Microvesicles and Exosomes: New Players in Metabolic and Cardiovascular Disease

C LAWSON and others EVs in cardiovascular 228:2 R57–R71 Review and metabolic diseases Microvesicles and exosomes: new players in metabolic and cardiovascular disease Charlotte Lawson1, Jose M Vicencio2, Derek M Yellon2 and Sean M Davidson2 Correspondence should be addresses 1Department of Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, to C Lawson London NW1 0TU, UK Email 2The Hatter Cardiovascular Institute, University College London, London WC1E 6HX, UK [email protected] Abstract The past decade has witnessed an exponential increase in the number of publications Keywords referring to extracellular vesicles (EVs). For many years considered to be extracellular debris, " diabetes EVs are now seen as novel mediators of endocrine signalling via cell-to-cell communication. " heart With the capability of transferring proteins and nucleic acids from one cell to another, they " endothelium have become an attractive focus of research for different pathological settings and are now " microvesicles regarded as both mediators and biomarkers of disease including cardio-metabolic disease. " nanoparticles They also offer therapeutic potential as signalling agents capable of targeting tissues or cells " exosomes with specific peptides or miRNAs. In this review, we focus on the role that microvesicles (MVs) " microRNA and exosomes, the two most studied classes of EV, have in diabetes, cardiovascular disease, endothelial dysfunction, coagulopathies, and polycystic ovary syndrome. We also provide an Journal of Endocrinology overview of current developments in MV/exosome isolation techniques from plasma and other fluids, comparing different available commercial and non-commercial methods. We describe different techniques for their optical/biochemical characterization and quantitation. We also review the signalling pathways that exosomes and MVs activate in target cells and provide some insight into their use as biomarkers or potential therapeutic agents. In summary, we give an updated focus on the role that these exciting novel nanoparticles offer for the endocrine community. Journal of Endocrinology (2016) 228, R57–R71 Introduction It is well established that patients with metabolic diseases, infarction, severe loss of cardiac function, and sub- in particular insulin resistance and type 2 diabetes mellitus sequently lead to the development of heart failure (T2DM), are more than twice as likely to develop (Hausenloy & Yellon 2013). A cluster of risk factors have accelerated cardiovascular disease (CVD) including ather- recently been defined by the American Diabetes Associ- osclerosis, stroke, and coronary artery disease (reviewed in ation and the American College of Cardiology Foundation Rask-Madsen & King (2013)). Coronary artery disease is a as reliable indicators of a patient’s risk for T2DM and CVD, major cause of morbidity and mortality worldwide, and and has been defined as cardiometabolic risk (CMR; is a leading cause of death in T2DM, with excess risk of Brunzell et al. 2008). These risks include obesity, fatality in women compared with men (Peters et al. 2014). hyperglycemia, hypertension, insulin resistance, and Extensive coronary artery disease can result in myocardial dyslipidemia. The presence of secondary CVD in patients http://joe.endocrinology-journals.org Ñ 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-15-0201 Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 12:46:41PM via free access Review C LAWSON and others EVs in cardiovascular 228:2 R58 and metabolic diseases with ischaemia-reperfusion (IR) or T2DM may be referred Both exosomes and MVs characteristically carry a to as cardio-metabolic disease (CMD). Given its increasing cargo, which they are able to deliver to cells in remote prevalence and severe consequences, new approaches are locations. The cargo can include genetic material such as needed to diagnose and treat CMD. mRNA, microRNA (miRNA), or even small amounts of Extracellular vesicles (EVs) are small (50 nm to 2 mm) DNA (Moldovan et al. 2013), and proteins including vesicles released from the surface of many different cell transcription factors, cytokines, and growth factors, have types into different bodily fluids, including plasma, milk, also been described. Importantly, MVs also carry cellular saliva, sweat, tears, semen, and urine. There are several receptors and transmembrane proteins on their surface classes of EV, including exosomes, microvesicles (MVs), characteristic of the cells from which they were released. and apoptotic bodies, which are produced by different This aids in their identification but also means that they mechanisms. Attracting perhaps the most attention can interact with specific target cells