The International Journal of Biochemistry & Cell Biology 50 (2014) 24–28
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Organelles in focus
Extracellular vesicles: New players in cardiovascular diseases Abderahim Gaceb a , Maria Carmen Martinez a , Ramaroson Andriantsitohaina a,b,∗ a b
INSERM UMR1063, Université d’Angers, Angers, France Centre Hospitalo-Universitaire d’Angers, Angers, France
a r t i c l e
i n f o
Article history: Received 15 November 2013 Received in revised form 14 January 2014 Accepted 26 January 2014 Available online 6 February 2014 Keywords: Extracellular vesicles Cardiovascular diseases Vascular cells Myocardium
a b s t r a c t Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Exchange of information between cells is attained through release of specific soluble signaling molecules or through direct cell-to-cell communication. In addition to these mechanisms, intercellular communication via extracellular vesicles (EV) has recently been identified as a conserved way during the evolution process. EV are particles heterogeneous in size (20–2000 nm) enclosed by a phospholipid bilayer, and released into extracellular medium of practically all cell types, both in vivo and in vitro. Up to now, three groups of EV have been mainly described depending on the mechanism of formation and physical characteristics: exosomes, microparticles and apoptotic bodies. Whereas exosomes are generated from the endosome-derived multivesicular bodies, both microparticles and apoptotic bodies are produced by budding from the plasma membrane (for review see Tual-Chalot et al., 2011; El Andaloussi et al., 2013). EV offer an elegant solution to cells to exchange biomolecules since, in one vesicle, it is possible to found lipids, proteins (receptors and enzymes), second messengers, mRNA, miRNA, and cell organelle fractions or proteins identified by proteomic analysis (Fig. 1). This leads to a new concept of EV as new cell organelles. Despite of these mix of components, EV possess specialized functions and play a key role in several pathologies
by regulating coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. The content and/or the number of EV depend on the cells they originate, the stimulus of production and the mechanism of vesicle generation. While quantification of the number of EV generated in vitro from a given number of cells is easy, the numeration of EV released by cells in vivo represents a huge challenge. This is particularly the case for non-circulating cells such as endothelial or smooth muscle cells. Nevertheless, Kanazawa et al. (2003) have shown that, in healthy subjects, 104 platelets can release 245 ± 11 microparticles whereas 104 monocytes release only 46 ± 7 microparticles. Other authors have shown that the number of platelet microparticles is significantly increased in diabetic patients (507 ± 15 per 104 platelets) (Ogata et al., 2005). Also, we have shown that the number of microparticles derived from activated leukocytes (CD62L+ ) is increased in obstructive sleep apnea patients when compared to healthy subjects (being 75 and 45 microparticles released from 5 × 103 leukocytes, respectively) (Priou et al., 2010). The mechanism of EV biogenesis represents very important criteria commonly used to classify different populations of EV. Thereby, two modes of EV biogenesis, “calcium-dependent” and “calcium-independent biogenesis” can be distinguished.
1.1. Calcium-dependent mechanism ∗ Corresponding author at: INSERM UMR 1063 “Stress oxidant et pathologies métaboliques”, Institut de Biologie en Santé, 4 rue Larrey, F-49933 Angers, France. Tel.: +33 2 44 68 85 80; fax: +33 2 44 68 85 85. E-mail address: [email protected] (R. Andriantsitohaina). 1357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2014.01.018
This mechanism includes microparticle and apoptotic body formation. It has been demonstrated that shedding of microparticles from the cell plasma membrane into the extracellular space is initiated by an increase in the cytosolic concentration of calcium
A. Gaceb et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 24–28
25
Multivesicular body Nuclear fragments
0.5-2 µm 0.02-0.1 µm
Endoplasmic reticulum 0.5-1 µm
Mitochondria
Microparticles
MFGE8 MHC molecules Tetraspanin Flotillin
EV
Exosomes
Nucleosomes
Apoptotic bodies
miRNA
Integrins
mRNA Enzymes
Selectins Adhesion molecules
Nuclear fragments
Cytoskeleton Receptor
Phosphatidyslerine
Ligand
Fig. 1. Biogenesis and content of extracellular vesicles (EV). Exosomes are formed by the inward budding of the multivesicular body membrane, whereas both microparticles and apoptotic bodies are generated from plasma membrane. All three types of EV carry receptors, ligands, active enzymes, cytoskeleton-associated proteins, mRNA and miRNA (in blue). In brown, apoptotic bodies (diameter between 0.5 and 2 m) are characterized by phosphatidylserine expression and nuclear components (nucleosomes, histones and nuclear fragments). In purple, microparticles (0.5–1 m) carry phosphatidylserine, integrins, selectins and other adhesion molecules. In green, exosomes (0.02–0.1 m) express tetraspanins, major histocompatibility complex molecules, flotillin and milk fat globule-epidermal growth factor 8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
