Extracellular vesicles in circulation Introduction Extracellular vesicles (EVs) are membrane limited vesicles, originating from cells and located in the extracellular space(1). The term extracellular vesicles comprises exosomes, microvesicles (MV) and apoptopic bodies (AB) and is chosen as a step towards standardization of terminology, as currently many more exotic terms are in use, such as oncosomes, minisomes, microparticles nanovesicles etc. The differences between exosomes, MVs and ABs lie in their size and origin. Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. Before secretion, they are present as the internal vesicles of an endosomal compartment, the multivesicular body (MVB) and are released upon exocytic fusion of this organelle with the plasma membrane(2). MVs are shed from the plasma membrane and have a diameter of 100-1000 nm(3) and ABs have a diameter of >1 µm and are formed by large blebs which occur during programmed cell death(4). Figure 1: Schematic representation of extracellular vesicles by Abcam®. Exosomes, exosome-like vesicle, microvesicles and membrane particles are membrane limited vesicles, originating from the cell and located in the extracellular space. Despite different origins inside the cell, they are all EVs and are able to act as carriers of information between cells. EVs is a synonym of membrane vesicles proposed by Thery et al, but EVs emphasizes the extracellular nature of the vesicles and this denomination is easier to distinguish from intracellular membrane vesicles(5). The emergence and growth of this bourgeoning research field is characterized by the recent formation of the International Society of Extracellular Vesicles (ISEV). Secretion of EVs differs between vesicle type and depends on the cell type. For example epsteinBarrvirus (EBV)-transformed B cell lines, primary dendritic cell (DC) cultures and tumor cells show constitutive secretion of EVs(6)., while other cells, like presynaptic nerve terminals(7) and erythrocytes(8) need a stimulus, like an increase in internal Ca2+, to stimulate exosome secretion. Another trigger for EVs is induced stress, such as hypoxia and shear stress, which often is regulated by the P53 protein(9). These factors do not only activate a cell to secrete EVs, but can also influence the molecular content of the vesicles. So why are these EVs so interesting? Recent studies indicate that these vesicles are used for communication between cells(10). This communication includes transmission of surface-membrane lipids, proteins and horizontal transfer of proteins and RNAs between cells. Transfer of these biomolecules can have profound effects on the activity of the acceptor cell. For example a number of studies have suggested a functional role for transfer of vesicles in the proliferation of tumors (11). Summarizing, functional transfer of EVs is important for the general homeostasis of cell populations in physiological conditions, but can also be a factor that aggravates pathological conditions(12). Since it is shown that EVs can act as carriers of RNA and proteins, it is very interesting to investigate their potential as drug delivery carriers to improve the efficacy of drugs. This technique could be used to enhance currently available pharmaceuticals, but can also be used to deliver potential new drugs, which are currently not suitable because of their fast clearance rate. Besides their potential as drug carriers, EVs can play an important role in diagnostic applications. It appears that every cell type has its own unique ‘fingerprint’ of protein and RNA distribution in their EVs and that this fingerprint can change depending on the (pathological) milieu. By screening the blood for elevated or altered levels of EVs and determining their origin it is possible to diagnose diseases in an early stage(13). Furthermore an interesting subject is to disrupt communication by EVs in order to alter disease outcome (14). These three topics are the