نقش میکرووزیکول ها (MVs) و اگزوزوم ها در بیماری های متابولیکی و قلب و عروق

ABSTRACT Microvesicles and exosomes: new players in metabolic and cardiovascular disease

It is well established that patients with metabolic diseases, in particular insulin resistance and type 2 diabetes mellitus (T2DM), are more than twice as likely to develop accelerated cardiovascular disease (CVD) including atherosclerosis, stroke, and coronary artery disease (reviewed in Rask-Madsen & King (2013)). Coronary artery disease is a major cause of morbidity and mortality worldwide, and is a leading cause of death in T2DM, with excess risk of fatality in women compared with men (Peters et al. 2014).

Extensive coronary artery disease can result in myocardial infarction, severe loss of cardiac function, and subsequently lead to the development of heart failure (Hausenloy & Yellon 2013). A cluster of risk factors have recently been defined by the American Diabetes Association and the American College of Cardiology Foundation as reliable indicators of a patient’s risk for T2DM and CVD, and has been defined as cardiometabolic risk (CMR; Brunzell et al. 2008). These risks include obesity, hyperglycemia, hypertension, insulin resistance, and dyslipidemia. The presence of secondary CVD in patients with ischaemia-reperfusion (IR) or T2DM may be referred to as cardio-metabolic disease (CMD). Given its increasing prevalence and severe consequences, new approaches are needed to diagnose and treat CMD. Extracellular vesicles (EVs) are small (50 nm to 2 mm) vesicles released from the surface of many different cell types into different bodily fluids, including plasma, milk, saliva, sweat, tears, semen, and urine. There are several classes of EV, including exosomes, microvesicles (MVs), and apoptotic bodies, which are produced by different mechanisms. Attracting perhaps the most attention recently have been exosomes (50–100 nm), a homogenous population of EV which are released from cells when multivesicular bodies (MVB; sometimes called multivesicular endosomes, MVE) fuse with the plasma membrane in a highly regulated process and release their contents. Cells can also produce a more heterogeneous population of EVs up to 2 mm in diameter called MVs, which are formed by budding and shedding of the cell membrane, a process that involves calcium dependent signalling and enzyme activity. Cells undergoing apoptosis also typically release EV of 1–5 mm in diameter which are referred to as apoptotic bodies (Dignat-George & Boulanger 2011, van der Pol et al. 2012, Colombo et al. 2014) (Fig. 1).

In some literature, MVs isolated by centrifugation are referred to as ‘microparticles,’ particularly those isolated from platelets or endothelial cells. For clarity, this review will refer to EVs simply as exosomes or MV on the basis of the mechanism of their cellular production and their size range – an approach that has been taken by others (Thery et al. 2009), with the caveat that most isolation methods do not provide a pure populations of vesicles. It is important to note that the size ranges of EVs may overlap and in particular, the size of MVs could overlap with the exosomal size range. Where a mixture of exosomes and MV is likely, for example when plasma vesicles are isolated by high speed (w100 000 g) ultracentrifugation, we refer to them more broadly as EV. These EV are sometimes also referred to as ‘exosome-like vesicles’.

One of the characteristic markers of all EVs is the presence on the outer surface of phosphatidylserine (PS), due to loss of membrane asymmetry during blebbing (apoptotic bodies) or budding (MV) and inward folding of the membrane during vesicle formation in MVBs (exosomes). This can be identified by binding of labelled annexin V, a reagent often used for flow cytometric analysis of apoptotic cells. However, more recently several groups have identified MVs lacking PS on the outer membrane, suggesting that this is not essential for MV formation (Larson et al. 2012, Hou et al. 2014).

Both exosomes and MVs characteristically carry a cargo, which they are able to deliver to cells in remote locations. The cargo can include genetic material such as mRNA, microRNA (miRNA), or even small amounts of DNA (Moldovan et al. 2013), and proteins including transcription factors, cytokines, and growth factors, have also been described. Importantly, MVs also carry cellular receptors and transmembrane proteins on their surface characteristic of the cells from which they were released. This aids in their identification but also means that they can interact with specific target cells instigating signalling cascades via receptor interactions (rececrine signalling – akin to cell–cell interactions) and also increasing specificity of cargo delivery. On the other hand, exosomes are characteristically decorated with markers including Alix, HSP70, and the tetraspanins CD9 and CD63, which may be associated with beta-2 integrin binding and intercellular communication. Although these are commonly used as markers of exosomes, they are not exclusive to exosomes and may be found on other EVs. Furthermore, not all EVs express CD63 and different sub-populations of exosomes may express different markers (Thery et al. 2009). It is important to consider that exosomes do not necessarily express the same marker proteins as their parent cells. For example, we found that the common endothelial marker CD144 is absent on exosomes from human umbilical vein endothelial cells (HUVECs) (Fig. 2). Recent work has further defined plasma EV and exosome surface marker expression by using extensive antibody profiling which showed that exosomes can express surface membrane markers such as CD146, CD4, CD3, and CD45 (Jorgensen et al. 2015). There is some evidence that the protein and RNA content of exosomes depends on the state of the source cell (de Jong et al. 2012).

The mechanism behind the formation of exosomes and selective packaging of proteins, lipids, and RNA is not completely understood but is gradually becoming revealed. The Endosomal Sorting Complex Responsible for Transport pathway does not seem to be required for exosome biogenesis, although some components are involved in their formation, particularly Alix (Trajkovic et al. 2008, Baietti et al. 2012, Raposo & Stoorvogel 2013). Other molecules that are enriched in exosomes such as tetraspanins and ceramide have also been implicated in exosome biogenesis. For example, inhibitors of neutral sphingomyelinase, an enzyme involved in ceramide production, inhibits exosome production (Trajkovic et al. 2008). Less well understood is the mechanism of exosome release. Certain members of the Rab GTPase family are required for efficient release of exosomes, although the exact members involved appears to depend on the cell type and experimental design, and may reflect different subtypes of exosomes relating to the stage (early or late) of endosome/MVB formation (Colombo et al. 2014).