resulting from the influx of extracellular calcium. The increase of calcium ions activates calpain which leads to degradation of various cytoskeleton-associated proteins. Thus, the result of dynamic interplay between phospholipid redistribution and cytoskeleton reorganization leads to the membrane budding and EV release. In addition, microparticle release is often, but not always, preceded by the loss of membrane asymmetry resulting from local perturbation of the bilayer structure leading to phosphatidylserine exposure at the membrane surface that could contribute to plasma membrane destabilization and blebbing (for review see Martinez et al., 2011). Apoptotic bodies are generated during apoptosis resulting from calcium-sensitive factors compartmentalized in various intracellular organelles including endoplasmic reticulum and mitochondria. In this case, membrane blebbing is, in part, mediated, by actin–myosin interaction. Phosphorylation of myosin light chain by Rho kinase I, becomes constitutively active upon cleavage by caspase 3 and induces a net increase in membrane blebbing (Akers et al., 2013). 1.2. Calcium-independent mechanism Exosomes are originated from the endosomal membrane cell compartment, and their release is subsequent to the exocytosis of multivesicular bodies into the extracellular space, after fusion with the plasma membrane. This mechanism is dependent on cytoskeleton activation, but independent of cytosolic calcium concentration. Independently of the mechanism implicated in the EV generation, all types of EV are generated from a selective cellular process
leading to significantly different selective enrichment of specific proteins, mRNA and miRNA. This is not the case of membrane particles from unspecific cell degradation. For instance, monocytes selectively package and secrete miRNA-150 into exosomes and deliver them to endothelial recipient cells where exmiRNA150 modulates endothelial cell functions (Zhang et al., 2010). Also, microparticles from activated and apoptotic T cells harbor the morphogen Sonic hedgehog whereas those from apoptotic T cells do not (Martínez et al., 2006). Altogether, these findings illustrate that EVs are easily distinguishable from membrane particles from unspecific cell degradation. The biological information is transmitted either by direct interaction between the vesicle membrane and the membrane of the recipient cell implicating ligand–receptor binding, fusion of the vesicle and target cell membranes, transfer of membrane constituents without fusion or by internalization of the vesicle content by the recipient cell. Consequently, vesicles can potentially be used for therapy, prognosis, and biomarkers for health and disease. Here, we summarize the role of EV, independently of their types, on the maintenance of homeostasis of the cardiovascular system as well as their involvement in the development and maintenance of cardiovascular diseases. 2. EV and homeostasis of the cardiovascular system Although the physiological role of EV is difficult to demonstrate, evidences show that EV possess specific effects due to their intrinsic composition and in addition, they may participate in regulating the
26
A. Gaceb et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 24–28
Fig. 2. Effects of extracellular vesicles (EV) in vascular cells and cardiomyocytes. (A) Circulating EV induce endothelial dysfunction by directly acting on endothelial cells and decreasing nitric oxide (NO) production, and increasing expression of pro-inflammatory proteins (ICAM-1, E-selectin, integrins). Also, EV increase plasmatic oxidative stress, NO, prostacyclin (PGI2 ) and reactive oxygen species (ROS) leading to vascular hyporeactivity. EV from atherosclerotic plaque induce ICAM overexpression on endothelial cells favoring monocyte adhesion and inflammation. (B) Progenitors-derived EV generated during myocardial ischemia/reperfusion carry glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK), and muscle pyruvate kinase (PKm2), which correct the deficit of glycolytic enzymes in the myocardium. Also, the surface protein CD73 present in EV increase survival signaling through the activation of reperfusion injury salvage kinases (RISK).
function of different circulating cells. Thus, EV can regulate and participate to coagulation, tissue repair and stem cell maintenance. For instance, during the process of coagulation, one of the major functions of blood platelets is to provide a membrane surface that accelerates blood coagulation and promotes the formation of the fibrin network in the hemostatic plug (Tans et al., 1991). Thus, specific procoagulant activity of the platelet-derived EV membranes is approximately 50- to 100-fold higher than that of activated platelets and this is due to enhanced surface densities of phosphatidylserine, CD61, CD62P and factor X bound (Sinauridze et al., 2007). Also, EV release can be considered as the major secretory pathway for rapid IL-1beta secretion from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins (MacKenzie et al., 2001). Regarding tissue repair, EV can participate in the development of blood vessel network during the physiological process of wound healing. Indeed, EV derived from endothelial cells, containing metalloproteinase proteins (MMP-2 and MMP-9), promote matrix degradation, thereby promoting the formation of new blood vessels (Taraboletti et al., 2002). Finally, EV induce efficient cell differentiation of stem cells participating in the maintenance of stem cell niche and repairing of injured tissue. In this context, Camussi et al. (2013) have shown that EV regulate the bidirectional exchange of genetic information from stromal to stem cells. EV may reprogram not only the phenotype of stem cells to acquire features of the injured tissue cells, but also they may induce de-differentiation of cells which have survived injury allowing tissue regeneration.