drivers of this upcoming research field and in this review a brief summary of the current research and opportunities will be given of EVs. Although EVs are found in all body fluids(15), this review will focus on EVs in circulation, because it is the most prevalent fluid in the body and has the most therapeutical relevance. Vesicles in circulation In the bloodstream EVs from four different cell types mainly are present, namely red blood cells, endothelial cells (ECs) leukocytes, and platelets. Celltype Quantities in Healthy Type of EVs Individuals(16) Platelet(17, 18) 237 × 106/L Endothelial(19) Specific Markers Effects Common Markers Exosomes Microvesicles Apoptopic blebs Glycoprotein IIb/IIIa Inflammation Coagulation Angiogenesis tsg101αv integrin (CD51) 64 × 106/L Exosomes Microvesicles Apoptopic blebs Inflammation E-selectin and Vascular Coagulation Endothelial Cadherin Angiogenesis Leukocyte(20) 46 × 106/L Exosomes Microvesicles Apoptopic blebs Protein Tyrosine Inflammation Phosphatase Receptor Coagulation Type C Angiogenesis CD14 Erythrocytes (21, 22) 28 × 106/L Microvesicles Glycophorin A Coagulation Table 1: Overview of the cell lines and their vesicles which are mainly present in the bloodstream. HSP 70 ESCRT cd 63 alix Effects attributed to these EVs are activation of the plasma coagulation system and modulation of the vascular tone. Furthermore several studies suggest prothrombotic, pro-inflammatory and proangiogenic activities(23). Platelets Already in 1967, Wolf showed that EVs from platelets, named platelet dust, had the same effect as whole activated platelets(24). Platelet EVs (PEVs) are well known for their procoagulant activity, which is increased by the presence of anionic phospholipids, particularly phosphatidylserine (PS) and tissue factor (TF)(25). PS is a phospholipid which normally resides at the inner layer of the cell membrane. It is capable to form an electrostatic interaction between positively ɣ-carboxyglutamic acid (GLA) domains in clotting proteins like coagulation factor VIIa (26).TF is a transmembrane glycoprotein which serves as a receptor and catalytic cofactor for coagulation factor VIIa and this complex is the major trigger of the coagulation protease cascade(27). TF also plays an important role in tumor survival and progression. It is shed into circulation on tumor derived EVs and promotes intravasation and orchestrates thrombin-, platelet- and fibrin-dependent tumor progression(28). PEVs have also been shown to contribute to vascular contractions, by metabolizing arachidonic acid to tritiated TXB2. Furthermore a study showed that arachidonic acid is able to transfer from ECs to PEVs. Without PEVs, no TXB2 was formed and this is likely because cyclooxygenase is needed, which is present in the EVs(29). Endothelial cells Besides PEVs, lots of research is focused on endothelial-derived EVs (EEVs) and especially their angiogenic properties. Angiogenesis plays an important role in cancer(30) and many other diseases, it is a tightly regulated process that involves endothelial cell survival, proliferation, migration, differentiation and morphological changes, such as tube formation. EEVs have been shown to influence vascular network formation of human umbilical vein endothelial cells (HUVECs). Effects included decreases in total capillary length, number of meshes and the amount of branching points and an increase in mesh area(31). EEVs also affect vascular tone by modulating the NO and PGI2 pathway (32). Some effects of EEVs seem to be dose-dependent as is shown on HUVEC invasiveness(33) and on plasminogen activation(34), in which the latter showed a saturable effect, where high dosages lead to effect inhibition. A clear review about EEVs and their role is written by Leroyer et al(35). Leukocytes Vascular tone is also affected by EVs originating from lymphocytes(36). Besides influencing endothelial cells, EVs from DCs have potent immunoregulatory properties. EVs from immature