instigating signalling recently have been exosomes (50–100 nm), a homogenous cascades via receptor interactions (rececrine signalling – population of EV which are released from cells when akin to cell–cell interactions) and also increasing speci- multivesicular bodies (MVB; sometimes called multivesi- ficity of cargo delivery. On the other hand, exosomes are cular endosomes, MVE) fuse with the plasma membrane in characteristically decorated with markers including Alix, a highly regulated process and release their contents. Cells HSP70, and the tetraspanins CD9 and CD63, which may can also produce a more heterogeneous population of EVs be associated with beta-2 integrin binding and intercel- up to 2 mm in diameter called MVs, which are formed by lular communication. Although these are commonly used budding and shedding of the cell membrane, a process as markers of exosomes, they are not exclusive to that involves calcium dependent signalling and enzyme exosomes and may be found on other EVs. Furthermore, activity. Cells undergoing apoptosis also typically release not all EVs express CD63 and different sub-populations EV of 1–5 mm in diameter which are referred to as of exosomes may express different markers (Thery et al. apoptotic bodies (Dignat-George & Boulanger 2011, van 2009). It is important to consider that exosomes do not der Pol et al. 2012, Colombo et al. 2014)(Fig. 1). necessarily express the same marker proteins as their In some literature, MVs isolated by centrifugation are parent cells. For example, we found that the common referred to as ‘microparticles,’ particularly those isolated endothelial marker CD144 is absent on exosomes from from platelets or endothelial cells. For clarity, this review human umbilical vein endothelial cells (HUVECs) (Fig. 2). will refer to EVs simply as exosomes or MV on the basis of Recent work has further defined plasma EV and exosome Journal of Endocrinology the mechanism of their cellular production and their size surface marker expression by using extensive antibody range – an approach that has been taken by others (Thery profiling which showed that exosomes can express surface et al. 2009), with the caveat that most isolation methods membrane markers such as CD146, CD4, CD3, and CD45 do not provide a pure populations of vesicles. It is (Jorgensen et al. 2015). There is some evidence that the important to note that the size ranges of EVs may overlap protein and RNA content of exosomes depends on the and in particular, the size of MVs could overlap with the state of the source cell (de Jong et al. 2012). exosomal size range. Where a mixture of exosomes and The mechanism behind the formation of exosomes MV is likely, for example when plasma vesicles are isolated and selective packaging of proteins, lipids, and RNA is not by high speed (w100 000 g) ultracentrifugation, we refer completely understood but is gradually becoming to them more broadly as EV. These EV are sometimes also revealed. The Endosomal Sorting Complex Responsible referred to as ‘exosome-like vesicles’. for Transport pathway does not seem to be required for One of the characteristic markers of all EVs is the exosome biogenesis, although some components are presence on the outer surface of phosphatidyl serine (PS), involved in their formation, particularly Alix (Trajkovic due to loss of membrane asymmetry during blebbing et al. 2008, Baietti et al. 2012, Raposo & Stoorvogel 2013). (apoptotic bodies) or budding (MV) and inward folding Other molecules that are enriched in exosomes such as of the membrane during vesicle formation in MVBs tetraspanins and ceramide have also been implicated in (exosomes). This can be identified by binding of labelled exosome biogenesis. For example, inhibitors of neutral annexin V, a reagent often used for flow cytometric sphingomyelinase, an enzyme involved in ceramide analysis of apoptotic cells. However, more recently several production, inhibits exosome production (Trajkovic et al. groups have identified MVs lacking PS on the outer 2008). Less well understood is the mechanism of exosome membrane, suggesting that this is not essential for MV release. Certain members of the Rab GTPase family are formation (Larson et al. 2012, Hou et al. 2014). required for efficient release of exosomes, although the http://joe.endocrinology-journals.org Ñ 2016 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-15-0201 Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 12:46:41PM via free access Review C LAWSON and others EVs in cardiovascular 228:2 R59 and metabolic diseases A 700 Extracellular vesicles 600 Microvesicles 500 Exosomes 400 300 200 indexed in pubmed indexed Number of publications 100 0 1955 1960 1965 1970 1972 1974 1976 1978 1980 1982 1984

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