3. EV as main actors in cardiovascular diseases In view of the essential role of EV on physiological processes, it is not surprising that, in pathological situations, EV can participate in the development and maintenance of cardiovascular diseases acting on both vascular and cardiac cells (Fig. 2). 3.1. EV and vascular wall cells Numerous studies have shown that EV, mainly microparticles, interact with cells from the vascular wall, mainly endothelial cells and smooth muscle cells. Elevated levels of total circulating microparticles and/or some microparticle subsets are mostly detected by flow cytometry in diseases such as myocardial infarction, diabetes, end-stage renal failure, metabolic syndrome and obstructive sleep apnea (Boulanger et al., 2001; Sabatier et al., 2002; Amabile et al., 2005; Agouni et al., 2008; Priou et al., 2010) and are frequently correlated with the severity of the diseases. EV from patients with these diseases induce endothelial dysfunction by (i) decreasing NO signaling by reducing the activity of endothelial NO synthase via an increased phosphorylation of the enzyme at its inhibitory site (Ser495) (Martin et al., 2004; Agouni et al., 2008; Priou et al., 2010) and/or by decreasing NO bioavailability (Boulanger et al., 2001), (ii) decreasing cyclic GMP production (Amabile et al., 2005), (iii) increasing protein nitration on endothelial cells (Agouni et al., 2008) and (iv) enhancing plasmatic oxidative stress markers (Helal et al., 2011). These effects are responsible for the reduced
A. Gaceb et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 24–28
endothelial vasodilatation of vessels exposed to EV ex vivo or those taken from EV-injected mice. Furthermore, EV from human atherosclerotic plaques enhance inflammatory responses by favoring the adhesion of monocytes to endothelial cells via the transfer of ICAM-1 molecules leading to atherosclerotic plaque progression (Rautou et al., 2011). EV from obstructive sleep apnea syndrome patients increase expression of pro-inflammatory proteins such as E-selectin, integrin alpha5 and ICAM-1 in human endothelial cells (Priou et al., 2010). EV can also achieve smooth muscle cells (Tesse et al., 2005) and modify vascular contraction. Injection of EV from metabolic syndrome patients in mice induce an up-regulation of inducible NO synthase without changes in cyclo-oxygenase 1 and cyclo-oxygenase-2 expression resulting in an overproduction of vasodilator mediators (NO and prostacyclin) leading to vascular hypo-reactivity in the mouse aorta (Agouni et al., 2011). In addition, in mouse aorta, enhanced oxidative stress induced by injection EV from metabolic syndrome patients is associated with enhanced expression of the NADPH-oxidase subunits, gp91phox and p47phox . Most interestingly, EV-induced vascular hypo-reactivity is abolished when the Fas/FasL pathway is neutralized. Metabolic syndrome EV also markedly increased MCP-1 mRNA and protein levels, without changing the mRNA levels of other pro-inflammatory cytokines in the mouse aorta. These data provide valuable information in our understanding of some of the paracrine roles that EV play as vectors of trans-cellular messengers in promoting vascular dysfunction during metabolic syndrome. On the other hand, EV from septic patients are able to stimulate the release of anti-inflammatory cytokine such as interleukin (IL)10 and the vasoconstrictor metabolites including thromboxane A2. These effects of EV probably participate in the protective effect of EV in counteracting vascular hyporeactivity observed at the early phase of septic shock (Mostefai et al., 2008). EV also participate in atherosclerosis. Indeed, EV secreted by endothelial cells stimulated by high shear-stress are enriched in miR-143/145. Once transferred to smooth muscle cells, these vesicles reduce the expression of miR-143/145 targets such as ELK1 and CAMK2d, two central regulators of smooth muscle cell phenotype, and promote de-differentiation and repress proliferation of smooth muscle cells, an event associated with atherosclerosis progression. These data provide evidence that atheroprotective stimuli such as shear stress, through activation of Rho kinases and ERK1/2 pathways (Vion et al., 2013), induce EV release carrying atheroprotective miRNA which may represent a therapeutical strategy against atherosclerosis (Hergenreider et al., 2012). Interestingly, EV derived from apoptotic endothelial cells generated during atherosclerosis contain miR-126, which controls endothelial cell signaling and provides atheroprotective effects in vivo. Indeed, in the ApoE−/− model of atherosclerosis, the administration of apoptotic endothelial cell-derived EV limits atherosclerosis progression by reducing of plaque area and macrophage infiltration in the vessel wall, facilitates the mobilization of progenitor cells via the production of chemokine CXCL12, an antiapoptotic survival factor, leading to stabilization of atheroma plaques (Zernecke et al., 2009). miR-126 content of EV from apoptotic endothelial cells is reduced in diabetic patients and this might explain the impaired peripheral angiogenic signaling in patients with diabetes (Zampetaki and Mayr, 2012). 3.2. EV and their effects on the myocardium Studies regarding the effects of EV on the myocardium are scarce and concern mainly exosomes from different origin including those from cardiomyocytes. Injured myocardium can generate EV enriched with miR-1 and miR-133a reported to induce cardiac hypertrophy by targeting