DCs have shown immunosuppressive therapeutic effects(37), while EVs from mature DCs (DEVs) can be used as immunostimulating vaccines(38). After capturing antigens, DCs incorporate MHC-antigenic peptide complexes in DEVs. After migration to regional lymph nodes, DEVs can present the antigenpeptide complexes to immature DCs, which thereby acquire the ability to stimulate CD4+ and CD8+ T cells. Erythrocytes As is shown in table 1 erythrocytes mostly lack the formation of exosomes and ABs. Their predecessor, reticulocytes, do secrete exosomes, since these EVs are part of the remodeling process, that accounts for the volume shrink, which occurs during erythrocyte maturation(39). MVs are shed from erythrocytes and have, like PEVs, procoagulant activity because of the presence of PS. Besides being procoagulant, it is hypothesized that these EVs are able to transfer their PS to healthy nucleated cells which leads to ‘falsely marked’ apoptotic cells(40). Furthermore it is hypothesized that erythrocyte EVs may not only serve to remove modified hemoglobin, but may also help to eliminate premature removal molecules from otherwise functional erythrocytes(41). Besides vesicle secretion intended for clearance, studies have shown miRNA in erythrocytes(42). Given the general view of erythrocytes lacking protein translation this could imply miRNA regulation through erythrocyte EVs. RNA’s in EVs One of the most intriguing aspects of EVs is their ability to functionally transfer miRNAs and mRNA. The first evidence of encapsulation of mRNAs into EVs was reported by Valadi et al(43) who stated that mast cell exosomes containing mRNA and miRNA from mouse, are transferred to both human and other murine cell lines. After this transferral, new murine proteins were found in the recipient cells, demonstrating that exosomal mRNA can be passed into other cells. Thus, it was established that the message delivered on from the donor cells to the neighboring cells via exosomes, does not simply mirror the transcriptional status of the donor cell. Another study identified and defined the profile of exosome-encapsulated miRNAs circulating in the plasma of healthy individuals, where they found that 37 miRNAs were expressed at significantly different levels between plasma EVs and peripheral blood mononuclear cells, and that the majority of these miRNAs were predicted to regulate cellular differentiation of blood cells and metabolic pathways(44). A third study showed that EVs from human bone marrow-derived mesenchymal stem cells and stem cell residing in the liver contain selective patterns of expression of miRNAs, which could be transferred to target cells, where they down regulated their targets(45). A table with examples of miRNAs involved in cell-to-cell communication is compiled by Redis et al(46). miRNA miR-126 miR-146a (plant) miR-150 miR-223 EBV-miRNAs (BARTs) Cell-to-cell Target Endothelial cells to vascular RGS16 cells Rice plant cells to small intestinal epithelial cells to LDLRAP1 human hepatoma cells Monocyte leukemia cells to c-Myb HMEC-1 cells Tumor-associated macrophages to breast cancer cells (SKBR3, MDA-MB-231) EBV-positive nasopharyngeal carcinoma cells to endothelial cells (HUVECs) Clinical and significance therapeutic ref Activation of tissue repair and angiogenesis (47) Decreased levels of LDL (lowdensity lipoprotein) (48) Promote cell migration (49) The Mef/βRegulation of invasiveness of catenin breast cancer cells pathway (50) CXCL11 and Repression LMP1 immunostimulatory genes (51) Table 2: Examples of miRNAs involved in cell-to-cell communication(46). of MiRNAs are known to regulate the expression of hundreds of target mRNAs, thereby modulating a large number of processes, some of which are described in the table (e.g. angiogenesis, apoptosis and tumor growth). These and other miRNAs might be used to knock down or over express certain proteins in target cells to achieve therapeutic effects, for example by over expressing