Archives of Biochemistry and Biophysics 561 (2014) 38–45

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Archives of Biochemistry and Biophysics

journal homepage: www.elsevier .com/ locate /yabbi

Review

Extracellular vesicles: Specialized bone messengers

http://dx.doi.org/10.1016/j.abb.2014.05.0110003-9861/� 2014 Published by Elsevier Inc.

⇑ Corresponding author. Fax: +31 (10)7032603.E-mail addresses: j.morhayim@erasmusmc.nl (J. Morhayim), m.baroncelli@

erasmusmc.nl (M. Baroncelli), j.vanleeuwen@erasmusmusmc.nl (J.P. van Leeuwen).1 Abbreviations used: EVs, extracellular vesicles; MSCs, mesenchymal stem cells;

MVBs, multivesicular bodies; ESCRT, endosomal sorting complex required forreceptor transport.

Jess Morhayim, Marta Baroncelli, Johannes P. van Leeuwen ⇑Department of Internal Medicine, Erasmus University Medical Center, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history:Received 3 March 2014and in revised form 16 April 2014Available online 22 May 2014

Keywords:Extracellular vesiclesIntercellular communicationBioactive cargoBone

Mammalian cells actively secrete factors that contribute to shape their microenvironment. These factorseither travel freely or they are enclosed within the lipid bilayer of extracellular vesicles (EVs), and regu-late the function of neighboring and distant cells. EVs are secreted by a wide spectrum of cell types andare found in various biological fluids. They convey their message by mediating the horizontal transfer ofbioactive molecules, such as proteins, mRNAs and miRNAs, between cells. Recent studies showed the vitalroles of EVs in a wide range of physiological and pathophysiological processes. In this review, we high-light the recent developments in the newly emerging EV field, including their biogenesis, molecular con-tent and function. Moreover, we discuss the role of EVs in bone biology and their promising applicationsin diagnosis, drug development and regenerative therapy.

� 2014 Published by Elsevier Inc.

Introduction

Multicellular organisms developed complex communicationnetworks to regulate biological activities, and consequently main-tain physiological homeostasis. Interruptions in intercellularcommunication lead to pathological complications and diseaseprogression. Cells exchange information with each other via immo-bilized molecules as well as secreted factors. Traditionally, secretedfactors include small soluble molecules such as neurotransmitters,chemokines, cytokines and hormones that can either act over shortdistances and affect the neighboring cells in a paracrine manner ortravel long distances in an endocrine manner. The last couple ofdecades have witnessed the unprecedented dedication of scientiststo the study of extracellular vesicles (EVs)1 as novel mediators ofintercellular communication.

EVs had long been regarded as cellular debris until Wolf andcolleagues described their biological significance in coagulationin 1967 [1]. Later same year, Anderson and Bonucci reported thediscovery of matrix vesicles as specialized EVs involved in biomin-eralization of the bone matrix [2,3]. Since then, EVs have beendescribed to have a broad range of functions in development,immunology, angiogenesis and stem cell biology, as well as diseaseprogression. EVs are considered to be bioactive organelles that

carry genetic information in forms of lipids, proteins and nucleicacids between cells, and have effects on diverse molecular func-tions, such as signaling and regulation of gene expression of thetarget cells [4–8]. EVs embrace a heterogeneous group of vesicles,including microvesicles, microparticles, ectosomes, exosomes,shedding vesicles, apoptotic bodies and many others releasedunder different biological circumstances. Even though the differentnames reflect their diversity in terms of biogenesis, structure, con-tent and function, there are still conflicts over the definition andcharacterization of different vesicular structures. For simplicity,EVs are often categorized in three classes based on the well-defined processes for EV biogenesis: small vesicles (10–100 nm)released by exocytosis (exosomes, exosome-like vesicles), biggervesicles (100–1000 nm) formed by budding from the plasmamembrane (microvesicles, shed vesicles, matrix vesicles) and bigvesicles (0.8–5 lm) released from dying cells (apoptotic bodies)(Fig. 1) [8–10].

Cells release EVs either constitutively during their growth orupon activation by biological stimuli. Apoptotic and diseased cellsalso release EVs providing valuable information about the healthstate of the cell [10,11]. EVs were detected in all studied cell typesso far and most biological fluids, including blood, urine and breastmilk [12–14]. Osteoblasts, the bone forming cells, also secrete EVsthat have well characterized roles in mineralization [15]. There isincreasing knowledge about the biological functions of EVs in otherbone-related processes. Particularly, the regenerative role of mes-enchymal stem cell (MSC) EVs makes them promising therapeuticagents for bone regenerative medicine [16–18]. In this chapter, wewill review the various roles of EVs in diverse aspects of intercellu-lar communication and highlight their function in bone biology.