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mRNAs associated with myotrophin and the TGF- signaling pathway (Bagnall et al., 2012). Since this type of EV is significantly increased in patients with acute myocardial infarction and angina pectoris (Kuwabara et al., 2011), they may be used as a potential marker for cardiomyocyte death. Interestingly, internalization of progenitors-derived EV, mainly from mesenchymal stem cells, reduces the consequences of ischemia/reperfusion during myocardial infarct (Lai et al., 2013). Mesenchymal-derived EV protect against both the deleterious effect during myocardial ischemia/reperfusion injury resulting from ATP deficit and the initiation of apoptosis by correcting the deficit of glycolytic enzymes in the myocardium. In fact, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK), and muscle pyruvate kinase (PKm2), which generate either ATP or NADH, are enzymatically active in mesenchymal-derived EV could potentially increase glycolytic flux and ATP production in reperfused myocardium. In addition, the surface protein CD73 present in EV may overcome the proapoptotic proteome of reperfused myocardium by increasing survival signaling through the activation of reperfusion injury salvage kinases (RISK). Furthermore, mouse cardiac progenitor-derived EV protect cardiomyocytes against apoptosis from ischemia in an acute mouse myocardial ischemia/reperfusion model (Chen et al., 2013). Although the exact mechanism implicated in these effects is not described, these authors have shown that cardiac progenitorderived EV inhibit caspase 3/7 activation in vitro cardiomyocytes (Chen et al., 2013). These results suggest that progenitor-derived EV may be used in the next future as a potential therapeutic tool for cardioprotection. 4. Future outlook The mechanisms of the formation of EV are still largely unexplored, and the distinction between several types of vesicles and their isolation is still a goal to be attained. Recently, improvement of detection devices allows distinguishing vesicles smaller than 100 nm (Van der Pol et al., 2013) indicating that probably hitherto unknown effects will soon be described. EV are a means of transport of biological molecules and emerge as new players mediating deleterious or beneficial effects in cardiovascular diseases and in other pathologies. To better understand the biological effects of EV the analyses of the content of vesicles by transcriptome, miRNA and proteome studies are needed. In the near future, EV may serve as potential clinical tools for new therapy by engineering of vesicles which consist of under-express or over-express a target of interest (mRNA, miRNA, protein) in the vesicles, to monitor the messages carried by these vesicles, in the aim of targeting a cell with a definite purpose. References Agouni A, Ducluzeau PH, Benameur T, Faure S, Sladkova M, Duluc L, et al. Microparticles from patients with metabolic syndrome induce vascular hyporeactivity via Fas/Fas-ligand pathway in mice. PLoS ONE 2011;6:e27809. Agouni A, Lagrue-Lak-Hal AH, Ducluzeau PH, Mostefai HA, Draunet-Busson C, Leftheriotis G, et al. Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome. Am J Pathol 2008;173:1210–9. Akers JC, Gonda D, Kim R, Carter BS, Chen CC. Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol 2013;113:1–11. Amabile N, Guérin AP, Leroyer A, Mallat Z, Nguyen C, Boddaert J, et al. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 2005;16:3381–8. Bagnall RD, Tsoutsman T, Shephard RE, Ritchie W, Semsarian C. Global microRNA profiling of the mouse ventricles during development of severe hypertrophic cardiomyopathy and heart failure. PLoS ONE 2012;7:e44744. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, et al. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 2001;104:2649–52.
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Camussi G, Deregibus MC, Cantaluppi V. Role of stem-cell-derived microvesicles in the paracrine action of stem cells. Biochem Soc Trans 2013;41:283–7. Chen L, Wang Y, Pan Y, Zhang L, Shen C, Qin G, et al. Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury. Biochem Biophys Res Commun 2013;431:566–71. El Andaloussi S, Mäger I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013;12:347–57. Helal O, Defoort C, Robert S, Marin C, Lesavre N, Lopez-Miranda J, et al. Increased levels of microparticles originating from endothelial cells, platelets and erythrocytes in subjects with metabolic syndrome: relationship with oxidative stress. Nutr Metab Cardiovasc Dis 2011;21:665–71. Hergenreider E, Heydt S, Tréguer K, Boettger T, Horrevoets AJ, Zeiher AM, et al. Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nat Cell Biol 2012;14:249–56. Kanazawa S, Nomura S, Kuwana M, Muramatsu M, Yamaguchi K, Fukuhara S. Monocyte-derived microparticles may be a sign of vascular complication in patients with lung cancer. Lung Cancer 2003;39:145–9. Kuwabara Y, Ono K, Horie T, Nishi H, Nagao K, Kinoshita M, et al. Increased microRNA1 and microRNA-133a levels in serum of patients with cardiovascular disease indicate myocardial damage. Circ Cardiovasc Genet 2011;4:446–54. Lai RC, Yeo RW, Tan KH, Lim SK. Mesenchymal stem cell exosome ameliorates reperfusion injury through proteomic complementation. Regen Med 2013;8:197–209. MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA, Surprenant A. Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 2001;15:825–35. Martin S, Tesse A, Hugel B, Martinez MC, Morel O, Frayssinet J-M, et al. Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression. Circulation 2004;109:1653–9. Martínez MC, Larbret F, Zobairi F, Coulombe J, Debili N, Vainchenker W, et al. Transfer of differentiation signal by membrane microvesicles harboring hedgehog morphogens. Blood 2006;108:3012–20. Martinez MC, Tual-Chalot S, Leonetti D, Andriantsitohaina R. Microparticles: targets and tools in cardiovascular disease. Trends Pharmacol Sci 2011;32:659–65. Mostefai HA, Meziani F, Mastronardi ML, Agouni A, Heymes C, Sargentini C, et al. Circulating microparticles from patients with septic shock exert protective role in vascular function. Am J Respir Crit Care Med 2008;178:1148–55. Ogata N, Imaizumi M, Nomura S, Shozu A, Arichi M, Matsuoka M, et al. Increased levels of platelet-derived microparticles in patients with diabetic retinopathy. Diabetes Res Clin Pract 2005;68:193–201.
