Extracellular vesicles in organ and systems function in health and disease

This special issue of The Journal of Physiology entitled ‘Extracellular vesicles in organ and systems function in health and disease’ originated from a focused symposium held at Experimental Biology in April 2021 sponsored by The Physiological Society and the American Physiological Society. The original symposium focused on extracellular vesicles (EVs) as targets for cardiovascular (CV) health, examining the role of EVs as couriers of cellular information as well as their therapeutic potential. Contributors provided a range of perspectives on the benefits and limitations of using EVs in biomedical applications. This special issue has widened the scope of the Experimental Biology symposium to move beyond the original CV focus and to cover the role and translational potential of EVs across multiple body systems. Articles include two Symposium Reviews, eight Topical Reviews, six original research papers along with a Journal Club article and a Perspective article. The combination of these contributions from leading experts across the field of EV research provides valuable insight into our current understanding of EVs, and importantly, appraises the potential of EVs for future clinical application. The opening review in this special issue provides a succinct evaluation of our current knowledge of the role that EVs play in physiological and pathophysiological processes across multiple systems, organs and cell types (Alqurashi et al., 2023). There is a useful summary of the classification of EVs according to their biological origin, the most commonly known being exosomes (derived from the endosomal pathway), ectosomes (derived via budding from the plasma membrane) and apoptotic vesicles (derived from apoptotic cells). Current recommendations also suggest classifying EVs according to size if their biological origin is unclear. What is clear from this overview is the sheer diversity of EV subtypes in existence, with the likelihood of more subpopulations emerging in the future. The growing translational potential of EVs as carriers of biological material, as well as drugs, is evident, and this review critically appraises the challenges and perspectives underlying the clinical application of EVs in the future. Importantly, the authors highlight the need for improved understanding of the fate of EVs in vivo, as well as the need for improved protocols for EV production and monitoring (Alqurashi et al., 2023). Given the current technical challenges surrounding EV production and use for clinical applications highlighted by Alqurashi et al. (2023), it is timely that one of the Symposium Reviews focuses on the potential for adapting bioengineering approaches to enhance the features of EVs, specifically exosomes (Malekian et al., 2023). This review highlights some of the properties of exosomes that, upon modification, could enable their use for drug delivery. These properties include modification of donor cells via transfection to express specific surface proteins, exosome–liposome hybridisation to alter the lipid profile of exosomes, and surface ligand modification to improve targeted delivery. The review provides a detailed summary of the numerous recent approaches used for exosome bioengineering and raises the possibility of using a combination of modification techniques for advanced therapeutic targeting. As such, it provides a useful technical resource for investigators across the EV field. Four of the review articles in this special issue focus on EVs and the CV system. Two of these reviews are from contributions to the original Experimental Biology symposium. These reviews cover not only the role that EVs play in CV health and disease, but also their potential scope as biomarkers for CV pathologies and as therapeutic tools of the future. The authors provide valuable insight into the complexity of EV-mediated communication across the CV system as well as the impact that EV cargo can have on CV function. There is detailed consideration of the strengths and limitations of current strategies for using EVs as therapeutics for CV disease, also outlined by Alqurashi et al. (2023), and this provides the reader with an awareness of the key challenges for future EV research focus. EV-mediated communication between different cell types of the CV system is essential to maintain healthy cellular function but can also lead to pathological consequences. Martins-Marques and Girao (2023) provide a timely overview of how EVs released from different CV cell populations can influence neighbouring cellular function. EVs released from cardiomyocytes, cardiac fibroblasts, endothelial cells and macrophages are among the cell types considered, and it is fascinating to reflect upon the potential that EVs have in cardioprotection. The authors also consider the evidence for EVs mediating cardiac remodelling processes across a range of CV pathologies including myocardial infarction, cardiac hypertrophy and fibrosis, and atrial fibrillation. Aligned with this is the potential that EVs have as biomarkers for pathology, as well as predictors of risk. This builds upon a previous review by Osteikoetxea et al. (2016) who consider the importance of the cellular source and quantity of EVs released from the CV system, suggesting that EVs released from specific CV cell types and in specific numbers may ultimately be cardioprotective or pathogenic. In a complementary Topical Review, Neves et al. (2023) emphasise the correlation between circulating exosomes and risk and severity of CV disease. This review provides a current understanding of the role of specific micro (mi)RNAs in cardioprotection and pathology and as disease biomarkers. The attractive features of exosomes (low immunogenicity and toxicity as well as strong ability to cross cell membranes) lend themselves well to clinical application. The authors raise the challenges