ExtracellularVesicles

Cancer

Nervous System

Stem Cell Biology

Inflammation

Coagulation

Angiogenesis

Bone Remodeling

Immune ResponseDevelopment

Donor Cell

Apoptotic Cell Target Cell

i

ii

iii

iv

Apoptotic Bodies (0.8-5 µm)

Microvesicles (100-1000 nm)

Exosomes (10-100 nm)

Fig. 1. Schematic representation of EV release from donor cells and the interaction with the target cells. EVs are released either by exocytosis of the multivesicular bodies ordirect budding from the plasma membrane. Apoptotic bodies are released from breakdown of the apoptotic cells. EVs interact with their targets via (i) signal transductionmediated by docking at the plasma membrane of the target cell and/ or via releasing the bioactive cargo upon (ii) fusion or (iii–iv) endocytosis followed by fusing with thedelimiting membrane of the endosomal compartment. Interaction with target cells leads to a broad range of biological functions. Interaction with the target cell is only shownfor microvesicles but exosomes and apoptotic bodies utilize similar mechanisms.

J. Morhayim et al. / Archives of Biochemistry and Biophysics 561 (2014) 38–45 39

Isolation and characterization

With the rapid development of EV research, the optimizationand standardization of isolation techniques have become of utmostimportance to improve the understanding of EV biology. Currently,the most commonly applied EV isolation method relies on differen-tial ultracentrifugation protocol developed by Thery and colleaguesin 2006 [19]. According to this protocol, biological fluids or theconditioned media from cultured cells are subjected to a series ofcentrifugation and ultracentrifugation steps to pellet the EVs. Cellsbreak open at high speeds, and as a consequence cellular organ-elles and big protein complexes are co-isolated with EVs. There-fore, it is very important to start with a low centrifugation speedto remove floating and/or dead cells, and perform serial centrifuga-tion steps in increasing speeds to remove the contaminants in astep-wise fashion. Furthermore, it is crucial that the purified EVpellet is free of bovine serum contaminants, such as serum proteinsand vesicles. For cell cultures it is common to use EV-depletedserum and/or serum-free medium treatment before EV collection.This becomes, indeed, more challenging with biological fluids.The duration and speed of each centrifugation step varies betweendifferent protocols based on the EV source. Big microvesicles andapoptotic bodies are commonly pelleted using 10,000g centrifuga-tion, while smaller microvesicles and exosomes require high speedcentrifugation at about 100,000g [6,19]. The crude EV pellet can besubjected to further purification by sucrose density gradient orhigh performance liquid chromatography to remove possible con-taminants. There have been attempts to separate EVs with differ-ent sizes based on their sediment density by sucrose densitygradient. Recent studies have reported the sediment densities as

the following: 1.11–1.21 g/ml for exosomes [20–22], 1.25–1.30 g/ml for microvesicles [23], and 1.18–1.28 g/ml for apoptotic bodies[24]. However, some small microvesicles can have even lighterdensities than exosomes making the separation of the differentEV populations very difficult using the conventional techniques [6].

Purified EVs are commonly characterized by microscopic, bio-chemical and fluidic analyses. EVs can be visualized by electronmicroscopy, which gives clues about their size and morphology(Fig. 2A). In the past, researchers used electron microscopy to ver-ify the presence of exosomes based on their cup shape morphol-ogy; however, it was recently discovered that this was due todehydration during sample processing [25,26]. Atomic forcemicroscopy, on the other hand, allows samples to be in their nativestate, and hence it proves to be a better alternative to study themorphology of different EVs (Fig. 2B)[27]. Other common methodsto study EVs include western blot and flow cytometry analysesusing known vesicle markers. On-going studies are focusing onidentifying markers specific to unique EV classes. A major chal-lenge of EV characterization is the accurate quantification. ELISAand immunoadsorption are possible methods to determine theabundance of EVs using known markers; however, they do not givecomprehensive information about the total EV concentration [28].Conventional flow cytometry is a useful technique to quantify bigEVs, while accurate quantification of EVs smaller than 250 nm isdifficult. Van der Vlist and colleagues developed a high-resolutionflow cytometry-based technique that can be used to detect smallerEVs based on their fluorescence intensity [29]. Recently, severalcompanies developed techniques that allow multi-parameter anal-ysis of EVs of certain size ranges providing greater accuracy thanfluidic-based techniques. Nanoparticle tracking analysis measures

(A) Topography – Scan Forward (B)

Mea

n fi

t 226

nm

Fig. 2. EV morphology and topography. EVs isolated from mineralizing human fetal pre-osteoblasts (SV-HFO cells) were fixed with glutaraldehyde and analyzed by (A)transmission electron microscopy (TEM), scale bar: 500 nm, and (B) atomic force microscopy (non-contact mode), scale bar: 223 nm. Both images show that EVs areheterogeneous in size and have spherical morphology.