Priou P, Gagnadoux F, Tesse A, Mastronardi ML, Agouni A, Meslier N, et al. Endothelial dysfunction and circulating microparticles from patients with obstructive sleep apnea. Am J Pathol 2010;177:974–83. Rautou PE, Leroyer AS, Ramkhelawon B, Devue C, Duflaut D, Vion AC, et al. Microparticles from human atherosclerotic plaques promote endothelial ICAM1-dependent monocyte adhesion and transendothelial migration. Circ Res 2011;108:335–43. Sabatier F, Darmon P, Hugel B, Combes V, Sanmarco M, Velut JG, et al. Type 1 and type 2 diabetic patients display different patterns of cellular microparticles. Diabetes 2002;51:2840–5. Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya OV, et al. Platelet microparticle membranes have 50- to 100-fold higher specific procoagulant activity than activated platelets. Thromb Haemost 2007;97: 425–34. Tans G, Rosing J, Thomassen MC, Heeb MJ, Zwaal RF, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991;77:2641–8. Taraboletti G, D’Ascenzo S, Borsotti P, Giavazzi R, Pavan A, Dolo V. Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicle-associated components by endothelial cells. Am J Pathol 2002;160:673–80. Tesse A, Martínez MC, Hugel B, Chalupsky K, Muller CD, Meziani F, et al. Upregulation of proinflammatory proteins through NF-kappaB pathway by shed membrane microparticles results in vascular hyporeactivity. Arterioscler Thromb Vasc Biol 2005;25:2522–7. Tual-Chalot S, Leonetti D, Andriantsitohaina R, Martínez MC. Microvesicles: intercellular vectors of biological messages. Mol Interv 2011;11:88–94. Van der Pol E, Coumans F, Varga Z, Krumrey M, Nieuwland R. Innovation in detection of microparticles and exosomes. J Thromb Haemost 2013;11(Suppl. 1): 36–45. Vion AC, Ramkhelawon B, Loyer X, Chironi G, Devue C, Loirand G, et al. Shear stress regulates endothelial microparticle release. Circ Res 2013;112:1323–33. Zampetaki A, Mayr M. MicroRNAs in vascular and metabolic disease. Circ Res 2012;110:508–22. Zhang Y, Liu D, Chen X, Li J, Li L, Bian Z, et al. Secreted monocytic miR-150 enhances targeted endothelial cell migration. Mol Cell 2010;39:133–44. Zernecke A, Bidzhekov K, Noels H, Shagdarsuren E, Gan L, Denecke B, et al. Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Sci Signal 2009;2:ra81.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Organelles in focus
Extracellular vesicles: New players in cardiovascular diseases Abderahim Gaceb a , Maria Carmen Martinez a , Ramaroson Andriantsitohaina a,b,∗ a b
INSERM UMR1063, Université d’Angers, Angers, France Centre Hospitalo-Universitaire d’Angers, Angers, France
a r t i c l e
i n f o
Article history: Received 15 November 2013 Received in revised form 14 January 2014 Accepted 26 January 2014 Available online 6 February 2014 Keywords: Extracellular vesicles Cardiovascular diseases Vascular cells Myocardium
a b s t r a c t Extracellular vesicles, particles released by all cell types, represent a new way to convey information between cells such as proteins, second messengers, and genetic information to modify the phenotype and function of the target cells. Recent data suggest that extracellular vesicles play a crucial role in both physiology and pathology, including coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. Thus extracellular vesicles participate in the processes of cardiovascular diseases from atherosclerosis, myocardial infarction to heart failure. Consequently, extracellular vesicles can potentially be exploited for therapy, prognosis, and biomarkers for health and disease. This review focuses on the role of extracellular vesicles in the development of cardiovascular diseases, as well as the deleterious and beneficial effects that they may provide in vascular cells and myocardium. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Exchange of information between cells is attained through release of specific soluble signaling molecules or through direct cell-to-cell communication. In addition to these mechanisms, intercellular communication via extracellular vesicles (EV) has recently been identified as a conserved way during the evolution process. EV are particles heterogeneous in size (20–2000 nm) enclosed by a phospholipid bilayer, and released into extracellular medium of practically all cell types, both in vivo and in vitro. Up to now, three groups of EV have been mainly described depending on the mechanism of formation and physical characteristics: exosomes, microparticles and apoptotic bodies. Whereas exosomes are generated from the endosome-derived multivesicular bodies, both microparticles and apoptotic bodies are produced by budding from the plasma membrane (for review see Tual-Chalot et al., 2011; El Andaloussi et al., 2013). EV offer an elegant solution to cells to exchange biomolecules since, in one vesicle, it is possible to found lipids, proteins (receptors and enzymes), second messengers, mRNA, miRNA, and cell organelle fractions or proteins identified by proteomic analysis (Fig. 1). This leads to a new concept of EV as new cell organelles. Despite of these mix of components, EV possess specialized functions and play a key role in several pathologies
by regulating coagulation, angiogenesis, cell survival, modulation of the immune response, and inflammation. The content and/or the number of EV depend on the cells they originate, the stimulus of production and the mechanism of vesicle generation. While quantification of the number of EV generated in vitro from a given number of cells is easy, the numeration of EV released by cells in vivo represents a huge challenge. This is particularly the case for non-circulating cells such as endothelial or smooth muscle cells. Nevertheless, Kanazawa et al. (2003) have shown that, in healthy subjects, 104 platelets can release 245 ± 11 microparticles whereas 104 monocytes release only 46 ± 7 microparticles. Other authors have shown that the number of platelet microparticles is significantly increased in diabetic patients (507 ± 15 per 104 platelets) (Ogata et al., 2005). Also, we have shown that the number of microparticles derived from activated leukocytes (CD62L+ ) is increased in obstructive sleep apnea patients when compared to healthy subjects (being 75 and 45 microparticles released from 5 × 103 leukocytes, respectively) (Priou et al., 2010). The mechanism of EV biogenesis represents very important criteria commonly used to classify different populations of EV. Thereby, two modes of EV biogenesis, “calcium-dependent” and “calcium-independent biogenesis” can be distinguished.
