2877
J Physiol 594.11 (2016) pp 2877–2880
EDITORIAL
Extracellular vesicles in cardiovascular disease: focus on vascular calcification Elena Aikawa Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
The Journal of Physiology
Email: [email protected]
This issue of The Journal of Physiology brings together four timely review articles (Kapustin & Shanahan, 2016; 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 & Aikawa, 2013; 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 & Stoorvogel, 2013). The society actively advocates for more accurate and standardized purification methods,
particularly used in the development of medical biomarkers and vaccines, and has recently outlined the minimal experimental requirement guidelines for the characterization of extracellular vesicle preparation (Lotvall et al. 2014). Controversy still exists in the field regarding the classification used for vesicular structures released by various cells, which are collectively called extracellular vesicles. The current criteria for the classification of extracellular vesicles involves their biogenesis, size, morphology, lipid/protein ratio and cellular origin. Depending on size and type, extracellular vesicles are broadly classified as exosomes (10–100 nm in diameter), ectosomes (or shedding microvesicles, 50–1000 nm in diameter) and apoptotic bodies (1000–5000 nm in diameter). Extracellular vesicles are also released during the physiological mineralization processes in bones (originally termed matrix vesicles), a discovery made in the mid-1960s by Anderson (1967) and Bonucci (1967). This is another category that should be added to vesicle classification. Matrix vesicles are small membranous structures (30–300 nm in diameter) surrounded by a lipid bilayer, produced by the blebbing of plasma membrane of osteoblasts, chondrocytes and odontoblasts, and they have a high calcification potential (New & Aikawa, 2013). While calcifying extracellular vesicles, involved in ectopic vascular calcification, display morphological similarities to bone-derived matrix vesicles, and were thus presumed to be of similar origin (Kapustin et al. 2011; 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 physio-
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
logical 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 & Shanahan, 2016; 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, ´ N´emeth, Sodar, Vukman, and Buz´as 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
DOI: 10.1113/JP272112
2878 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.
Editorial 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 & Shanahan (2016) provides new and exciting findings from their recent study, which established that extracellular vesicles that originate through the exosomal pathway serve as novel players in pathological vascular calcification. This group showed that mineral imbalance found in patients with chronic kidney disease stimulates the secretion of exosomes by ‘synthetic’ smooth muscle cells probably through cytoskeletal remodelling and by activation of sphingomyelin phosphodiesterase 3 (SMPD3) (Kapustin et al. 2015). Calcium imbalance induces changes in exosome composition, resulting in the accumulation of phosphatidylserine, annexin 6 and MMP-2, which converts exosomes into loci for calcification. It is critical to mention that major differences exist between two currently established in vitro models of smooth muscle cell calcification, which may reflect different pathologies. The model used in the Kapustin study utilizes a high-calcium/phosphate medium that remains predisposed to spontaneous, alkaline phosphatase (ALP)-independent mineral formation. This culture model mimics the pathogenesis of medial calcification in patients with chronic kidney disease (Kapustin et al. 2011; 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).
J Physiol 594.11
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 18 F-labelled sodium fluoride showed accumulation of the 18 F 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
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.11
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. References Aikawa E, Aikawa M, Libby P, Figueiredo JL, Rusanescu G, Iwamoto Y, Fukuda D, Kohler RH, Shi GP, Jaffer FA & Weissleder R (2009). Arterial and aortic valve calcification abolished by elastolytic cathepsin S deficiency in chronic renal disease. Circulation 119, 1785–1794. Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH, Jaffer FA, Aikawa M & Weissleder R (2007a). Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation 116, 2841–2850. Aikawa E, Nahrendorf M, Sosnovik D, Lok VM, Jaffer FA, Aikawa M & Weissleder R (2007b). Multimodality molecular imaging identifies proteolytic and osteogenic activities in early aortic valve disease. Circulation 115, 377–386. Anderson HC (1967). Electron microscopic studies of induced cartilage development and calcification. J Cell Biol 35, 81–101. Balaj L, Atai NA, Chen W, Mu D, Tannous BA, Breakefield XO, Skog J & Maguire CA (2015). Heparin affinity purification of extracellular vesicles. Sci Rep 5, 10266. Bonucci E (1967). Fine structure of early cartilage calcification. J Ultrastruct Res 20, 33–50. Brownlee Z, Lynn KD, Thorpe PE & Schroit AJ (2014). A novel “salting-out” procedure for the isolation of tumor-derived exosomes. J Immunol Methods 407, 120–126. Buzas EI, Gyorgy B, Nagy G, Falus A & Gay S (2014). Emerging role of extracellular vesicles in inflammatory diseases. Nat Rev Rheumatol 10, 356–364. Dweck MR, Chow MW, Joshi NV, Williams MC, Jones C, Fletcher AM, Richardson H, White A, McKillop G, van Beek EJ, Boon NA, Rudd JH & Newby DE (2012). Coronary arterial 18 F-sodium fluoride uptake: a novel marker of plaque biology. J Am Coll Cardiol 59, 1539–1548. Fertig ET, Gherghiceanu M & Popescu LM (2014). Extracellular vesicles release by cardiac telocytes: electron microscopy and electron tomography. J Cell Mol Med 18, 1938–1943.