of technical application for drug delivery since loading exosomes with the desired cargo (drugs, miRs, proteins) may interfere with exosome structure and therefore ability to target the desired host. They highlight this as a key area of focus in need of development in order to design effective exosome-based drug delivery methods in the future. Delving further into the specific cargo that may mediate CV function, there is a Topical Review included in this special issue that focuses specifically on the role of non-coding (nc)RNAs in CV disease (Collado et al., 2023). These ncRNAs include long non-coding (lnc)RNAs, miRNAs and circular (circ)RNAs. The review contains two very useful diagrams outlining the many and varied effects of EV-derived ncRNAs in both atherosclerosis progression and coronary artery disease, as well as ischaemic heart disease and cardiac fibrosis. The accompanying text for both diagrams provides a wide range of recent references covering the complex effects of EV-derived ncRNAs on different cells of the CV system and cell–cell communication. As previously highlighted by Martins-Marques and Girao (2023) and Neves et al. (2023), this review reiterates the need for improved techniques to characterise EV size, morphology and concentration. The authors present a useful summary of the advantages and disadvantages of the different classical methods for EV isolation, including differential centrifugation, density gradient centrifugation, ultracentrifugation, size-exclusion chromatography, precipitation and immunocapture. Importantly, the review highlights recent and new approaches, including atomic force microscopy, and Raman spectroscopy, the latter enabling characterisation of size, morphology and content all in one. The theme of technical approaches to characterise, image and monitor EVs comprises the second Symposium Review and this provides a wide range of current and emerging strategies (Ranjan et al., 2023). The authors highlight the challenges associated with designing EVs as therapeutics, including their short half-life and adverse off-target effects. Efficient targeting is a key challenge and one that the authors consider in the context of the CV system. Connexin 43, a widely expressed gap junction protein (prevalent in cardiomyocytes), has potential in both the delivery of exosomal contents to cells and, when present as an exosomal protein, in protection against cardiotoxic drugs. This reinforces the importance of connexin 43 in EV communication as previously outlined by Martins-Marques and Girao (2023). Ranjan et al. (2023) also report that recent studies using cardiosphere-derived exosomes and stem cell-derived exosomes have shown potential in cardiac repair, but they stress that delivering the number required for therapeutic purposes is demanding and there is still uncertainty around their actual content/cargo. The ability to track exosomes in vivo provides another challenge to researchers and the review by Ranjan et al. (2023) covers a range of techniques that are currently used, such as covalent binding of tagged molecules to the exosome surface, genetic modulation, metabolic labelling for real-time imaging and surface radiolabelling. The authors highlight that current methodology does not enable tracking of different exosome sub-populations carrying different cargo and secreted by the same cell in health and disease. The current limitations in EV-based clinical applications for CV pathologies rely on advances in these technical approaches and this review provides clear insight into where these challenges lie. The potential that EVs may have in the treatment of acute ischaemic stroke is another area covered by a timely Topical Review (Fullerton et al., 2023). The authors highlight the importance that EVs derived from mesenchymal stem cells may have in restoring neurological function following acute ischaemic stroke. In a similar vein to research covered in Neves et al. (2023) relating to CV pathology, this review details the role that individual EV-derived miRNAs (including miRNA-124, miRNA-126, miRNA-98, miRNA-146 and miRNA-181a) may play in stroke recovery. It is clear that significant alterations occur in circulating EV miRNA in people who have experienced stroke, and the authors discuss those miRNAs that could have potential as both diagnostic and prognostic tools for ischaemic stroke. As emphasised previously by Ranjan et al. (2023), the authors highlight potential limitations in methodological and analytical approaches in EV isolation and characterisation, for example differences in timing of sampling, EV isolation and storage procedures as well as the importance of accounting for how treatment for stroke may alter EV physiology. That said, even taking these limitations into account, there do appear to be common signatures of miRNA changes in EVs following stroke. This review indicates the advantages of using EV-mediated neuroregenerative therapy over the use of stem cells. Since EVs can be delivered systemically (they can cross the blood–brain barrier), this is favourable for design of future therapeutic approaches, and current preclinical models look promising. The therapeutic potential of EVs in the context of liver pathology is another fascinating area covered by a Topical Review (Srinivas et al., 2023). The authors present current information on liver EV biomarkers that show therapeutic promise across conditions including viral hepatitis, fatty liver disease and cancer. Importantly, the information on biomarkers outlined in this review mainly stems from large prospective trials and not from pilot studies. This provides insight into the translational potential that EVs may have in the hepatic system. There is some evidence from laboratory studies that targeted modulation of EV function may reduce certain liver pathologies. For example, the use of GW4869, which is a pharmacological inhibitor of EV release, has shown promise