1.1. Calcium-dependent mechanism ∗ Corresponding author at: INSERM UMR 1063 “Stress oxidant et pathologies métaboliques”, Institut de Biologie en Santé, 4 rue Larrey, F-49933 Angers, France. Tel.: +33 2 44 68 85 80; fax: +33 2 44 68 85 85. E-mail address: [email protected] (R. Andriantsitohaina). 1357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2014.01.018
This mechanism includes microparticle and apoptotic body formation. It has been demonstrated that shedding of microparticles from the cell plasma membrane into the extracellular space is initiated by an increase in the cytosolic concentration of calcium
A. Gaceb et al. / The International Journal of Biochemistry & Cell Biology 50 (2014) 24–28
25
Multivesicular body Nuclear fragments
0.5-2 µm 0.02-0.1 µm
Endoplasmic reticulum 0.5-1 µm
Mitochondria
Microparticles
MFGE8 MHC molecules Tetraspanin Flotillin
EV
Exosomes
Nucleosomes
Apoptotic bodies
miRNA
Integrins
mRNA Enzymes
Selectins Adhesion molecules
Nuclear fragments
Cytoskeleton Receptor
Phosphatidyslerine
Ligand
Fig. 1. Biogenesis and content of extracellular vesicles (EV). Exosomes are formed by the inward budding of the multivesicular body membrane, whereas both microparticles and apoptotic bodies are generated from plasma membrane. All three types of EV carry receptors, ligands, active enzymes, cytoskeleton-associated proteins, mRNA and miRNA (in blue). In brown, apoptotic bodies (diameter between 0.5 and 2 m) are characterized by phosphatidylserine expression and nuclear components (nucleosomes, histones and nuclear fragments). In purple, microparticles (0.5–1 m) carry phosphatidylserine, integrins, selectins and other adhesion molecules. In green, exosomes (0.02–0.1 m) express tetraspanins, major histocompatibility complex molecules, flotillin and milk fat globule-epidermal growth factor 8. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
resulting from the influx of extracellular calcium. The increase of calcium ions activates calpain which leads to degradation of various cytoskeleton-associated proteins. Thus, the result of dynamic interplay between phospholipid redistribution and cytoskeleton reorganization leads to the membrane budding and EV release. In addition, microparticle release is often, but not always, preceded by the loss of membrane asymmetry resulting from local perturbation of the bilayer structure leading to phosphatidylserine exposure at the membrane surface that could contribute to plasma membrane destabilization and blebbing (for review see Martinez et al., 2011). Apoptotic bodies are generated during apoptosis resulting from calcium-sensitive factors compartmentalized in various intracellular organelles including endoplasmic reticulum and mitochondria. In this case, membrane blebbing is, in part, mediated, by actin–myosin interaction. Phosphorylation of myosin light chain by Rho kinase I, becomes constitutively active upon cleavage by caspase 3 and induces a net increase in membrane blebbing (Akers et al., 2013). 1.2. Calcium-independent mechanism Exosomes are originated from the endosomal membrane cell compartment, and their release is subsequent to the exocytosis of multivesicular bodies into the extracellular space, after fusion with the plasma membrane. This mechanism is dependent on cytoskeleton activation, but independent of cytosolic calcium concentration. Independently of the mechanism implicated in the EV generation, all types of EV are generated from a selective cellular process
leading to significantly different selective enrichment of specific proteins, mRNA and miRNA. This is not the case of membrane particles from unspecific cell degradation. For instance, monocytes selectively package and secrete miRNA-150 into exosomes and deliver them to endothelial recipient cells where exmiRNA150 modulates endothelial cell functions (Zhang et al., 2010). Also, microparticles from activated and apoptotic T cells harbor the morphogen Sonic hedgehog whereas those from apoptotic T cells do not (Martínez et al., 2006). Altogether, these findings illustrate that EVs are easily distinguishable from membrane particles from unspecific cell degradation. The biological information is transmitted either by direct interaction between the vesicle membrane and the membrane of the recipient cell implicating ligand–receptor binding, fusion of the vesicle and target cell membranes, transfer of membrane constituents without fusion or by internalization of the vesicle content by the recipient cell. Consequently, vesicles can potentially be used for therapy, prognosis, and biomarkers for health and disease. Here, we summarize the role of EV, independently of their types, on the maintenance of homeostasis of the cardiovascular system as well as their involvement in the development and maintenance of cardiovascular diseases. 2. EV and homeostasis of the cardiovascular system Although the physiological role of EV is difficult to demonstrate, evidences show that EV possess specific effects due to their intrinsic composition and in addition, they may participate in regulating the
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Fig. 2. Effects of extracellular vesicles (EV) in vascular cells and cardiomyocytes. (A) Circulating EV induce endothelial dysfunction by directly acting on endothelial cells and decreasing nitric oxide (NO) production, and increasing expression of pro-inflammatory proteins (ICAM-1, E-selectin, integrins). Also, EV increase plasmatic oxidative stress, NO, prostacyclin (PGI2 ) and reactive oxygen species (ROS) leading to vascular hyporeactivity. EV from atherosclerotic plaque induce ICAM overexpression on endothelial cells favoring monocyte adhesion and inflammation. (B) Progenitors-derived EV generated during myocardial ischemia/reperfusion carry glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK), and muscle pyruvate kinase (PKm2), which correct the deficit of glycolytic enzymes in the myocardium. Also, the surface protein CD73 present in EV increase survival signaling through the activation of reperfusion injury salvage kinases (RISK).