Table 1The most commonly identified EV proteins described in ExoCartaa.

Protein family Gene name

Heat shock proteins HSPA8HSP90AA1HSP90AB1

Tetraspanins CD9CD63CD81

Metabolic enzymes ALDOAENO1GAPDHLDHAPGK1PKM2

Annexins ANXA2ANXA5

Cytoskeletal and associated proteins ACTBACTG1CFL1MSN

Elongation factors EEF1A1EEF2

14-3-3 Family proteins YWHAEYWHAZ

Other ALBPDCD6IPSDCBP

a ExoCarta: http://www.exocarta.org/.

40 J. Morhayim et al. / Archives of Biochemistry and Biophysics 561 (2014) 38–45

concentration and absolute size distribution of particles as small as50 nm based on their Brownian motion in fluids [27,30]. qNano isanother method that uses nanopores to measure size distributionof EVs bigger than 100 nm [31].

Molecular composition

EVs are small cell fractions consisting of a lipid bilayer enclosedlumen that contains vesicular cargo, which is a selective combina-tion of lipids, proteins and nucleic acids essential for EV structureand function. Even though the parent cell and EV biogenesis arethe contributing factors to cargo differences, most of the structuralcargo is shared among EVs released by different cells. Comprehen-sive understanding of the molecular composition of different EVpopulations is crucial to understand their molecular function. Pro-teomics, lipidomics, microarrays and deep sequencing analyseshave been applied to characterize the molecular content of EVsfrom diverse cellular sources. ExoCarta and EVpedia are the twonew databases that list the entire lipid, protein and RNA contentof EVs described in the literature [32–37].

LipidsEVs are formed by a specific selection of lipids organized in a

bilayer membrane that provides structure and protects the bioac-tive cargo from degradation before they reach their targets. MostEVs are enriched with structural lipids, such as cholesterol, sphin-gomyelin and phosphatidylserine, compared to the cellular plasmamembrane [38–40]. The mode of biogenesis greatly affects thelipid composition of a given EV class. Exosomes are enriched withendosomal phospholipid bis-monoacylglycero-phosphate, whereasmicrovesicles contain plasma membrane levels of phosphatidyl-choline and are devoid of phospholipid bis-monoacylglycero-phos-phate [41,42]. Lipids are not only the structural elements of EVsbut they also contribute to their bioactivity. Vesicular lipids playpivotal roles in EV biogenesis, release, interaction with other cellsand biological function. Lipid rafts formed by cholesterol andsphingomyelin are shown to be important for clathrin pit forma-tion that mediates exosome biogenesis [43]. These lipid rafts canalso act as signaling complexes along with the lipid-associated pro-teins. Furthermore, exosomes contain prostaglandins and leukotri-enes important to mediate cell signaling [44,45]. Transfer ofvesicular lipids to recipient cells may lead to changes in cellhomeostasis caused by the accumulation of lipids and their associ-ated enzymes. For instance, T cells release EVs that upon uptake bymonocytes result in cholesterol accumulation leading to pheno-typic alterations [22].

ProteinsAs with lipids, EVs are enriched with proteins that contribute to

the biogenesis, structure, motility and the biological function ofEVs. The most commonly identified protein cargo includes cyto-skeletal proteins, tetraspanins, membrane and nuclear receptors,heat shock proteins, proteases, adhesion molecules, signaling mol-ecules, metabolic enzymes, annexins, transporters and ion chan-nels. Table 1 lists the top 25 identified EV proteins as describedby ExoCarta [32,34]. Despite the large number of shared proteinsamong different EV classes, the mode of biogenesis determinesthe enrichment of endosomal-associated proteins and plasmamembrane proteins in exosomes and microvesicles, respectively[5,46]. Tetraspanins that cluster at the site of exocytosis andplasma membrane budding act as sorting machineries to targetproteins into EVs [47,48]. Chaperones such as heat shock proteins

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coupled to tetraspanins also aid in sorting lumen proteins. Cyto-skeleton proteins are crucial for EV release, structure, and motility[49]. EVs are particularly enriched in cellular markers based on thecell of origin. For instance, tumor EVs are enriched with metallo-proteinases and other proteolytic enzymes involved in the diges-tion of extracellular matrix necessary for invasion and tumorprogression [50,51].