in viral hepatitis, where EVs can increase viral transmission to uninfected cells. SHP099 (a SHP2 inhibitor) can reduce the release of pro-fibrogenic exosomes, thus potentially reducing liver fibrosis. The limitations of these approaches though lie in the fact that many of these EV inhibitors are not selective and can target EVs derived from both healthy and diseased cells. That said, the use of EVs as vehicles for treating liver disease has seen promise with recent nucleic acid-loaded EVs used for siRNA gene silencing in hepatic cancer. The authors also highlight the recent use of artificial exosome-mimetic nanovesicles, which overcome the low yields often seen from EVs isolated under physiological conditions. In an original research paper, Chen et al. (2023) investigate the role that macrophage microvesicles (MMVs) play in intercellular communication in lung disease. MMVs carry and transfer a variety of nucleic acids, proteins and lipids between cells and this study examines the role that circRNAs play in chronic lung injury, specifically injury induced by methamphetamine (MA) abuse. This paper is one of the first to examine the role of these lncRNAs and the authors specifically investigate the role of circ_YTHDF2 in MMV-mediated communication between macrophages and alveolar endothelial cells. The study uses a combination of primary material (a mouse model of chronic lung injury induced by MA, from which bronchoalveolar lavage fluid provides a source of MMVs) and macrophage and endothelial cells. Results suggest that MMVs are the predominant form of EVs extracted from both lavage fluid and from macrophages in culture. Following long-term treatment with MA (6 weeks for mice and up to 24 h for cells in culture), an increased release of MMVs is apparent and, interestingly, increased uptake of MMVs by endothelial cells in culture. A heatmap from a circRNA microarray identifies circ_YTHDF2 as the circRNA most significantly altered in MMVs following MA treatment. Knockdown of circ_YTHDF2 in control MMVs results in increased levels of tumour necrosis factor α, interleukin 6 and intercellular adhesion molecule 1, indicating that circ_YTHDF2 regulates the immunoreactivity of MMVs. By exploring the effects of these MMVs on alveolar endothelial cells, the authors demonstrate that si-circ_YTHDF2 MMVs exacerbate MA-induced inflammatory effects and endothelial to mesenchymal cell transition of aortic endothelial cells. Overexpression of this circRNA reverses these effects and the authors demonstrate this in their MA-induced mouse model of lung injury. This study effectively demonstrates the importance that circRNAs within EVs may have in mediating lung pathology and supports the review by Collado et al. (2023) emphasising the role that these lncRNAs may play in pathological processes. In terms of lung pathology, Chen et al. (2023) highlight the need for further research on other circRNAs that could be involved in this process. It is clear from the papers included in this special issue that our knowledge of EVs is growing exponentially. One area where our current understanding of EVs remains limited, yet could hold tremendous clinical potential, is that of pregnancy. In a Topical Review, Farrelly et al. (2023) cover the current literature investigating the role of placental and maternal EVs across a range of pregnancy complications, including pre-eclampsia, preterm birth, fetal growth restriction and complications arising from gestational diabetes. This review serves as an important source of our research knowledge in this area and highlights the gaps that still exist. During pregnancy, the placenta enables bidirectional EV trafficking, and there is clear evidence for placental EVs to enter maternal cells and vice versa. There is growing evidence for the possibility of using EVs as biomarkers to predict and/or diagnose complications or disease. Reported alterations in EV miRNA profile in gestational diabetes as well as pre-eclampsia could enable predictive diagnosis. In a possible role as therapeutics, there is evidence that EVs could alleviate endothelial dysfunction observed in pre-eclampsia. EVs are involved in normal induction of labour at term and therefore could play a role in the mechanisms involved in preterm birth. This is the leading cause of perinatal morbidity and mortality, and therefore there is an urgent need for improved understanding of the processes involved and strategies for intervention. The review provides important information derived from a mouse model study where administration of EVs with pro-inflammatory cargo to pregnant mice at gestational day 15 resulted in premature birth. Studies in humans have found that the number, size distribution and cargo content of placental EVs isolated during first and second trimesters of pregnancy vary significantly between pregnancies that go to term and those that are preterm. These results provide more insight into an area where there is a real lack of effective treatment, and information provided in this review serves as an important resource for basic researchers and clinicians in the field of obstetrics. A number of articles in this special issue examine the roles that exercise and age play in EV physiology and health. There is a Topical Review outlining the potential role of EVs in healthy ageing and five original research papers. These research papers explore the impact of exercise on EV physiology and pathophysiology, in aged and young individuals, as well as methodology for separation and identification of EVs from individuals undergoing different types of exercise training. One study also reports upon the therapeutic potential for EVs in recovering aged muscle after disuse. Taken together, these articles provide a valuable source for exercise physiologists as a range of perspectives on how EVs released during exercise could influence health in older individuals and those with clinical