Extracellular vesicles in cardiovascular ... - Wiley Online Library

2895

J Physiol 594.11 (2016) pp 2895–2903

SYMPOSIUM REVIEW

Extracellular vesicles in cardiovascular calcification: expanding current paradigms Jona B. Krohn1 , Joshua D. Hutcheson2 , Eduardo Mart´ınez-Mart´ınez1 and Elena Aikawa1,2 1 2

Center for Excellence in Vascular Biology, Harvard Medical School, Boston, MA, USA Center for Interdisciplinary Cardiovascular Sciences, Harvard Medical School, Boston, MA, USA

Homeostatic State

Vascular smooth muscle cell

The Journal of Physiology

Non-calcifying EVs

Osteogenic differentiation

Fetuin-A MGP miR30,125b, 126,143/5,155

Calcifying EVs Disease State

EV

– OM + Annexins Ca2+ Pi ALP

MVB

MV

Calcification of collagen matrix

Osteogenic differentiation

Jona B. Krohn is a student of medicine at the University of Heidelberg, Germany. Following clinical experience at Loyola University Stritch School of Medicine in Chicago, USA and Medical University of Vienna, Austria, he joined the laboratory of Dr Elena Aikawa in 2014 to investigate the mechanisms of extracellular vesicle release in vascular calcification. Elena Aikawa is an Associate Professor of Medicine at Harvard Medical School, Principle Investigator at the Centre for Excellence in Vascular Biology and Director of the Vascular Biology Program at the Centre for Interdisciplinary Cardiovascular Sciences at Brigham and Women’s Hospital. Dr Aikawa is an Editorial Board Member of several scientific journals and has authored over 150 manuscripts on cardiovascular pathobiology. Her current research focuses on the mechanisms of vascular calcification and calcific aortic valve disease.

This review was presented at the symposium “Extracellular vesicles, exosomes and microparticles in cardiovascular disease”, which took place at Physiology 2015, Cardiff, UK between 6–8 July 2015. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

DOI: 10.1113/JP271338

2896

J. B. Krohn and others

J Physiol 594.11

Abstract Vascular calcification is a major contributor to the progression of cardiovascular disease, one of the leading causes of death in industrialized countries. New evidence on the mechanisms of mineralization identified calcification-competent extracellular vesicles (EVs) derived from smooth muscle cells, valvular interstitial cells and macrophages as the mediators of calcification in diseased heart valves and atherosclerotic plaques. However, the regulation of EV release and the mechanisms of interaction between EVs and the extracellular matrix leading to the formation of destabilizing microcalcifications remain unclear. This review focuses on current limits in our understanding of EVs in cardiovascular disease and opens up new perspectives on calcific EV biogenesis, release and functions within and beyond vascular calcification. We propose that, unlike bone-derived matrix vesicles, a large population of EVs implicated in cardiovascular calcification are of exosomal origin. Moreover, the milieu-dependent loading of EVs with microRNA and calcification inhibitors fetuin-A and matrix Gla protein suggests a novel role for EVs in intercellular communication, adding a new mechanism to the pathogenesis of vascular mineralization. Similarly, the cell type-dependent enrichment of annexins 2, 5 or 6 in calcifying EVs posits one of several emerging factors implicated in the regulation of EV release and calcifying potential. This review aims to emphasize the role of EVs as essential mediators of calcification, a major determinant of cardiovascular mortality. Based on recent findings, we pinpoint potential targets for novel therapies to slow down the progression and promote the stability of atherosclerotic plaques. (Received 30 September 2015; accepted after revision 30 November 2015; first published online 29 January 2016) Corresponding author E. Aikawa: Harvard Medical School, Cardiovascular Medicine, Brigham and Women’s Hospital, 77 Ave Louis Pasteur, NRB-741, Boston, MA 02115, USA. Email: [email protected] Abstract figure legend Vessel wall-derived extracellular vesicles (EVs) are selectively loaded with calcification inhibitors fetuin-A, matrix Gla protein (MGP) and anti-osteogenic microRNA (miRNA) (green box) or pro-calcific annexins, alkaline phosphatase (ALP), calcium (Ca2+ ) and inorganic phosphate (Pi ) (red box). Calcifying conditions (e.g. culture in osteogenic media, OM) increase absolute EV release and EV calcific potential by shifting the balance towards increased expression of pro-calcific factors and suppression of calcification inhibitors in EVs. EVs may originate from the exosomal pathway (1), as multivesicular bodies (MVB; observed but not confirmed in smooth muscle cells, SMCs) (2), or by budding off the cell membrane (not confirmed in SMCs) (3). While the exocytosis pathway is an established mechanism in SMC-mediated calcific EV release, the exact conditions and potential context specificity of these pathways of EV biogenesis are still unclear. Under physiological conditions, non-calcifying EVs transfer inhibitory factors and regulatory miRNA as a form of paracrine signalling, preventing osteogenic differentiation of adjacent cells. Under calcifying conditions, however, calcification-competent EVs are sequestered in the fibrillar matrix, nucleating calcium phosphate mineral. Dysregulated paracrine signalling resulting in an imbalance of calcification inhibitors and miRNA leads to increased osteogenic differentiation of vessel wall cells, expediting vascular calcification. Abbreviations ALP, alkaline phosphatase; EV, extracellular vesicle; miRNA, microRNA; MV, matrix vesicle; MVB, multivesicular body; SMC, smooth muscle cell; VEC, valvular endothelial cell; VIC, valvular interstitial cell; TEM, transmission electron microscopy.