miR-15a/16-1, to inhibit leukemia cell growth(52). Binding of EVs to their target cells EVs are able to bind and fuse to their target cells. Binding of monocyte EVs to activated platelets occurs through a molecular bridge. After activation, platelets translocate a vascular cell adhesion molecule, P-selectin, to the plasma membrane. P-selectin is a member of the selectin family of adhesion molecules and recognizes a lineage-specific carbohydrate as well as a protein component. P-selectin glycoprotein ligand-1 (PSGL-1) is P-selectins dominant ligand in vivo(53) and this ligand is localized on EVs. Besides a docking function, P-selectin has also been shown to have a signaling function, which up regulates TF expression(54). Molecules present on blood-borne exosomes are lactadherin, CD11a, CD54, PS and tetraspanins CD9 and CD81. These molecules can bind to αv/β3 integrin, CD11a and CD54 on DCs, followed by internalization of the EVs(55). After internalization, exosomes are sorted into recycling endosomes and then through late endosomes/lysosomes. This leads to irreversible maturation, Antigen presentation and ends with apoptotic cell death(56). Immature DCs are better capable to endocytose vesicles than mature DCs. Besides DCs, also specialized splenic macrophages and kupffer cells are able to bind blood-borne exosomes. Target modification Besides natural occurring ligands, studies have shown it is possible to induce targeting by modifying the EVs. A recently developed technique is to fuse targeting peptides to the extra-exosomal N terminus of a protein found abundantly in exosomal membranes(57). In this study they fused three different peptides to murine lysosome-associated membrane protein 2b (Lamp2b), which is a type 1 integral membrane protein with a large luminal domain which is heavily glycosylated(58). The peptides used were a FLAG epitope, rabies viral glycoprotein (RVG) peptide and a muscle-specific peptide (MSP). The first peptide fused to Lamp2b was FLAG. This is a small peptide consisting of 8 nucleotides which can be targeted with commercial available antibodies(59). This peptide was used to determine the topology of Lamp2b and a pulldown assay showed the epitope is localized to the external exosomal surface, but FLA is not used as a targeting peptide. The second peptide, The 29- amino-acid RVG peptide, specifically binds to the acetylcholine receptor expressed by neuronal cells and provides a non invasive approach for the delivery across the blood-brain barrier(60) and the third peptide, MSP, is a heptapeptide (ASSLNIA) which is identified by in vivo phage display and which has an increased binding affinity for skeletal and cardiac muscle(61). Besides (ASSLNIA), even more specific MSPs have been found, which can target for example specifically laryngeal muscle surface receptors(62). Other targeting peptides are being investigated as well. Many viruses are specialized in binding and transducing information to host cells. Proteins which are responsible for this effect can be incorporated in vesicles and this is called pseudotyping. An often used protein is the spike glycoprotein of the vesicular stomatitis virus (VSV-G). Over expression of VSV-G in human cells induces the release of fusogenic vesicles named gesicles. These gesicles have a number of similarities, like the sites of budding(63), the molecular content and the targeting signals(64), with EVs. Gesicle formation is , at its core, a form of EV biogenesis(65). VSV-G is able to attach to a broad range of host cells(66) and these gesicles incorporate proteins from their producer cells and are able to deliver them to recipient cells. This protein delivery technique has been used to transfer membrane, cytoplasmic and nuclear proteins in precise amounts (67). Nuclear proteins were efficient in activating transcription, but for increased performances a fusion to the H-RAS c terminus was needed, since this motif is farnelysed in vivo and redirects