Editorial Harding CV, Heuser JE & Stahl PD (2013). Exosomes: looking back three decades and into the future. J Cell Biol 200, 367–371. Hjortnaes J, Butcher J, Figueiredo JL, Riccio M, Kohler RH, Kozloff KM, Weissleder R & Aikawa E (2010). Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: a role for inflammation. Eur Heart J 31, 1975–1984. Hutcheson JD, Goettsch C, Bertazzo S, Maldonado N, Ruiz JL, Goh W, Yabusaki K, Faits T, Bouten C, Franck G, Quillard T, Libby P, Aikawa M, Weinbaum S & Aikawa E (2016). Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques. Nat Mater 15, 335–343. Hutcheson JD, Goettsch C, Pham T, Iwashita M, Aikawa M, Singh SA & Aikawa E (2014). Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol. J Extracell Vesicles 3, 25129. Joshi NV, Vesey AT, Williams MC, Shah AS, Calvert PA, Craighead FH, Yeoh SE, Wallace W, Salter D, Fletcher AM, van Beek EJ, Flapan AD, Uren NG, Behan MW, Cruden NL, Mills NL, Fox KA, Rudd JH, Dweck MR & Newby DE (2014). 18 F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial. Lancet 383, 705–713. Kapustin AN & Shanahan CM (2016). Emerging roles for vascular smooth muscle cell exosomes in calcification and coagulation. J Physiol 594, 2905–2914. Kapustin AN, Chatrou ML, Drozdov I, Zheng Y, Davidson SM, Soong D, Furmanik M, Sanchis P, De Rosales RT, Alvarez-Hernandez D, Shroff R, Yin X, Muller K, Skepper JN, Mayr M, Reutelingsperger CP, Chester A, Bertazzo S, Schurgers LJ & Shanahan CM (2015). Vascular smooth muscle cell calcification is mediated by regulated exosome secretion. Circ Res 116, 1312–1323. Kapustin AN, Davies JD, Reynolds JL, McNair R, Jones GT, Sidibe A, Schurgers LJ, Skepper JN, Proudfoot D, Mayr M & Shanahan CM (2011). Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization. Circ Res 109, e1–e12. Krohn JB, Hutcheson JD, Martinez-Martinez E & Aikawa E (2016a). Extracellular vesicles in cardiovascular calcification: expanding current paradigms. J Physiol 594, 2895–2903. Krohn JB, Hutcheson JD, Martinez-Martinez E, Irvin WS, Bouten CV, Bertazzo S, Bendeck MP & Aikawa E (2016b). Discoidin domain receptor-1 regulates calcific extracellular vesicle release in vascular smooth muscle cell fibrocalcific response via transforming growth factor-β signaling. Arterioscler Thromb Vasc Biol 36, 525–533.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
2879 Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW & Thery C (2014). Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3, 26913. Martin SS, Blaha MJ, Blankstein R, Agatston A, Rivera JJ, Virani SS, Ouyang P, Jones SR, Blumenthal RS, Budoff MJ & Nasir K (2014). Dyslipidemia, coronary artery calcium, and incident atherosclerotic cardiovascular disease: implications for statin therapy from the multi-ethnic study of atherosclerosis. Circulation 129, 77–86. New SE & Aikawa E (2013). Role of extracellular vesicles in de novo mineralization: an additional novel mechanism of cardiovascular calcification. Arterioscler Thromb Vasc Biol 33, 1753–1758. New SE, Goettsch C, Aikawa M, Marchini JF, Shibasaki M, Yabusaki K, Libby P, Shanahan CM, Croce K & Aikawa E (2013). Macrophage-derived matrix vesicles: an alternative novel mechanism for microcalcification in atherosclerotic plaques. Circ Res 113, 72–77. Osteikoetxea X, N´emeth A, Sodar B, Vukman K & Buzas E (2016). Extracellular vesicles in cardiovascular diseases: are they Jedi or Sith? J Physiol 594, 2881–2894. Raposo G & Stoorvogel W (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200, 373–383. Ruiz JL, Weinbaum S, Aikawa E & Hutcheson JD (2016). Zooming in on the genesis of atherosclerotic plaque microcalcifications. J Physiol 594, 2915–2927. Sitar S, Kejzar A, Pahovnik D, Kogej K, Tusek-Znidaric M, Lenassi M & Zagar E (2015). Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation. Anal Chem 87, 9225–9233. Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M & Giebel B (2011). Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87, 146–150. Vaidyanathan R, Naghibosadat M, Rauf S, Korbie D, Carrascosa LG, Shiddiky MJ & Trau M (2014). Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing. Anal Chem 86, 11125–11132. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ & Lotvall JO (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9, 654–659.