Introduction

Vascular calcification predicts cardiovascular morbidity and mortality (Tolle et al. 2015). Clinical studies using advanced imaging modalities to visualize coronary artery calcium have found a strong correlation between calcific burden, progression of arteriosclerosis and overall survival (Budoff et al. 2007). However, the relationship between calcification and atherosclerotic plaque stability determining the risk of plaque rupture and subsequent cardiovascular events remains controversial (Otsuka et al. 2014). Emerging evidence identifies size and location

of calcific deposits as two major variables categorizing vascular calcification with respect to its effect on plaque stability (Hutcheson et al. 2014). While large calcifications detectable by coronary computed tomography (CT) may stabilize the plaque (Lin et al. 2006), microcalcifications in the collagen-poor fibrous cap that are not detectable by conventional imaging methods (Aikawa et al. 2007; Dweck et al. 2012) have been shown to promote plaque rupture by exerting mechanical stress on the surrounding tissue (Kelly-Arnold et al. 2013). Clinical data support the theory of a negative correlation between atherosclerotic calcification density and cardiovascular C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

J Physiol 594.11

Extracellular vesicles in calcification

risk (Criqui et al. 2014), thus emphasizing the need for a more comprehensive understanding of the mechanisms involved in calcific plaque formation. Ultrastructural analyses have identified the presence of extracellular vesicles (EVs) in calcified human aortic valves (Fig. 1F) and medial arterial calcifications (Kim, 1976) as well as in atherosclerotic intimal plaques (New et al. 2013). EVs released by smooth muscle cells (SMCs) (Kapustin et al. 2011) and macrophages (Fig. 1D) (New et al. 2013) in the vessel wall interact with fibrillar collagen and may thus serve as nucleating foci for calcific mineral crystallization in early calcification; however, the mechanisms of interaction between EVs and collagen driving the formation of either macro- or microcalcifications remain unknown. Analyses of calcifying EVs identified a subset of proteins enriched in SMC-derived EVs that may facilitate their potential to calcify. Among the EV-specific proteins, alkaline phosphatase, members of the annexin family, and matrix metalloproteinase-2 (MMP-2) were found to play a prominent role in the process of matrix calcification, releasing required substrates and forming intermediates known to trigger calcium phosphate precipitation (Kapustin et al. 2011). Despite extensive work on the structural makeup of EVs implicated in vascular calcification, our knowledge of the cellular mechanisms governing EV release and the role of EVs in cell–matrix homeostasis is limited. This review aims to discuss our current understanding of EVs in vascular calcification and the impact of EV–matrix interaction on the formation of calcific mineral and stability of atherosclerotic plaques. Moreover, we provide new perspectives on the origin and identity of calcifying EVs and the regulatory mechanisms involved in EV release and accumulation, opening a field of novel therapeutic targets for ectopic cardiovascular calcification. The controversy of vesicle origin

Comparative analyses of the plasma membranes of EVs and their putative releasing cells in different physiological and pathological contexts have raised several theories on vesicle origin. In physiological mineralization processes, epiphyseal chondrocytes and bone osteoblasts were shown to release EVs (originally termed matrix vesicles (MVs)) from budding at specific sites of their plasma membrane (Leach et al. 1995). This MV-specific process may thus account for the differential enrichment of acidic phospholipids and glycosylphosphatidyl-inositol (GPI)-anchored alkaline phosphatase among numerous other elements on the MV membrane (Golub, 2009). While calcifying EVs involved in cardiovascular calcification exhibit structural similarity to osteoblast-derived MVs (Fig. 1E) and were thus assumed to be of similar origin (Kapustin et al. 2011), more recent C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

2897

work on the mechanisms of EV release in the context of pathological calcification points at the existence of the exosomal pathway as the origin of SMC-derived EVs (Kapustin et al. 2015). Proteomics of SMC-derived MVs revealed the presence of specific Rab GTPases (Kapustin et al. 2011), a family of proteins implicated in regulated vesicular trafficking (Stenmark, 2009). Interestingly, a recent study on EV biogenesis in vascular SMCs using fluorescently labelled fetuin-A showed EVs originating through multivesicular body and endosomal stages, suggesting SMC-derived EVs to be the product of regulated exocytosis (Kapustin et al. 2015). Kapustin et al. found that the observed EVs bear structural resemblance to exosomes released from non-mineralizing cell lines, and that specific inhibition of exosome release in vascular SMCs abrogated their mineralization capacity in vitro. The biogenesis of EVs by mineralizing cells is observed to follow two distinguishable pathways: a process of direct EV budding from the plasma membrane, and an endosomal pathway via multivesicular bodies fusing with the plasma membrane and releasing membrane-contained EVs termed exosomes (Fig. 1A–C). Present evidence in the literature propounds a cell-specific release mechanism, with plasma membrane-derived MVs predominant in chondrocytes (Kirsch et al. 1997) and CD68-positive macrophages (New et al. 2013) (Fig. 1D), and exosomes mediating SMC calcification (Kapustin et al. 2015). The exosomal pathway can be further divided into a multivesicular body (MVB)–plasma membrane fusion mechanism (Kapustin et al. 2015) and the direct release of MVB-like structures into the extracellular space (Fig. 1B and C), both mechanisms implicated in SMC-mediated calcification. Moreover, the presence of exosomes was observed in the context of bone mineralization (Xiao et al. 2007; Shapiro et al. 2015), underlining the importance of exosomes as emerging components of calcification in both physiological and pathological contexts. In calcified vasculature from dialysis patients, the formation of multivesicular bodies and expression of exosome-specific marker CD63 (Pols & Klumperman, 2009) preceded and co-localized with calcific deposition. However, vesicles isolated from human atherosclerotic plaques bear only a slight resemblance to the EVs isolated from SMCs (Kapustin et al. 2015) and macrophages (New et al. 2013) in vitro (Mayr et al. 2009). This finding is best explained by the versatile origin of EVs found in human atherosclerotic plaques, deriving mostly from leukocytes red blood cells and SMCs (Leroyer et al. 2007). Taken together, the present evidence on EV origin in vascular calcification strengthens the hypotheses that (1) EV formation follows different pathways of origin that may occur within specific cell types or simultaneously within the same cell population, and (2) calcification-competent EVs of exosomal origin have a role in processes beyond calcification.

2898


Calcifying extracellular vesicles and the emerging role of microRNA: intercellular communication gone awry?

The emerging role of microRNA (miRNA) in intercellular communication and cardiovascular disease has given rise to a potential new perspective on EV-mediated vascular calcification. miRNAs regulate biological processes by binding to specific sequences on the 3 -untranslated region of messenger RNA (mRNA) to influence mRNA stability and protein synthesis (Bartel, 2009). The presence of circulating miRNA has been verified both in an EV-bound form (Valadi et al. 2007) and as an EV-independent protein complex (Arroyo et al. 2011), and compared to SMCs, miRNAs are concentrated in EVs and exosomes, making up as much as 50% of total RNA content (Chaturvedi et al. 2015). Several studies point towards a selective loading mechanism of miRNA into EVs (Diehl et al. 2012; Hergenreider et al. 2012; Lo Cicero et al. 2015), accounting for the vast differences of miRNA subsets present in SMCs and EVs (Chaturvedi et al. 2015). Of the dysregulated miRNAs loaded into calcifying EVs by SMCs, a significant number was found to target genes involved in osteogenic differentiation. miRNAs 30, 125-b, 143, 145 and 155 all influence the expression of a specific set of osteogenic markers such as Smad1, RUNX-2, ALP and osterix (Mizuno et al. 2008; Goettsch et al. 2011; Wu et al. 2012; Chen et al. 2013),and altered concentrations