function of different circulating cells. Thus, EV can regulate and participate to coagulation, tissue repair and stem cell maintenance. For instance, during the process of coagulation, one of the major functions of blood platelets is to provide a membrane surface that accelerates blood coagulation and promotes the formation of the fibrin network in the hemostatic plug (Tans et al., 1991). Thus, specific procoagulant activity of the platelet-derived EV membranes is approximately 50- to 100-fold higher than that of activated platelets and this is due to enhanced surface densities of phosphatidylserine, CD61, CD62P and factor X bound (Sinauridze et al., 2007). Also, EV release can be considered as the major secretory pathway for rapid IL-1beta secretion from activated monocytes and may represent a more general mechanism for secretion of similar leaderless secretory proteins (MacKenzie et al., 2001). Regarding tissue repair, EV can participate in the development of blood vessel network during the physiological process of wound healing. Indeed, EV derived from endothelial cells, containing metalloproteinase proteins (MMP-2 and MMP-9), promote matrix degradation, thereby promoting the formation of new blood vessels (Taraboletti et al., 2002). Finally, EV induce efficient cell differentiation of stem cells participating in the maintenance of stem cell niche and repairing of injured tissue. In this context, Camussi et al. (2013) have shown that EV regulate the bidirectional exchange of genetic information from stromal to stem cells. EV may reprogram not only the phenotype of stem cells to acquire features of the injured tissue cells, but also they may induce de-differentiation of cells which have survived injury allowing tissue regeneration.
3. EV as main actors in cardiovascular diseases In view of the essential role of EV on physiological processes, it is not surprising that, in pathological situations, EV can participate in the development and maintenance of cardiovascular diseases acting on both vascular and cardiac cells (Fig. 2). 3.1. EV and vascular wall cells Numerous studies have shown that EV, mainly microparticles, interact with cells from the vascular wall, mainly endothelial cells and smooth muscle cells. Elevated levels of total circulating microparticles and/or some microparticle subsets are mostly detected by flow cytometry in diseases such as myocardial infarction, diabetes, end-stage renal failure, metabolic syndrome and obstructive sleep apnea (Boulanger et al., 2001; Sabatier et al., 2002; Amabile et al., 2005; Agouni et al., 2008; Priou et al., 2010) and are frequently correlated with the severity of the diseases. EV from patients with these diseases induce endothelial dysfunction by (i) decreasing NO signaling by reducing the activity of endothelial NO synthase via an increased phosphorylation of the enzyme at its inhibitory site (Ser495) (Martin et al., 2004; Agouni et al., 2008; Priou et al., 2010) and/or by decreasing NO bioavailability (Boulanger et al., 2001), (ii) decreasing cyclic GMP production (Amabile et al., 2005), (iii) increasing protein nitration on endothelial cells (Agouni et al., 2008) and (iv) enhancing plasmatic oxidative stress markers (Helal et al., 2011). These effects are responsible for the reduced
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endothelial vasodilatation of vessels exposed to EV ex vivo or those taken from EV-injected mice. Furthermore, EV from human atherosclerotic plaques enhance inflammatory responses by favoring the adhesion of monocytes to endothelial cells via the transfer of ICAM-1 molecules leading to atherosclerotic plaque progression (Rautou et al., 2011). EV from obstructive sleep apnea syndrome patients increase expression of pro-inflammatory proteins such as E-selectin, integrin alpha5 and ICAM-1 in human endothelial cells (Priou et al., 2010). EV can also achieve smooth muscle cells (Tesse et al., 2005) and modify vascular contraction. Injection of EV from metabolic syndrome patients in mice induce an up-regulation of inducible NO synthase without changes in cyclo-oxygenase 1 and cyclo-oxygenase-2 expression resulting in an overproduction of vasodilator mediators (NO and prostacyclin) leading to vascular hypo-reactivity in the mouse aorta (Agouni et al., 2011). In addition, in mouse aorta, enhanced oxidative stress induced by injection EV from metabolic syndrome patients is associated with enhanced expression of the NADPH-oxidase subunits, gp91phox and p47phox . Most interestingly, EV-induced vascular hypo-reactivity is abolished when the Fas/FasL pathway is neutralized. Metabolic syndrome EV also markedly increased MCP-1 mRNA and protein levels, without changing the mRNA levels of other pro-inflammatory cytokines in the mouse aorta. These data provide valuable information in our understanding of some of the paracrine roles that EV play as vectors of trans-cellular messengers in promoting vascular dysfunction during metabolic syndrome. On the other hand, EV from septic patients are able to stimulate the release of anti-inflammatory cytokine such as interleukin (IL)10 and the vasoconstrictor metabolites including thromboxane A2. These effects of EV probably participate in the protective effect of EV in counteracting vascular hyporeactivity observed at the early phase of septic shock (Mostefai et al., 2008). EV also participate in atherosclerosis. Indeed, EV secreted by endothelial cells stimulated by high shear-stress are enriched in miR-143/145. Once transferred to smooth muscle cells, these vesicles reduce the expression of miR-143/145 targets such as ELK1 and CAMK2d, two central regulators of smooth