2880 Vengrenyuk Y, Carlier S, Xanthos S, Cardoso L, Ganatos P, Virmani R, Einav S, Gilchrist L & Weinbaum S (2006). A hypothesis for vulnerable plaque rupture due to stress-induced debonding around cellular microcalcifications in thin fibrous caps. Proc Natl Acad Sci USA 103, 14678–14683.
Editorial Vliegenthart R, Oudkerk M, Hofman A, Oei HH, van Dijck W, van Rooij FJ & Witteman JC (2005). Coronary calcification improves cardiovascular risk prediction in the elderly. Circulation 112, 572–577.
J Physiol 594.11
Additional information Competing interests
None declared.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.11 (2016) pp 2877–2880
EDITORIAL
Extracellular vesicles in cardiovascular disease: focus on vascular calcification Elena Aikawa Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
The Journal of Physiology
Email: [email protected]
This issue of The Journal of Physiology brings together four timely review articles (Kapustin & Shanahan, 2016; 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 & Aikawa, 2013; 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 & Stoorvogel, 2013). The society actively advocates for more accurate and standardized purification methods,
particularly used in the development of medical biomarkers and vaccines, and has recently outlined the minimal experimental requirement guidelines for the characterization of extracellular vesicle preparation (Lotvall et al. 2014). Controversy still exists in the field regarding the classification used for vesicular structures released by various cells, which are collectively called extracellular vesicles. The current criteria for the classification of extracellular vesicles involves their biogenesis, size, morphology, lipid/protein ratio and cellular origin. Depending on size and type, extracellular vesicles are broadly classified as exosomes (10–100 nm in diameter), ectosomes (or shedding microvesicles, 50–1000 nm in diameter) and apoptotic bodies (1000–5000 nm in diameter). Extracellular vesicles are also released during the physiological mineralization processes in bones (originally termed matrix vesicles), a discovery made in the mid-1960s by Anderson (1967) and Bonucci (1967). This is another category that should be added to vesicle classification. Matrix vesicles are small membranous structures (30–300 nm in diameter) surrounded by a lipid bilayer, produced by the blebbing of plasma membrane of osteoblasts, chondrocytes and odontoblasts, and they have a high calcification potential (New & Aikawa, 2013). While calcifying extracellular vesicles, involved in ectopic vascular calcification, display morphological similarities to bone-derived matrix vesicles, and were thus presumed to be of similar origin (Kapustin et al. 2011; 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 physio-
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
logical 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 & Shanahan, 2016; 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, ´ N´emeth, Sodar, Vukman, and Buz´as 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
DOI: 10.1113/JP272112
2878 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.