proteins to the cell membrane. Another study which used over expression of VSV-G, tried to include a second targeting peptide, namely biotin acceptorpeptide- transmembrane domain (BAP-TM). This genetically engineered receptor allows binding to biotinylated ligands via a streptavidin bridge(68). After incubation with streptavidin-conjugated magnetic beads, it is possible to attract exosomes with a magnetic field, which can be used for specific targeting(69). These and other techniques could be used to target drug loaded vesicles to their destinations, which will lead to great improvements to their pharmacokinetics. Conclusion Only recently have we learned that EVs are more than waste and cell debris. EVs are continuously present in the bloodstream, where they provide communication between cells and help to maintain homeostasis. Besides maintaining homeostasis they also play an important role in pathological states and the development of cancer. Here we discussed the most important and prevalent EVs in circulation. Evs from platelets, ECs, leukocytes and erythrocytes are always present and this should be taken in mind when developing experimental, diagnostic or therapeutic tests. Several important effects of EVs originating from these cell types are highlighted and their RNA content is discussed. Furthermore, target binding is elucidated and target modification is discussed. By fusing targeting proteins or pseudotyping, the binding of EVs to their target cells can be improved. These techniques can further improve the delivery of miRNAs/mRNAs or therapeutic proteins. All in all this upcoming research field is very promising in understanding the cancer microenvironment and can give rise to many breakthroughs in the field of diagnostics, gene silencing and drug delivery. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Gyorgy, B., Szabo, T. G., Pasztoi, M., Pal, Z., Misjak, P., Aradi, B., Laszlo, V., Pallinger, E., Pap, E., Kittel, A., Nagy, G., Falus, A., and Buzas, E. I. (2011) Membrane vesicles, current state-ofthe-art: emerging role of extracellular vesicles, Cell Mol Life Sci 68, 2667-2688. Stoorvogel, W., Kleijmeer, M. J., Geuze, H. J., and Raposo, G. (2002) The biogenesis and functions of exosomes, Traffic 3, 321-330. Distler, J. H., Pisetsky, D. S., Huber, L. C., Kalden, J. R., Gay, S., and Distler, O. (2005) Microparticles as regulators of inflammation: novel players of cellular crosstalk in the rheumatic diseases, Arthritis Rheum 52, 3337-3348. Lane, J. D., Allan, V. J., and Woodman, P. G. (2005) Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells, J Cell Sci 118, 4059-4071. Thery, C., Ostrowski, M., and Segura, E. (2009) Membrane vesicles as conveyors of immune responses, Nat Rev Immunol 9, 581-593. Fevrier, B., and Raposo, G. (2004) Exosomes: endosomal-derived vesicles shipping extracellular messages, Curr Opin Cell Biol 16, 415-421. Sudhof, T. C., and Rizo, J. (2011) Synaptic vesicle exocytosis, Cold Spring Harb Perspect Biol 3. Savina, A., Furlan, M., Vidal, M., and Colombo, M. I. (2003) Exosome release is regulated by a calcium-dependent mechanism in K562 cells, J Biol Chem 278, 20083-20090. Yu, X., Harris, S. L., and Levine, A. J. (2006) The regulation of exosome secretion: a novel function of the p53 protein, Cancer Res 66, 4795-4801. Lee, T. H., D'Asti, E., Magnus, N., Al-Nedawi, K., Meehan, B., and Rak, J. (2011) Microvesicles as mediators of intercellular communication in cancer--the emerging science of cellular 'debris', Semin Immunopathol 33, 455-467. Janowska-Wieczorek, A., Wysoczynski, M., Kijowski, J., Marquez-Curtis, L., Machalinski, B., Ratajczak, J., and Ratajczak, M. Z. (2005) Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer, Int J Cancer 113, 752-760. Cocucci, E., Racchetti, G., and Meldolesi, J. (2009) Shedding microvesicles: artefacts no more, Trends Cell Biol 19, 43-51. Simpson, R. J., Lim, J. W., Moritz, R. L., and Mathivanan, S. (2009) Exosomes: proteomic insights