have allowed the isolation and investigation of individual (sub)micrometer-sized vesicles. Such single-vesicle experiments have revealed large heterogeneities in the structure and function of membrane components of single vesicles, which were hidden in ensemble studies. These results have opened enormous possibilities for bioanalysis and biotechnological applications involving unprecedented miniaturization at the nanometer and attoliter range. This review will cover important developments toward single-vesicle analysis and the central discoveries made in this exciting field of research.
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Affiliation(s)
- Horst Pick
- Institute of Chemical Sciences and Engineering , Ecole Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne , Switzerland
| | - Ana Catarina Alves
- Institute of Chemical Sciences and Engineering , Ecole Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne , Switzerland
| | - Horst Vogel
- Institute of Chemical Sciences and Engineering , Ecole Polytechnique Fédérale de Lausanne (EPFL) , CH-1015 Lausanne , Switzerland
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Abstract
Exosomes are secreted extracellular vesicles (EVs) that carry micro RNAs and other factors to reprogram cancer cells and tissues affected by cancer. Exosomes are exchanged between cancer cells and other tissues, often to prepare a premetastatic niche, escape immune surveillance, or spread multidrug resistance. Only a few studies investigated the function of lipids in exosomes although their lipid composition is different from that of the secreting cells. Ceramide is one of the lipids critical for exosome formation, and it is also enriched in these EVs. New research suggests that lipids in the exosomal membrane may organize and transmit "mobile rafts" that turn exosomes into extracellular signalosomes spreading activation of cell signaling pathways in oncogenesis and metastasis. Ceramide may modulate the function of mobile rafts and their effect on these cell signaling pathways. The critical role of lipids and, in particular, ceramide for formation, secretion, and function of exosomes may lead to a radically new understanding of cancer biology and therapy.
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Affiliation(s)
- Ahmed Elsherbini
- Department of Physiology, University of Kentucky, Lexington, KY, United States
| | - Erhard Bieberich
- Department of Physiology, University of Kentucky, Lexington, KY, United States
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37
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Autissier E, Li H, Goepfert PA, Reeves RK. Short Communication: Apoptotic Membrane Microparticles Quantified by Fluorescent Bead-Based Assay Are Elevated in HIV and SIV Infections. AIDS Res Hum Retroviruses 2018; 34:446-448. [PMID: 29486582 DOI: 10.1089/aid.2018.0011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Apoptotic membrane microparticles (MMPs) derived from dying cells of multiple cell origins are highly immunostimulatory and are indicative of global immune activation and cell death in a variety of diseases. In this study, we developed a flow cytometric bead assay to quantify annexin-V+ apoptotic (MMPs) in plasma from humans and rhesus macaques. With a combination of flow cytometry and pan-fluorescent beads, MMPs were enumerated in plasma specimens by adding a constant ratio of beads to initial fluid volumes and then calculating MMP/mL based on MMP-to-bead ratios. Using this straightforward assay, we found that circulating MMP quantifications were highly reproducible and similar in number between normal rhesus macaques and humans subjects. However, MMPs increased two- to threefold during HIV and simian immunodeficiency virus (SIV) infections and were positively associated with T cell immune activation. Collectively, we present a rapid bead-based assay for both humans and macaque models to quantify MMPs that could be an instigator and predictor of immune activation, which is a primary source of HIV/SIV disease.
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Affiliation(s)
- Estelle Autissier
- Division of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts
| | - Haiying Li
- Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
| | - Paul A. Goepfert
- Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama
| | - R. Keith Reeves
- Division of Immunology, New England Primate Research Center, Harvard Medical School, Southborough, Massachusetts
- Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
- Ragon Institute of Massachusetts General Hospital, MIT, and Harvard, Cambridge, Massachusetts
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Extracellular vesicles: A new therapeutic strategy for joint conditions. Biochem Pharmacol 2018; 153:134-146. [PMID: 29427625 DOI: 10.1016/j.bcp.2018.02.004] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2017] [Accepted: 02/05/2018] [Indexed: 02/07/2023]
Abstract
Extracellular vesicles (EVs) are attracting increasing interest since they might represent a more convenient therapeutic tool with respect to their cells of origin. In the last years much time and effort have been expended to determine the biological properties of EVs from mesenchymal stem cells (MSCs) and other sources. The immunoregulatory, anti-inflammatory and regenerative properties of MSC EVs have been demonstrated in in vitro studies and animal models of rheumatoid arthritis or osteoarthritis. This cell-free approach has been proposed as a possible better alternative to MSC therapy in autoimmune conditions and tissue regeneration. In addition, EVs show great potential as biomarkers of disease or delivery systems for active molecules. The standardization of isolation and characterization methods is a key step for the development of EV research. A better understanding of EV mechanisms of action and efficacy is required to establish the potential therapeutic applications of this new approach in joint conditions.
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