Editorial 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 & Shanahan (2016) provides new and exciting findings from their recent study, which established that extracellular vesicles that originate through the exosomal pathway serve as novel players in pathological vascular calcification. This group showed that mineral imbalance found in patients with chronic kidney disease stimulates the secretion of exosomes by ‘synthetic’ smooth muscle cells probably through cytoskeletal remodelling and by activation of sphingomyelin phosphodiesterase 3 (SMPD3) (Kapustin et al. 2015). Calcium imbalance induces changes in exosome composition, resulting in the accumulation of phosphatidylserine, annexin 6 and MMP-2, which converts exosomes into loci for calcification. It is critical to mention that major differences exist between two currently established in vitro models of smooth muscle cell calcification, which may reflect different pathologies. The model used in the Kapustin study utilizes a high-calcium/phosphate medium that remains predisposed to spontaneous, alkaline phosphatase (ALP)-independent mineral formation. This culture model mimics the pathogenesis of medial calcification in patients with chronic kidney disease (Kapustin et al. 2011; 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).
J Physiol 594.11
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 18 F-labelled sodium fluoride showed accumulation of the 18 F 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
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.11
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. References Aikawa E, Aikawa M, Libby P, Figueiredo JL, Rusanescu G, Iwamoto Y, Fukuda D, Kohler RH, Shi GP, Jaffer FA & Weissleder R (2009). Arterial and aortic valve calcification abolished by elastolytic cathepsin S deficiency in chronic renal disease. Circulation 119, 1785–1794. Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH, Jaffer FA, Aikawa M & Weissleder R (2007a). Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation 116, 2841–2850. Aikawa E, Nahrendorf M, Sosnovik D, Lok VM, Jaffer FA, Aikawa M & Weissleder R (2007b). Multimodality molecular imaging identifies proteolytic and osteogenic activities in early aortic valve disease. Circulation 115, 377–386. Anderson HC (1967). Electron microscopic studies of induced cartilage development and calcification. J Cell Biol 35, 81–101. Balaj L, Atai NA, Chen W, Mu D, Tannous BA, Breakefield XO, Skog J & Maguire CA (2015). Heparin affinity purification of extracellular vesicles. Sci Rep 5, 10266. Bonucci E (1967). Fine structure of early cartilage calcification. J Ultrastruct Res 20, 33–50. Brownlee Z, Lynn KD, Thorpe PE & Schroit AJ (2014). A novel “salting-out” procedure for the isolation of tumor-derived exosomes. J Immunol Methods 407, 120–126. Buzas EI, Gyorgy B, Nagy G, Falus A & Gay S (2014). Emerging role of extracellular vesicles in inflammatory diseases. Nat Rev Rheumatol 10, 356–364. Dweck MR, Chow MW, Joshi NV, Williams MC, Jones C, Fletcher AM, Richardson H, White A, McKillop G, van Beek EJ, Boon NA, Rudd JH & Newby DE (2012). Coronary arterial 18 F-sodium fluoride uptake: a novel marker of plaque biology. J Am Coll Cardiol 59, 1539–1548. Fertig ET, Gherghiceanu M & Popescu LM (2014). Extracellular vesicles release by cardiac telocytes: electron microscopy and electron tomography. J Cell Mol Med 18, 1938–1943.
Editorial Harding CV, Heuser JE & Stahl PD (2013). Exosomes: looking back three decades and into the future. J Cell Biol 200, 367–371. Hjortnaes J, Butcher J, Figueiredo JL, Riccio M, Kohler RH, Kozloff KM, Weissleder R & Aikawa E (2010). Arterial and aortic valve calcification inversely correlates with osteoporotic bone remodelling: a role for inflammation. Eur Heart J 31, 1975–1984. Hutcheson JD, Goettsch C, Bertazzo S, Maldonado N, Ruiz JL, Goh W, Yabusaki K, Faits T, Bouten C, Franck G, Quillard T, Libby P, Aikawa M, Weinbaum S & Aikawa E (2016). Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques. Nat Mater 15, 335–343. Hutcheson JD, Goettsch C, Pham T, Iwashita M, Aikawa M, Singh SA & Aikawa E (2014). Enrichment of calcifying extracellular vesicles using density-based ultracentrifugation protocol. J Extracell Vesicles 3, 25129. Joshi NV, Vesey AT, Williams MC, Shah AS, Calvert PA, Craighead FH, Yeoh SE, Wallace W, Salter D, Fletcher AM, van Beek EJ, Flapan AD, Uren NG, Behan MW, Cruden NL, Mills NL, Fox KA, Rudd JH, Dweck MR & Newby DE (2014). 