and diagnostic potential, Expert Rev Proteomics 6, 267-283. Abid Hussein, M. N., Boing, A. N., Sturk, A., Hau, C. M., and Nieuwland, R. (2007) Inhibition of microparticle release triggers endothelial cell apoptosis and detachment, Thromb Haemost 98, 1096-1107. Simpson, R. J., Jensen, S. S., and Lim, J. W. (2008) Proteomic profiling of exosomes: current perspectives, Proteomics 8, 4083-4099. Berckmans, R. J., Nieuwland, R., Boing, A. N., Romijn, F. P., Hack, C. E., and Sturk, A. (2001) Cell-derived microparticles circulate in healthy humans and support low grade thrombin generation, Thromb Haemost 85, 639-646. Heijnen, H. F., Schiel, A. E., Fijnheer, R., Geuze, H. J., and Sixma, J. J. (1999) Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules, Blood 94, 3791-3799. Sprague, D. L., Elzey, B. D., Crist, S. A., Waldschmidt, T. J., Jensen, R. J., and Ratliff, T. L. (2008) Platelet-mediated modulation of adaptive immunity: unique delivery of CD154 signal by platelet-derived membrane vesicles, Blood 111, 5028-5036. Dignat-George, F., and Boulanger, C. M. (2011) The many faces of endothelial microparticles, Arterioscler Thromb Vasc Biol 31, 27-33. Lacroix, R., Plawinski, L., Robert, S., Doeuvre, L., Sabatier, F., Martinez de Lizarrondo, S., Mezzapesa, A., Anfosso, F., Leroyer, A., Poullin, P., Jourde, N., Njock, M. S., Boulanger, C., Angles-Cano, E., and Dignat-George, F. (2012) Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis, Haematologica. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. Nantakomol, D., Dondorp, A. M., Krudsood, S., Udomsangpetch, R., Pattanapanyasat, K., Combes, V., Grau, G. E., White, N. J., Viriyavejakul, P., Day, N. P., and Chotivanich, K. (2011) Circulating red cell-derived microparticles in human malaria, J Infect Dis 203, 700-706. van Beers, E. J., Schaap, M. C., Berckmans, R. J., Nieuwland, R., Sturk, A., van Doormaal, F. F., Meijers, J. C., and Biemond, B. J. (2009) Circulating erythrocyte-derived microparticles are associated with coagulation activation in sickle cell disease, Haematologica 94, 1513-1519. Simak, J., and Gelderman, M. P. (2006) Cell membrane microparticles in blood and blood products: potentially pathogenic agents and diagnostic markers, Transfus Med Rev 20, 1-26. Wolf, P. (1967) The nature and significance of platelet products in human plasma, Br J Haematol 13, 269-288. Owens, A. P., 3rd, and Mackman, N. (2011) Microparticles in hemostasis and thrombosis, Circ Res 108, 1284-1297. van de Waart, P., Bruls, H., Hemker, H. C., and Lindhout, T. (1983) Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles, Biochemistry 22, 2427-2432. Ruf, W., and Mueller, B. M. (1996) Tissue factor in cancer angiogenesis and metastasis, Curr Opin Hematol 3, 379-384. Ruf, W., Yokota, N., and Schaffner, F. (2010) Tissue factor in cancer progression and angiogenesis, Thromb Res 125 Suppl 2, S36-38. Pfister, S. L. (2004) Role of platelet microparticles in the production of thromboxane by rabbit pulmonary artery, Hypertension 43, 428-433. Carmeliet, P., and Jain, R. K. (2000) Angiogenesis in cancer and other diseases, Nature 407, 249-257. Mezentsev, A., Merks, R. M., O'Riordan, E., Chen, J., Mendelev, N., Goligorsky, M. S., and Brodsky, S. V. (2005) Endothelial microparticles affect angiogenesis in vitro: role of oxidative stress, Am J Physiol Heart Circ Physiol 289, H1106-1114. Brodsky, S. V., Zhang, F., Nasjletti, A., and Goligorsky, M. S. (2004) Endothelium-derived microparticles impair endothelial function in vitro, Am J Physiol Heart Circ Physiol 286, H1910-1915. Taraboletti, G., D'Ascenzo, S., Borsotti, P., Giavazzi, R., Pavan, A., and Dolo, V. (2002) Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicle-associated components by endothelial cells, Am J Pathol 160, 673-680. Lacroix, R., Sabatier, F., Mialhe, A., Basire, A., Pannell, R., Borghi, H., Robert, S., Lamy, E., Plawinski, L., Camoin-Jau, L., Gurewich, V., Angles-Cano, E., and Dignat-George, F. (2007) Activation