conditions. McIlvenna and Whitman (2023) review the role of small EVs (sEVs), such as exosomes and microparticles, in the ageing process, and explore how exercise may influence this. Cellular senescence, occurring as part of ageing, involves release of factors known as the senescence-associated secretory phenotype (SASP). The contribution that sEVs make to the SASP and the role of their cargo in this process is clear, particularly since evidence suggests changes can occur in sEV cargo that are not reflected in their host senescent cells. As well as contributing to cell senescence, sEVs derived from human induced pluripotent stem cells (iPSCs) can also reverse this process. The anti-oxidant capacity of sEVs from iPSCs may mediate this property, but the authors highlight the need to properly define the sEV cargo as well as ensure sEV isolation techniques are standardised. This is a recurring message throughout the literature and in this special issue (Alqurashi et al., 2023; Fullerton et al., 2023; Neves et al., 2023; Srinivas et al., 2023). It is clear that exercise can promote healthy ageing but the underlying mechanisms remain elusive. This review highlights that sEV cargos such as TMSB4X (important in muscle generation), glutathione peroxidase and superoxide dismutase (both antioxidants) are upregulated in sEVs following exercise. Importantly, the authors emphasise the value of using proteomic datasets to gather information on EV cargo and stress that, with more data being deposited, our understanding of the impact of sEV cargo in exercise and ageing will grow, guiding future research in this area. Five original research papers explore the impact of different types of exercise on EV physiology and the implications for health. Together, these studies give detailed insight into what is a vast and intriguing area of research within the EV field. McIlvenna et al. (2023) explore the effects of high intensity intermittent training (HIIT) on sEV content of both platelet-poor and platelet-free plasma. Echoing views from other papers in this special issue, the authors stress the importance of a standardised EV isolation technique to enable clear interpretation. In particular, they highlight the challenges of EV separation from plasma and the contaminating influence that remnant platelets may have. They have therefore incorporated assessment of sEVs using single particle analysis, which circumvents the need for sample purification, from both platelet poor and platelet free plasma. They report that HIIT exercise results in a significant increase in the number of circulating sEVs and alters the expression of common tetraspanin proteins. Interestingly, depletion of platelets influences the expression of tetraspanins before and after HIIT and the authors stress the advantages of using single particle analysis and awareness of platelet contribution to sEV isolation. Another important factor to consider when examining the impact of exercise on EV physiology is the possibility that resting EV physiology may differ across individuals with different histories of exercise. This could affect interpretation of research data. Darragh et al. (2023) isolated sEVs across three groups of individuals (all male) with different histories of exercise training. This included those who were or The study used methods of EV including tracking analysis, and of selective EV and targeted on EVs isolated from Results suggest that the number and metabolic profile of EVs from resting across all three groups is This study provides valuable information for future research on EVs in exercise. The authors emphasise that future examine sub-populations of EVs across both and for a more analysis, but for exercise these results upon the need for EV physiology across different groups at research papers in this special issue focus on the impact that exercise can have on circulating EVs in individuals with chronic conditions such as diabetes or in This is an area by et al. the vast potential that muscle with The authors raise not only the of EVs as of muscle function but also of how exercise may promote health via this In this special issue, et al. (2023) demonstrate that acute exercise can the of and circulating to large EVs in has that EV numbers increase in and diabetes and this increase may disease risk. In with EV number, acute exercise also (a of and metabolic The authors highlight that exercise may reduce disease risk via an This the for more research on effects across different EV subpopulations in and circulating miRNAs as and associated with EVs have shown to with et al., and it be fascinating to investigate their potential as biomarkers for risk and how they are with exercise. In to the effects exercise can have on EVs in individuals with chronic there is also evidence that exercise may have a impact on ageing via modulation of EV physiology. et al. 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In this research paper, the authors examine the role of muscle cells in muscle health. These cells and therefore as a system against muscle a mouse model of the authors used single cell analysis with assessment to that the of isolated from muscle is a that involves healthy with the authors demonstrate that of small EVs isolated from these can reverse remodelling and improve physiology in mouse muscle following The authors proteomic analysis of small EVs from and this provides insight into changes observed across the the authors stress that further research is required to the mechanisms that are for the EV effects. The value of this study in the possibility that EVs may promote muscle in individuals who are to exercise and reiterates the message previously by (2023). Taken together, the contributions to this special issue enhance our current understanding in the field of EV The Topical Reviews are a valuable for researchers across different of physiology. 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