Extracellular vesicles in cardiovascular disease: focus on vascular calcification

This issue of The Journal of Physiology brings together four timely review articles (Kapustin Krohn et al. 2016a; Osteikoetxea et al. 2016; Ruiz et al. 2016) that discuss for the first time in The Journal the role of extracellular vesicles in cardiovascular calcification. The goal of this issue is to highlight some of the key advances and controversies in the extracellular vesicle field with a focus on vascular calcification. Cardiovascular calcification is a growing burden in ageing societies of the Western world. Vascular calcification not only remains a risk factor for cardiovascular events, but also itself contributes to cardiovascular risk. Indeed, recent data indicate that calcium score acts as a better predictor of acute cardiovascular events than lipid measurements (Vliegenthart et al. 2005; Martin et al. 2014) and that microcalcifications in the thin atherosclerotic plaque cap contribute to plaque destabilization (Vengrenyuk et al. 2006) and subsequent rupture, leading to myocardial infarction and stroke. Furthermore, emerging evidence suggests that vascular cell-derived extracellular vesicles may serve as a continuous source of damaging microcalcifications in atherosclerotic plaques (New New et al. 2013; Kapustin et al. 2015; Hutcheson et al. 2016; Krohn et al. 2016b). Hence, the role of extracellular vesicles in the formation of cardiovascular microcalcifications and their genesis and growth merit future investigation. Discoveries in past decades signifying the role of extracellular vesicles in intercellular communication and signaling led to tremendous advances in the field of exosome biology, resulting in the formation of the International Society for Extracellular Vesicles in 2011 (Harding et al. 2013; Raposo New et al. 2013), emerging evidence based on their biogenesis and mechanisms of release suggests that they are of exosomal origin (Kapustin et al. 2015). Recent studies demonstrated that pathological extracellular vesicles contributing to calcification in the cardiovascular system originate from smooth muscle cells and macrophages (Kapustin et al. 2011, 2015; New et al. 2013; Hutcheson et al. 2016; Krohn et al. 2016b), particularly in atherosclerosis and chronic kidney disease, conditions associated with phosphate and calcium imbalance. The present view from our laboratory is that in a normal environment vascular cells release extracellular vesicles that serve a physiological function and preserve homeostasis, but in a pathological environment due to changes in cytoskeletal orientation or impairment in vesicle trafficking and cargo loading, certain vesicles acquire properties that promote calcification potential. This issue of The Journal of Physiology begins with an overview by Osteikoetxea et al. (2016) of extracellular vesicles and their biological roles in cardiovascular disease. The three articles that follow (Kapustin Ruiz et al. 2016; Krohn et al. 2016a) collectively summarize the most up-to-date knowledge on the role of calcifying extracellular vesicles from two groups that primarily focus their research on ectopic cardiovascular calcification. In the first review, Drs Osteikoetxea, Németh, Sódar, Vukman, and Buzás from the Hungarian Academy of Sciences provide the most current classification of different subtypes of extracellular vesicles based on their biogenesis and size. The authors note that in addition to the differences in vesicle size, there are other important parameters that define their biological roles and should be taken into consideration when determining extracellular vesicle subtypes. In addition, they provide an overview on common techniques used to separate size-based extracellular vesicle subpopulations, including centrifugation (Valadi et al. 2007), precipitation (Brownlee et al. 2014), microfluidic-based devices (Vaidyanathan et al. 2014), affinity capture (Balaj et al. 2015), size exclusion chromatography (Sokolova et al. 2011) and field-flow fractionation (Sitar et al. 2015). The type of isolation typically depends on the origin of the sample, its volume and equipment availability. The isolation of the vesicles is usually followed by their detection and quantification. Several detection devices are currently available, including nanoparticle-tracking analysis (Hutcheson et al. 2014) and dynamic light scattering (Sitar et al. 2015). Additionally, several high-resolution microscopy methods are used to determine extracellular vesicle size and morphology (Buzas et al. 2014; Hutcheson et al. 2016). The authors correctly point out that due to the growing numbers of various technologies for vesicle isolation and detection, standardized protocols and controls need to be in place to assure that vesicle characterization meets the requirement guidelines (Lotvall et al. 2014) and that results remain comparable between different laboratories. The comprehensive summary of extracellular vesicles subtypes, as well as their isolation protocols and detection methods are provided at the end of each interrelated paragraph of this review article. Osteikoetxea and colleagues further discuss the possible reasons for discordant functions of extracellular vesicles, an important topic for the rest of this thematic issue, which focuses on the role of extracellular vesicles in pathological states. As recent evidence suggests, extracellular vesicles may play a role as extracellular communicators to improve organ function. Their role may also be detrimental in certain diseases, including cardiovascular calcification. The authors provide several explanations on potential causes for pathological functions of extracellular vesicles in the cardiovascular system, including (1) inherent differences of extracellular vesicles subtypes, (2) differences in the vesicle-releasing cell populations (e.g. protective vs. pathological), (3) differences in the functional states of vesicle-releasing cells (e.g. metabolically active vs. metabolically inactive), and (4) differences in postsynthetic modifications to molecules of extracellular vesicles (e.g. phosphorylation). It is important to note that specific populations of extracellular vesicles secreted by cells in a given functional state could have a combinatory effect when interacting with other cells, vesicles, or soluble mediators. The next three reviews from the Aikawa group at Brigham and Women's Hospital, Harvard Medical School, and Shanahan laboratory at King's College London, focus on extracellular vesicles in vascular calcification. Krohn et al. (2016a) discuss our current understanding of the origin and release mechanisms of extracellular vesicles and propose that subpopulations of the extracellular vesicles identified in calcified vasculature follow a different pathway of origin from bone-derived matrix vesicles. Unlike matrix vesicles implicated in physiological bone formation, which bleb off specialized domains on the cell membrane, extracellular vesicles found in intimal and medial vascular calcification may be generated via different pathways. Krohn et al.’s Abstract Figure shows this and includes (1) exosomes (EV) originated from multivesicular bodies through the exocytosis pathway, (2) vesicles produced by shedding from the plasma membrane (MV), and (3) a group of vesicles shielded by a plasma membrane (MVB) that are released into extracellular space as a cargo. These membrane-bound vesicles undergo dissolution of an external membrane followed by the release of individual vesicles into the extracellular matrix. While these types of vesicles remain understated in the literature, they have been recently described in telocytes (Fertig et al. 2014) and also observed in our in vivo and in vitro studies of calcifying cardiovascular tissue and smooth muscle cells growing in osteogenic medium. The review article from Kapustin Kapustin et al. 2015). On the other hand, in the following review article, Krohn and colleagues refer to studies in which they employed a β-glycerolphosphate, a specific ALP substrate promoting osteogenic changes seen in atherosclerotic plaques (Hutcheson et al. 2014, 2016; Krohn et al. 2016b). Indeed, culturing smooth muscle cells in these two different conditions results in the production of distinct phenotypes of extracellular vesicles: ALP-low in a high-calcium/phosphate condition and ALP-high in an osteogenic condition. The regulation of ALP activity in smooth muscle cell-derived extracellular vesicles, however, remains unclear and merits further investigation. In the last of this series of reviews, Ruiz et al. (2016) summarize the clinical impact of calcification on cardiovascular morbidity and mortality and discuss how extracellular vesicle-derived microcalcification influences atherosclerotic plaque stability. Our laboratory used a high-resolution structured illumination microscopy and spectroscopic analyses to directly visualize the formation of extracellular vesicle-derived microcalcification (Hutcheson et al. 2016). Using a three-dimensional collagen hydrogel model of the fibrous cap, Hutcheson et al. most recently demonstrated that extracellular vesicles released from vascular smooth muscle cells may aggregate and nucleate calcium crystals within a collagen fibrillar network, thus resembling mature microcalcifications observed in human atherosclerotic plaques (Hutcheson et al. 2016). It remains unclear, however, whether calcium crystals can grow directly from a single vesicle or vesicle aggregation and whether fusion should precede microcalcification formation. Since microcalcifications may directly and critically contribute to plaque rupture and subsequent acute cardiovascular events, it would be important to not only visualize their formation in vitro but also detect early calcific changes in at-risk patient populations. Early molecular imaging studies on calcification in a mouse model of atherosclerosis provided the first insight into the identification of microcalcification in atherosclerotic plaques and aortic valves using a near-infrared calcium tracer and optical imaging modalities (Aikawa et al. 2007a,b, 2009; Hjortnaes et al. 2010). Recent advances exploiting positron emission tomography/CT (PET/CT) with 18F-labelled sodium fluoride showed accumulation of the 18F tracer in active regions of mineralization in patients’ atherosclerotic plaques (Dweck et al. 2012), thus providing an important tool for the identification of spotty microcalcifications associated with adverse cardiovascular events (Joshi et al. 2014). Further investigations are needed to foster our current understanding of the mechanisms of extracellular vesicle-derived calcification. Future research may lead to a generation of a new targeted therapies aimed at the multiple points of intervention of microcalcification formation, and to the development of novel imaging agents and approaches for visualization of early calcification in diseased patient populations. None declared.