muscle cell phenotype, and promote de-differentiation and repress proliferation of smooth muscle cells, an event associated with atherosclerosis progression. These data provide evidence that atheroprotective stimuli such as shear stress, through activation of Rho kinases and ERK1/2 pathways (Vion et al., 2013), induce EV release carrying atheroprotective miRNA which may represent a therapeutical strategy against atherosclerosis (Hergenreider et al., 2012). Interestingly, EV derived from apoptotic endothelial cells generated during atherosclerosis contain miR-126, which controls endothelial cell signaling and provides atheroprotective effects in vivo. Indeed, in the ApoE−/− model of atherosclerosis, the administration of apoptotic endothelial cell-derived EV limits atherosclerosis progression by reducing of plaque area and macrophage infiltration in the vessel wall, facilitates the mobilization of progenitor cells via the production of chemokine CXCL12, an antiapoptotic survival factor, leading to stabilization of atheroma plaques (Zernecke et al., 2009). miR-126 content of EV from apoptotic endothelial cells is reduced in diabetic patients and this might explain the impaired peripheral angiogenic signaling in patients with diabetes (Zampetaki and Mayr, 2012). 3.2. EV and their effects on the myocardium Studies regarding the effects of EV on the myocardium are scarce and concern mainly exosomes from different origin including those from cardiomyocytes. Injured myocardium can generate EV enriched with miR-1 and miR-133a reported to induce cardiac hypertrophy by targeting
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mRNAs associated with myotrophin and the TGF- signaling pathway (Bagnall et al., 2012). Since this type of EV is significantly increased in patients with acute myocardial infarction and angina pectoris (Kuwabara et al., 2011), they may be used as a potential marker for cardiomyocyte death. Interestingly, internalization of progenitors-derived EV, mainly from mesenchymal stem cells, reduces the consequences of ischemia/reperfusion during myocardial infarct (Lai et al., 2013). Mesenchymal-derived EV protect against both the deleterious effect during myocardial ischemia/reperfusion injury resulting from ATP deficit and the initiation of apoptosis by correcting the deficit of glycolytic enzymes in the myocardium. In fact, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK), and muscle pyruvate kinase (PKm2), which generate either ATP or NADH, are enzymatically active in mesenchymal-derived EV could potentially increase glycolytic flux and ATP production in reperfused myocardium. In addition, the surface protein CD73 present in EV may overcome the proapoptotic proteome of reperfused myocardium by increasing survival signaling through the activation of reperfusion injury salvage kinases (RISK). Furthermore, mouse cardiac progenitor-derived EV protect cardiomyocytes against apoptosis from ischemia in an acute mouse myocardial ischemia/reperfusion model (Chen et al., 2013). Although the exact mechanism implicated in these effects is not described, these authors have shown that cardiac progenitorderived EV inhibit caspase 3/7 activation in vitro cardiomyocytes (Chen et al., 2013). These results suggest that progenitor-derived EV may be used in the next future as a potential therapeutic tool for cardioprotection. 4. Future outlook The mechanisms of the formation of EV are still largely unexplored, and the distinction between several types of vesicles and their isolation is still a goal to be attained. Recently, improvement of detection devices allows distinguishing vesicles smaller than 100 nm (Van der Pol et al., 2013) indicating that probably hitherto unknown effects will soon be described. EV are a means of transport of biological molecules and emerge as new players mediating deleterious or beneficial effects in cardiovascular diseases and in other pathologies. To better understand the biological effects of EV the analyses of the content of vesicles by transcriptome, miRNA and proteome studies are needed. In the near future, EV may serve as potential clinical tools for new therapy by engineering of vesicles which consist of under-express or over-express a target of interest (mRNA, miRNA, protein) in the vesicles, to monitor the messages carried by these vesicles, in the aim of targeting a cell with a definite purpose. References Agouni A, Ducluzeau PH, Benameur T, Faure S, Sladkova M, Duluc L, et al. Microparticles from patients with metabolic syndrome induce vascular hyporeactivity via Fas/Fas-ligand pathway in mice. PLoS ONE 2011;6:e27809. Agouni A, Lagrue-Lak-Hal AH, Ducluzeau PH, Mostefai HA, Draunet-Busson C, Leftheriotis G, et al. Endothelial dysfunction caused by circulating microparticles from patients with metabolic syndrome. Am J Pathol 2008;173:1210–9. Akers JC, Gonda D, Kim R, Carter BS, Chen CC. Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol 2013;113:1–11. Amabile N, Guérin AP, Leroyer A, Mallat Z, Nguyen C, Boddaert J, et al. Circulating endothelial microparticles are associated with vascular dysfunction in patients with end-stage renal failure. J Am Soc Nephrol 2005;16:3381–8. Bagnall RD, Tsoutsman T, Shephard RE, Ritchie W, Semsarian C. Global microRNA profiling of the mouse ventricles during development of severe hypertrophic cardiomyopathy and heart failure. PLoS ONE 2012;7:e44744. Boulanger CM, Scoazec A, Ebrahimian T, Henry P, Mathieu E, Tedgui A, et al. Circulating microparticles from patients with myocardial infarction cause endothelial dysfunction. Circulation 2001;104:2649–52.
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