18 F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial. Lancet 383, 705–713. Kapustin AN & Shanahan CM (2016). Emerging roles for vascular smooth muscle cell exosomes in calcification and coagulation. J Physiol 594, 2905–2914. Kapustin AN, Chatrou ML, Drozdov I, Zheng Y, Davidson SM, Soong D, Furmanik M, Sanchis P, De Rosales RT, Alvarez-Hernandez D, Shroff R, Yin X, Muller K, Skepper JN, Mayr M, Reutelingsperger CP, Chester A, Bertazzo S, Schurgers LJ & Shanahan CM (2015). Vascular smooth muscle cell calcification is mediated by regulated exosome secretion. Circ Res 116, 1312–1323. Kapustin AN, Davies JD, Reynolds JL, McNair R, Jones GT, Sidibe A, Schurgers LJ, Skepper JN, Proudfoot D, Mayr M & Shanahan CM (2011). Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization. Circ Res 109, e1–e12. Krohn JB, Hutcheson JD, Martinez-Martinez E & Aikawa E (2016a). Extracellular vesicles in cardiovascular calcification: expanding current paradigms. J Physiol 594, 2895–2903. Krohn JB, Hutcheson JD, Martinez-Martinez E, Irvin WS, Bouten CV, Bertazzo S, Bendeck MP & Aikawa E (2016b). Discoidin domain receptor-1 regulates calcific extracellular vesicle release in vascular smooth muscle cell fibrocalcific response via transforming growth factor-β signaling. Arterioscler Thromb Vasc Biol 36, 525–533.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
2879 Lotvall J, Hill AF, Hochberg F, Buzas EI, Di Vizio D, Gardiner C, Gho YS, Kurochkin IV, Mathivanan S, Quesenberry P, Sahoo S, Tahara H, Wauben MH, Witwer KW & Thery C (2014). Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 3, 26913. Martin SS, Blaha MJ, Blankstein R, Agatston A, Rivera JJ, Virani SS, Ouyang P, Jones SR, Blumenthal RS, Budoff MJ & Nasir K (2014). Dyslipidemia, coronary artery calcium, and incident atherosclerotic cardiovascular disease: implications for statin therapy from the multi-ethnic study of atherosclerosis. Circulation 129, 77–86. New SE & Aikawa E (2013). Role of extracellular vesicles in de novo mineralization: an additional novel mechanism of cardiovascular calcification. Arterioscler Thromb Vasc Biol 33, 1753–1758. New SE, Goettsch C, Aikawa M, Marchini JF, Shibasaki M, Yabusaki K, Libby P, Shanahan CM, Croce K & Aikawa E (2013). Macrophage-derived matrix vesicles: an alternative novel mechanism for microcalcification in atherosclerotic plaques. Circ Res 113, 72–77. Osteikoetxea X, N´emeth A, Sodar B, Vukman K & Buzas E (2016). Extracellular vesicles in cardiovascular diseases: are they Jedi or Sith? J Physiol 594, 2881–2894. Raposo G & Stoorvogel W (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200, 373–383. Ruiz JL, Weinbaum S, Aikawa E & Hutcheson JD (2016). Zooming in on the genesis of atherosclerotic plaque microcalcifications. J Physiol 594, 2915–2927. Sitar S, Kejzar A, Pahovnik D, Kogej K, Tusek-Znidaric M, Lenassi M & Zagar E (2015). Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation. Anal Chem 87, 9225–9233. Sokolova V, Ludwig AK, Hornung S, Rotan O, Horn PA, Epple M & Giebel B (2011). Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces 87, 146–150. Vaidyanathan R, Naghibosadat M, Rauf S, Korbie D, Carrascosa LG, Shiddiky MJ & Trau M (2014). Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing. Anal Chem 86, 11125–11132. Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ & Lotvall JO (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9, 654–659.
2880 Vengrenyuk Y, Carlier S, Xanthos S, Cardoso L, Ganatos P, Virmani R, Einav S, Gilchrist L & Weinbaum S (2006). A hypothesis for vulnerable plaque rupture due to stress-induced debonding around cellular microcalcifications in thin fibrous caps. Proc Natl Acad Sci USA 103, 14678–14683.
Editorial Vliegenthart R, Oudkerk M, Hofman A, Oei HH, van Dijck W, van Rooij FJ & Witteman JC (2005). Coronary calcification improves cardiovascular risk prediction in the elderly. Circulation 112, 572–577.
J Physiol 594.11
Additional information Competing interests
None declared.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