of plasminogen into plasmin at the surface of endothelial microparticles: a mechanism that modulates angiogenic properties of endothelial progenitor cells in vitro, Blood 110, 2432-2439. Leroyer, A. S., Anfosso, F., Lacroix, R., Sabatier, F., Simoncini, S., Njock, S. M., Jourde, N., Brunet, P., Camoin-Jau, L., Sampol, J., and Dignat-George, F. (2010) Endothelial-derived microparticles: Biological conveyors at the crossroad of inflammation, thrombosis and angiogenesis, Thromb Haemost 104, 456-463. Martin, S., Tesse, A., Hugel, B., Martinez, M. C., Morel, O., Freyssinet, J. M., and Andriantsitohaina, R. (2004) Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression, Circulation 109, 1653-1659. Yang, C., and Robbins, P. D. (2012) Immunosuppressive exosomes: a new approach for treating arthritis, Int J Rheumatol 2012, 573528. Morse, M. A., Garst, J., Osada, T., Khan, S., Hobeika, A., Clay, T. M., Valente, N., Shreeniwas, R., Sutton, M. A., Delcayre, A., Hsu, D. H., Le Pecq, J. B., and Lyerly, H. K. (2005) A phase I study of dexosome immunotherapy in patients with advanced non-small cell lung cancer, J Transl Med 3, 9. Spinella, P. C., Sparrow, R. L., Hess, J. R., and Norris, P. J. (2011) Properties of stored red blood cells: understanding immune and vascular reactivity, Transfusion 51, 894-900. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. Liu, R., Klich, I., Ratajczak, J., Ratajczak, M. Z., and Zuba-Surma, E. K. (2009) Erythrocytederived microvesicles may transfer phosphatidylserine to the surface of nucleated cells and falsely 'mark' them as apoptotic, Eur J Haematol 83, 220-229. Willekens, F. L., Werre, J. M., Groenen-Dopp, Y. A., Roerdinkholder-Stoelwinder, B., de Pauw, B., and Bosman, G. J. (2008) Erythrocyte vesiculation: a self-protective mechanism?, Br J Haematol 141, 549-556. Hamilton, A. J. (2010) MicroRNA in erythrocytes, Biochem Soc Trans 38, 229-231. Valadi, H., Ekstrom, K., Bossios, A., Sjostrand, M., Lee, J. J., and Lotvall, J. O. (2007) Exosomemediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9, 654-659. Hunter, M. P., Ismail, N., Zhang, X., Aguda, B. D., Lee, E. J., Yu, L., Xiao, T., Schafer, J., Lee, M. L., Schmittgen, T. D., Nana-Sinkam, S. P., Jarjoura, D., and Marsh, C. B. (2008) Detection of microRNA expression in human peripheral blood microvesicles, PLoS One 3, e3694. Collino, F., Deregibus, M. C., Bruno, S., Sterpone, L., Aghemo, G., Viltono, L., Tetta, C., and Camussi, G. (2010) Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs, PLoS One 5, e11803. Redis, R. S., Calin, S., Yang, Y., You, M. J., and Calin, G. A. Cell-to-cell miRNA transfer: From body homeostasis to therapy, Pharmacology &amp; Therapeutics. Zernecke, A., Bidzhekov, K., Noels, H., Shagdarsuren, E., Gan, L., Denecke, B., Hristov, M., Koppel, T., Jahantigh, M. N., Lutgens, E., Wang, S., Olson, E. N., Schober, A., and Weber, C. (2009) Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection, Sci Signal 2, ra81. Zhang, L., Hou, D., Chen, X., Li, D., Zhu, L., Zhang, Y., Li, J., Bian, Z., Liang, X., Cai, X., Yin, Y., Wang, C., Zhang, T., Zhu, D., Zhang, D., Xu, J., Chen, Q., Ba, Y., Liu, J., Wang, Q., Chen, J., Wang, J., Wang, M., Zhang, Q., Zhang, J., Zen, K., and Zhang, C. Y. (2012) Exogenous plant MIR168a specifically targets mammalian LDLRAP1: evidence of cross-kingdom regulation by microRNA, Cell Res 22, 107-126. Zhang, Y., Liu, D., Chen, X., Li, J., Li, L., Bian, Z., Sun, F., Lu, J., Yin, Y., Cai, X., Sun, Q., Wang, K., Ba, Y., Wang, Q., Wang, D., Yang, J., Liu, P., Xu, T., Yan, Q., Zhang, J., Zen, K., and Zhang, C. Y. (2010) Secreted monocytic miR-150 enhances targeted endothelial cell migration, Mol Cell 39, 133-144. Yang, M., Chen, J., Su, F., Yu, B., Lin, L., Liu, Y., Huang, J. D., and Song, E. (2011) Microvesicles secreted by macrophages shuttle invasion-potentiating microRNAs into breast cancer cells, Mol Cancer 10, 117. Pegtel, D. M., Cosmopoulos, K., Thorley-Lawson, D. A., van