J Physiol 594.11

of these miRNAs in EVs result in shifts in calcium and MAPK signalling pathways implicated in SMC-mediated calcification (Chaturvedi et al. 2015). The novel concept of an EV-mediated intercellular crosstalk has recently been shown to apply to other cell populations besides SMCs involved in cardiovascular disease. An additional dimension of SMC phenotype regulation comprises endothelial cell-derived EVs selectively loaded with miRNAs 126 (Zernecke et al. 2009) and 143/145 (Hergenreider et al. 2012) that mediate paracrine signalling across cells within the vessel wall. Furthermore, in a recent study we showed intracellular uptake and subsequent perinuclear enrichment of EVs in a sheep valvular interstitial cell model (Fig. 2), pointing towards a role of EVs in cardiovascular intercellular communication. Current knowledge of the content of vessel wall-derived EVs, including miRNA, chaperones, inflammatory factors and other signalling molecules (Kapustin et al. 2011), suggests a novel role of calcifying EVs in cell–cell communication. In the context of cardiovascular disease, this new evidence may expand today’s understanding of EVs in vascular calcification and the contribution of EVs to disease progression. We propose that EVs are in fact intercellular messengers erroneously caught in the extracellular matrix, adding a new dimension to EV-mediated calcific plaque formation. Firstly, as

A

B

C

D

E

F

CD68 Figure 1. Ultrastructural analysis of EVs implicated in cardiovascular disease suggests heterogeneous populations of different origin and calcification potential A–C, transmission electron microscopy (TEM) of valvular interstitial cells (VICs) from a calcified aortic valve (A) and SMCs stressed with calcifying media (B and C) shows cytosolic multivesicular body-like structures (arrowheads) that are subsequently released from the plasma membrane. Scale bar: 1 µm (A), 500 nm (B and C). D, immunogold TEM of a macrophage releasing CD68-positive MVs. Scale bar: 200 nm; adapted from New et al. (2013). E, TEM of human calcified aortic valve tissue reveals double membrane-enclosed EVs (arrowhead) nucleating mineral in between collagen fibres. Scale bar: 200 nm. F, density-dependent colour scanning electron microscopy (DDC-SEM) of a human calcified aortic valve with vesicle aggregates (arrowheads) in spatial association with larger calcific nodules (arrow). Scale bar: 2 µm.

C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society


J Physiol 594.11

released EVs become entrapped in the extracellular matrix within the plaque, they act as nucleating foci for the formation of microcalcifications. Secondly, the released EVs are prevented from reaching their target cells, causing dysregulated gene expression patterns that result in enhanced osteogenic differentiation. EVs thus initiate and propagate vascular calcific responses through two independent mechanisms. Cellular and extracellular regulatory mechanisms of vesicle release and accumulation

While the connection between EV release and calcific plaque formation has been demonstrated by several studies, our current understanding of the mechanisms governing EV release and accumulation in the extracellular matrix remains sparse. Similar to miRNA content of EVs, calcification-competent EVs derived from vascular SMCs contain a specific profile of enriched proteins different from non-calcifying EVs, suggesting a regulated packaging mechanism (Shanahan et al. 2011). On the cellular level, the release of exosomes from multivesicular bodies in SMCs is initiated by hydrolysis of membrane-bound sphingomyelins through

A

B

PKH26+DAPI

C

D

Exo-Green+DAPI

Figure 2. Transfer of VIC-derived EVs to valvular endothelial cell (VEC) cultures reveals intercellular vesicle transfer through the endosomal pathway A, fluorescently labelled VIC-derived EVs with membrane-bound PKH26 are taken up by VECs and progressively enriched in the perinuclear space, recapitulating retrograde transport of endosomes. C, fluorescence labelling of internal exosome proteins using Exo-Green shows similar patterns of perinuclear enrichment of exosome-contained proteins. B and D, PBS control. ×40, scale bar: 10 µm. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

2899

sphingomyelin phosphodiesterase-3 (SMPD3) (Kapustin et al. 2015). Interestingly, this reaction has also been independently implicated in the loading mechanism of miRNA into EVs (Hergenreider et al. 2012). Consistently, the study by Kapustin et al. shows that SMCs grown in calcifying media had increased gene expression of SMPD3, and selective inhibition of its enzymatic activity was found to significantly decrease the release of SMC-derived calcification-competent EVs. In a high-phosphate environment mimicking the pro-calcific conditions present in chronic kidney disease, the role of inflammation in vascular calcification was found to be implicated in exosome release through the effect of cytokines such as TGF-β1, IL-6 and IL-10, which decreased, and platelet-derived growth factor and tumour necrosis factor-α, which increased the rate of exocytosis in vascular SMCs. Several mechanisms of EV–matrix interaction were stipulated to regulate EV accumulation and vascular mineralization. Fetuin-A, matrix Gla protein (MGP) and S100A9, which are selectively enriched in EVs, have emerged in recently published reports as putative regulators of EV-mediated mineralization. Fetuin-A and MGP block calcification by inhibiting mineral nucleation (Reynolds et al. 2004). Conversely, the disruption of calcium homeostasis leading to increased release of calcifying EVs by SMCs is concomitant with decreased MGP content in EVs (Kapustin et al. 2011). In macrophage-derived EVs, S100A9 and annexin 5 were similarly induced upon calcium/phosphate stimulation and co-localized on the surface of EVs to form a nucleation complex for calcific mineral (New et al. 2013). Further in vivo analysis revealed upregulated expression of S100A9 and annexin 5 in calcified plaques of ApoE−/− mice and human atheromata, underlining the essential role of these EV-derived factors in the propagation of matrix mineralization. As new evidence of the EV-resident factors regulating extracellular matrix mineralization emerges, the mechanisms of EV–matrix interaction that determine the formation and spatial properties of extracellular mineral deposits remain poorly understood. In atherosclerotic plaque formation, the accumulation of collagen constituting vascular fibrotic response has been shown to sequentially precede calcification and may thus act as a scaffold to initiate mineralization (Hsu et al. 2004). A recent study on the role of collagen in calcifying EV accumulation identified an inverse relationship between collagen density and volume of resulting mineral deposits in the extracellular space, establishing a mechanism of EV–matrix interaction that may predict the stability of atherosclerotic plaques (Hutcheson et al. 2016). Moreover, our current research on the cell–matrix axis in calcifying EV release points towards an additional regulatory role of collagen on the cell level, activating feedback mechanisms

2900


to control quantitative EV release and the calcifying potential of EVs. Further studies are needed to disclose how the aforementioned factors are interconnected to regulate vascular fibrocalcific responses on the cell, matrix and EV level and what role these mechanisms play in the formation of calcified atherosclerotic plaques. Annexin complexes as nucleation sites for mineralization: a novel regulatory mechanism of calcification?

The connection between members of the annexin family and the process of cardiovascular calcification is a well-known concept that has been demonstrated in multiple contexts. However, the mechanisms by which annexins initiate and/or propagate mineralization of the vessel wall matrix are poorly characterized. Annexins are a family of tissue non-specific proteins with 12 members expressed in mammals (Genetos et al. 2014). Depending upon isoform and cell type, annexins may perform a dual-mode function as voltage-gated Ca2+ channels or Ca2+ -dependent anionic phospholipid binding proteins (Gillette & Nielsen-Preiss, 2004) contributing to essential cell functions, including exo- and endocytosis, plasma membrane organization, lipid raft generation, regulation of ion channels and cytokine release (Gerke et al. 2005). In an externalized form, certain annexins have been implicated in extracellular roles such as inflammation and apoptosis, coagulation and fibrinolysis, and enchondral ossification (Belluoccio et al. 2010). EVs derived from different cell types have shown selective enrichment of annexins 2, 5 (Fig. 3) and 6 with a specificity of annexin 5

A

cells NM

vesicles OM

NM

OM

annexin 2 annexin 5 GAPDH

B

Anx5 Anx5 β-actin Ctl

Ca/P

Figure 3. Differential expression of annexins 2 and 5 in calcification-competent EVs A, immunoblot of VIC-derived cell and EV lysates reveals predominant expression of annexin 2 in EVs under calcifying conditions. NM-normal media; OM-osteogenic media. B, Western blot for annexin 5 shows enrichment in macrophage-derived MVs that is verified by immunogold TEM. Scale bar: 200 nm; reproduced with permission from New et al. (2013).