Eijndhoven, M. A., Hopmans, E. S., Lindenberg, J. L., de Gruijl, T. D., Wurdinger, T., and Middeldorp, J. M. (2010) Functional delivery of viral miRNAs via exosomes, Proc Natl Acad Sci U S A 107, 6328-6333. Calin, G. A., Cimmino, A., Fabbri, M., Ferracin, M., Wojcik, S. E., Shimizu, M., Taccioli, C., Zanesi, N., Garzon, R., Aqeilan, R. I., Alder, H., Volinia, S., Rassenti, L., Liu, X., Liu, C. G., Kipps, T. J., Negrini, M., and Croce, C. M. (2008) MiR-15a and miR-16-1 cluster functions in human leukemia, Proc Natl Acad Sci U S A 105, 5166-5171. Yang, J., Hirata, T., Croce, K., Merrill-Skoloff, G., Tchernychev, B., Williams, E., Flaumenhaft, R., Furie, B. C., and Furie, B. (1999) Targeted gene disruption demonstrates that P-selectin glycoprotein ligand 1 (PSGL-1) is required for P-selectin-mediated but not E-selectinmediated neutrophil rolling and migration, J Exp Med 190, 1769-1782. Furie, B., and Furie, B. C. (2004) Role of platelet P-selectin and microparticle PSGL-1 in thrombus formation, Trends Mol Med 10, 171-178. Morelli, A. E., Larregina, A. T., Shufesky, W. J., Sullivan, M. L., Stolz, D. B., Papworth, G. D., Zahorchak, A. F., Logar, A. J., Wang, Z., Watkins, S. C., Falo, L. D., Jr., and Thomson, A. W. (2004) Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells, Blood 104, 3257-3266. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V. S., Davoust, J., and Ricciardi-Castagnoli, P. (1997) Maturation stages of mouse dendritic cells in growth factor-dependent long-term cultures, J Exp Med 185, 317-328. Alvarez-Erviti, L., Seow, Y., Yin, H., Betts, C., Lakhal, S., and Wood, M. J. (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes, Nat Biotechnol 29, 341-345. Lichter-Konecki, U., Moter, S. E., Krawisz, B. R., Schlotter, M., Hipke, C., and Konecki, D. S. (1999) Expression patterns of murine lysosome-associated membrane protein 2 (Lamp-2) transcripts during morphogenesis, Differentiation 65, 43-58. Einhauer, A., and Jungbauer, A. (2001) The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins, J Biochem Biophys Methods 49, 455-465. Kumar, P., Wu, H., McBride, J. L., Jung, K. E., Kim, M. H., Davidson, B. L., Lee, S. K., Shankar, P., and Manjunath, N. (2007) Transvascular delivery of small interfering RNA to the central nervous system, Nature 448, 39-43. Samoylova, T. I., and Smith, B. F. (1999) Elucidation of muscle-binding peptides by phage display screening, Muscle Nerve 22, 460-466. Flint, P. W., Li, Z. B., Lehar, M., Saito, K., and Pai, S. I. (2005) Laryngeal muscle surface receptors identified using random phage library, Laryngoscope 115, 1930-1937. Booth, A. M., Fang, Y., Fallon, J. K., Yang, J. M., Hildreth, J. E., and Gould, S. J. (2006) Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane, J Cell Biol 172, 923-935. Nguyen, D. G., Booth, A., Gould, S. J., and Hildreth, J. E. (2003) Evidence that HIV budding in primary macrophages occurs through the exosome release pathway, J Biol Chem 278, 5234752354. Shen, B., Wu, N., Yang, J. M., and Gould, S. J. (2011) Protein targeting to exosomes/microvesicles by plasma membrane anchors, J Biol Chem 286, 14383-14395. Akkina, R. K., Walton, R. M., Chen, M. L., Li, Q. X., Planelles, V., and Chen, I. S. (1996) Highefficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G, J Virol 70, 2581-2585. Mangeot, P. E., Dollet, S., Girard, M., Ciancia, C., Joly, S., Peschanski, M., and Lotteau, V. (2011) Protein transfer into human cells by VSV-G-induced nanovesicles, Mol Ther 19, 16561666. Tannous, B. A., Grimm, J., Perry, K. F., Chen, J. W., Weissleder, R., and Breakefield, X. O. (2006) Metabolic biotinylation of cell surface receptors for in vivo imaging, Nat Methods 3, 391-396. Maguire, C. A., Balaj, L., Sivaraman, S., Crommentuijn, M. H., Ericsson, M., Mincheva-Nilsson, L., Baranov, V., Gianni, D., Tannous, B. A., Sena-Esteves, M., Breakefield, X. O., and Skog, J. (2012) Microvesicle-associated AAV Vector as a Novel Gene Delivery System, Mol Ther 20, 960-971.