J Physiol 594.11

for macrophages (New et al. 2013) and annexin 6 for SMC-derived EVs (Kapustin et al. 2015). Furthermore, induction of MV-bound annexin 6 in calcifying media and reversal of extracellular matrix mineralization through annexin 6 knockdown suggests a stimulatory effect of annexin 6 on EV-mediated nucleation core formation. Both annexins 5 and 6 demonstrated a tendency to associate with phosphatidylserine (PS) to form complexes in lipid raft microdomains on the cell surface and in EVs. As mentioned earlier, a complex of annexin 5, PS and S100A9 on the surface of macrophage-derived MVs facilitates mineralization, and several annexin-deficiency models underlined a significant role of annexins 2 and 5 in the process of physiological calcification, osteogenic differentiation and matrix mineralization in the osteoblast lineage (Genetos et al. 2014). A recent focus on annexin 2 in mineralization-competent EVs revealed a notable role of annexin 2 in the recruitment of calcification regulators in EV biogenesis. In the process of physiological mineralization, annexin 2 has been ascribed an essential function in the organization and dynamics of lipid raft microdomains, specialized areas on the plasma membrane that are discussed as one of the potential origins of EVs (Gerke & Moss, 2002). In its alleged role as master regulator of EV loading, annexin 2 binds calcification inhibitor fetuin-A on the SMC membrane, leading to its endocytosis and thus preventing fetuin-A from being enriched in EVs (Chen et al. 2008). The hypothesis of annexin 2 as a propagator of vascular calcification is further supported by the co-expression of annexins 2 and 5 as well as ALP in calcifying EVs that was inversely correlated with the expression of fetuin-A. This observation made by Chen et al. in a vascular SMC model concurs with a study of annexin 2 in osteoblastic cells, where annexin 2 overexpression enhanced ALP activity and calcific deposition, and disruption of either annexin 2 or lipid rafts conversely abrogated mineralization (Gillette & Nielsen-Preiss, 2004). As a growing body of evidence corroborates the role of annexins in EV-mediated vascular calcification, our current understanding of the molecular mechanisms of annexin-mediated EV release and mineral nucleation remains nebulous. The previously described dual function of annexins as Ca2+ channels with a unique Ca2+ -dependent potential to bind phospholipids has moved into the focus of controversy in current literature. In vascular SMCs, EV-mediated mineralization was found to be independent of the Ca2+ channel activity of abundantly present annexin 6 (Kapustin et al. 2011), consistent with previous findings on an essential role of the physicochemical structure of membrane-bound annexins in mineral nucleation (Genge et al. 2007). In a mineralization model using cartilage and chondrocyte-derived MVs, however, Ca2+ influx through


J Physiol 594.11


annexins 2, 5 and 6 was deemed crucial to MV biogenesis and intraluminal crystal growth (Kirsch et al. 2000). Further investigation may disclose differential functions of annexins depending on the stage of EV formation and maturation that would explain present controversies in the function of annexins in EV-mediated vascular calcification. Concluding remarks and future perspectives

The introduction of EVs as mediators of cell-induced matrix mineralization marked the discovery of an essential mechanism in the pathogenesis of cardiovascular disease. However, as the connection between calcifying EVs and vascular calcification proceeds to gain recognition, a number of emerging questions regarding the mechanisms of vesicle release and deposition in the extracellular space remain unanswered. This review aims to pinpoint currently discussed controversies regarding the origin, release and ultrastructural makeup of EVs associated with pathological vascular calcification in the process of atherosclerotic plaque formation. Conclusively, we propose that a large subpopulation of the EVs found in calcified vascular lesions follow a different pathway of origin from MVs implicated in physiological bone formation. Unlike osteoblast- or chondrocyte-derived MVs, which are known to bleb off specialized domains on the cell membrane, EVs observed in atherosclerotic lesions and calcified aortic valves could be of at least two different origins: exosomes derived from multivesicular bodies through the exocytosis pathway, and EVs budding from the plasma membrane of its parental cell. Although the mechanisms of calcific EV release are still insufficiently understood, the recent emergence of novel regulators like fetuin-A, MGP, annexins and miRNA may be a major step forward in the search for potent therapeutic targets for cardiovascular disease. The novel concept of EVs as intercellular communicators fine-tuning the osteogenic phenotype of vessel wall SMCs through miRNA may particularly shift the focus of interest in future research on mechanisms and therapeutics of cardiovascular calcification. Pioneer investigations into these newly established targets yielded promising in vitro and in vivo results on pharmacological interventions of miRNA (Rayner et al. 2011; Schober et al. 2014) and annexins using the established Ca2+ channel blocker verapamil (Chen et al. 2010), reporting a significant impact on calcific MV biogenesis and atherosclerotic lesion formation. Future work aimed at filling the gaps in our current knowledge of EVs in calcific plaque formation may thus lead to the launch of a new generation of targeted therapies allowing us to effectively prevent vascular calcification and promote the structural stability of atherosclerotic plaques.


2901

References Aikawa E, Nahrendorf M, Figueiredo JL, Swirski FK, Shtatland T, Kohler RH, Jaffer FA, Aikawa M & Weissleder R (2007). Osteogenesis associates with inflammation in early-stage atherosclerosis evaluated by molecular imaging in vivo. Circulation 116, 2841–2850. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, Mitchell PS, Bennett CF, Pogosova-Agadjanyan EL, Stirewalt DL, Tait JF & Tewari M (2011). Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci USA 108, 5003–5008. Bartel DP (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233. Belluoccio D, Grskovic I, Niehoff A, Schlotzer-Schrehardt U, Rosenbaum S, Etich J, Frie C, Pausch F, Moss SE, Poschl E, Bateman JF & Brachvogel B (2010). Deficiency of annexins A5 and A6 induces complex changes in the transcriptome of growth plate cartilage but does not inhibit the induction of mineralization. J Bone Miner Res 25, 141–153. Budoff MJ, Shaw LJ, Liu ST, Weinstein SR, Mosler TP, Tseng PH, Flores FR, Callister TQ, Raggi P & Berman DS (2007). Long-term prognosis associated with coronary calcification: observations from a registry of 25,253 patients. J Am Coll Cardiol 49, 1860–1870. Chaturvedi P, Chen NX, O’Neill K, McClintick JN, Moe SM & Janga SC (2015). Differential miRNA expression in cells and matrix vesicles in vascular smooth muscle cells from rats with kidney disease. PLoS One 10, e0131589. Chen NX, Kiattisunthorn K, O’Neill KD, Chen X, Moorthi RN, Gattone VH 2nd, Allen MR & Moe SM (2013). Decreased microRNA is involved in the vascular remodeling abnormalities in chronic kidney disease (CKD). PLoS One 8, e64558. Chen NX, Kircelli F, O’Neill KD, Chen X & Moe SM (2010). Verapamil inhibits calcification and matrix vesicle activity of bovine vascular smooth muscle cells. Kidney Int 77, 436–442. Chen NX, O’Neill KD, Chen X & Moe SM (2008). Annexin-mediated matrix vesicle calcification in vascular smooth muscle cells. J Bone Miner Res 23, 1798–1805. Criqui MH, Denenberg JO, Ix JH, McClelland RL, Wassel CL, Rifkin DE, Carr JJ, Budoff MJ & Allison MA (2014). Calcium density of coronary artery plaque and risk of incident cardiovascular events. JAMA 311, 271–278. Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, Ziemann M, Helbing T, El-Osta A, Jowett JB & Peter K (2012). Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovasc Res 93, 633–644. 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 18F-sodium fluoride uptake: a novel marker of plaque biology. J Am Coll Cardiol 59, 1539–1548. Genetos DC, Wong A, Weber TJ, Karin NJ & Yellowley CE (2014). Impaired osteoblast differentiation in annexin A2and -A5-deficient cells. PLoS One 9, e107482.

2902


Genge BR, Wu LN & Wuthier RE (2007). In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine. J Biol Chem 282, 26035–26045. Gerke V, Creutz CE & Moss SE (2005). Annexins: linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol 6, 449–461. Gerke V & Moss SE (2002). Annexins: from structure to function. Physiol Rev 82, 331–371. Gillette JM & Nielsen-Preiss SM (2004). The role of annexin 2 in osteoblastic mineralization. J Cell Sci 117, 441–449. Goettsch C, Rauner M, Pacyna N, Hempel U, Bornstein SR & Hofbauer LC (2011). miR-125b regulates calcification of vascular smooth muscle cells. Am J Pathol 179, 1594–1600. Golub EE (2009). Role of matrix vesicles in biomineralization. Biochim Biophys Acta 1790, 1592–1598. Hergenreider E, Heydt S, Treguer K, Boettger T, Horrevoets AJ, Zeiher AM, Scheffer MP, Frangakis AS, Yin X, Mayr M, Braun T, Urbich C, Boon RA & Dimmeler S (2012). Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat Cell Biol 14, 249–256. Hsu HH, Tawfik O & Sun F (2004). Mechanism of dystrophic calcification in rabbit aortas: temporal and spatial distributions of calcifying vesicles and calcification-related structural proteins. Cardiovasc Pathol 13, 3–10. Hutcheson JD, Goettsch C, Bertazzo S, Maldonado N, Ruiz JL, Goh W, Yabusaki K, Faits T, Bouten C, Libby P, Aikawa M, Weinbaum S & Aikawa E (2016). High-resolution microscopic visualization of vascular calcification genesis, growth, and association with collagen in atherosclerotic plaques. Nat Mat 15, 335–343. Hutcheson JD, Maldonado N & Aikawa E (2014). Small entities with large impact: microcalcifications and atherosclerotic plaque vulnerability. Curr Opin Lipidol 25, 327–332. 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, e1–12. Kelly-Arnold A, Maldonado N, Laudier D, Aikawa E, Cardoso L & Weinbaum S (2013). Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries. Proc Natl Acad Sci USA 110, 10741–10746. Kim KM (1976). Calcification of matrix vesicles in human aortic valve and aortic media. Fed Proc 35, 156–162. Kirsch T, Harrison G, Golub EE & Nah HD (2000). The roles of annexins and types II and X collagen in matrix vesicle-mediated mineralization of growth plate cartilage. J Biol Chem 275, 35577–35583.

J Physiol 594.11

Kirsch T, Nah HD, Shapiro IM & Pacifici M (1997). Regulated production of mineralization-competent matrix vesicles in hypertrophic chondrocytes. J Cell Biol 137, 1149–1160. Leach RJ, Schwartz Z, Johnson-Pais TL, Dean DD, Luna M & Boyan BD (1995). Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: evidence for independent membrane biogenesis. J Bone Miner Res 10, 1614–1624. Leroyer AS, Isobe H, Leseche G, Castier Y, Wassef M, Mallat Z, Binder BR, Tedgui A & Boulanger CM (2007). Cellular origins and thrombogenic activity of microparticles isolated from human atherosclerotic plaques. J Am Coll Cardiol 49, 772–777. Lin TC, Tintut Y, Lyman A, Mack W, Demer LL & Hsiai TK (2006). Mechanical response of a calcified plaque model to fluid shear force. Ann Biomed Eng 34, 1535–1541. Lo Cicero A, Stahl PD & Raposo G (2015). Extracellular vesicles shuffling intercellular messages: for good or for bad. Curr Opin Cell Biol 35, 69–77. Mayr M, Grainger D, Mayr U, Leroyer AS, Leseche G, Sidibe A, Herbin O, Yin X, Gomes A, Madhu B, Griffiths JR, Xu Q, Tedgui A & Boulanger CM (2009). Proteomics, metabolomics, and immunomics on microparticles derived from human atherosclerotic plaques. Circ Cardiovasc Genet 2, 379–388. Mizuno Y, Yagi K, Tokuzawa Y, Kanesaki-Yatsuka Y, Suda T, Katagiri T, Fukuda T, Maruyama M, Okuda A, Amemiya T, Kondoh Y, Tashiro H & Okazaki Y (2008). miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation. Biochem Biophys Res Commun 368, 267–272. 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. Otsuka F, Sakakura K, Yahagi K, Joner M & Virmani R (2014). Has our understanding of calcification in human coronary atherosclerosis progressed? Arterioscler Thromb Vasc Biol 34, 724–736. Pols MS & Klumperman J (2009). Trafficking and function of the tetraspanin CD63. Exp Cell Res 315, 1584–1592. Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van Gils JM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA & Moore KJ (2011). Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest 121, 2921–2931. Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL & Shanahan CM (2004). Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD. J Am Soc Nephrol 15, 2857–2867. Schober A, Nazari-Jahantigh M, Wei Y, Bidzhekov K, Gremse F, Grommes J, Megens RT, Heyll K, Noels H, Hristov M, Wang S, Kiessling F, Olson EN & Weber C (2014). MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat Med 20, 368–376. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society

J Physiol 594.11


Shanahan CM, Crouthamel MH, Kapustin A & Giachelli CM (2011). Arterial calcification in chronic kidney disease: key roles for calcium and phosphate. Circ Res 109, 697–711. Shapiro IM, Landis WJ & Risbud MV (2015). Matrix vesicles: Are they anchored exosomes? Bone 79, 29–36. Stenmark H (2009). Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10, 513–525. Tolle M, Reshetnik A, Schuchardt M, Hohne M & van der Giet M (2015). Arteriosclerosis and vascular calcification: causes, clinical assessment and therapy. Eur J Clin Invest 45, 976–985. 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. Wu T, Zhou H, Hong Y, Li J, Jiang X & Huang H (2012). miR-30 family members negatively regulate osteoblast differentiation. J Biol Chem 287, 7503–7511. Xiao Z, Camalier CE, Nagashima K, Chan KC, Lucas DA, de la Cruz MJ, Gignac M, Lockett S, Issaq HJ, Veenstra TD, Conrads TP & Beck GR Jr (2007). Analysis of the extracellular matrix vesicle proteome in mineralizing osteoblasts. J Cell Physiol 210, 325–335.


2903

Zernecke A, Bidzhekov K, Noels H, Shagdarsuren E, Gan L, Denecke B, Hristov M, Koppel T, Jahantigh MN, Lutgens E, Wang S, Olson EN, Schober A & Weber C (2009). Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal 2, ra81.

Additional information Funding E.A. is funded by grants from the National Institutes of Health (NHLBI) (R01 HL114805, R01 HL109506). J.B.K. is funded by a Boehringer Ingelheim Fonds MD fellowship.

Acknowledgements The authors thank Dr Sergio Bertazzo for providing the image depicting extracellular vesicles using density-dependent colour scanning electron microscopy and Chelsea Swallom for editorial assistance.