Nucleic acidsThe discovery of EV-associated nucleic acids with regulatory

capacity greatly contributed to understand the role of EVs ingene-based cell communication. In late ‘90s, EVs co-isolated withviruses were already suggested to contain RNA [52]. A decade later,researchers found mRNA for chemokines and growth factors intumor EVs derived from different cancer cell lines [53]. In 2007,Valadi and colleagues showed that mast cell exosomes wereenriched with miRNAs [20]. Since then there have been a greatamount of research demonstrating the presence of functionalRNA molecules in EVs derived from cell cultures and biological flu-ids [54–59]. Bioanalyzer profiles showed that EVs were devoid ofthe cellular rRNA peaks, instead they were enriched with smallRNAs [20]. Deep sequencing analyses identified mainly the pres-ence of mRNAs and miRNAs but also other small non-coding RNAmolecules such as tRNA, vault RNA, YRNA, small interfering RNA,repeat sequences, structural RNA and RNA transcripts overlappingwith protein coding regions within EVs [60,61]. Turchinovich andcolleagues demonstrated that miRNAs associated with the outermembrane of EVs also had important implications in EV function[62]. Even though most EV preparations were devoid of DNA, sev-eral groups reported the presence of functional DNA in apoptoticbodies secreted by cancer cells as a way of transferring their acti-vated oncogenes [11,63].

Biogenesis and cargo loading

In spite of the recent progress in elucidation of EV biology, theexact process of EV biogenesis and cargo sorting is still not known;however the amount of data is fortunately increasing. EVs are gen-erated via diverse biological mechanisms triggered by microenvi-ronmental stimuli, cellular activation, stress, transformation andprogrammed cell death. We know at least three distinct mecha-nisms of EV biogenesis: exocytosis, direct budding from the plasmamembrane, and breakdown of dying cells, each leading to therelease of distinct EV classes.

Exosomes and exosome-like vesicles are small vesicles (10–100 nm) that originate from exocytosis of multivesicular bodies(MVBs) [64,65]. Vesicle budding into exosomal MVB lumen is stillunder investigation, nevertheless there are studies suggesting thatexosome formation follows a mechanism parallel to endosomaldegradation pathway. Cellular cargo destined for degradation inlysosomes are selectively sorted into vesicles that bud into MVBlumen under the control of endosomal sorting complex requiredfor receptor transport (ESCRT) and their associated proteins, suchas Alix and VPS4 [66,67]. ESCRT subunits ESCRT-0, -I, and -II recog-nize and control the destination of ubiquitinated cargo proteins atthe endosomal delimiting membrane. Silencing of ESCRT complexcomponents and/or their associated proteins reduced exosomesecretion suggesting that ESCRT-mediated sorting plays a role inexosome formation but possibly via a different mechanism thanthe degradation pathway [68]. Exosomes are also released in alipid-dependent manner independently from the ESCRT pathway,depending on cell type and stimuli. Trajkovic and colleaguesshowed that ceramides that were concentrated around lipid-raftsof oligodendroglial cells sort exosomal cargo into vesicles andmediate budding into the MVB lumen [69]. The importance of lip-ids is further supported by the findings showing that that exosomal

MVBs were rich in cholesterol and phosphatidylserine on the outerlipid leaflet, while degradative MVBs were cholesterol poor, butrich in lysobisphosphatidic acid [28]. There is evidence showingthat cells can produce exosomes independently of both ESCRTcomplex and ceramides. For instance, tetraspanins are also sug-gested to play a role in exosome sorting [48]. The process ofMVB fusion with plasma membrane and release of exosomes is stillunclear. It is noted that exosome release process is a cytoskeletondependent and p53-controlled process [70]. Furthermore, ESCRT-III subunit and GTPases, such as Rab5, Rab27 and Rab35, have beenimplicated to play roles in exosome release [6,71].

The molecular mechanisms of microvesicle and apoptotic bodybiogenesis are far less understood than exosome biogenesis. Directbudding from the plasma membrane releases a heterogeneous EVpopulation with a size range between 100 and 1000 nm [43]. TheseEVs are commonly referred to as microvesicles as well as shed ves-icles, and to a lesser extent microparticles. Microvesicles contain aspecific group of plasma membrane proteins suggesting that bud-ding occurs at specific parts of the membrane. As with exosomes,cholesterol-rich lipid rafts enriched with ceramides, cholesteroland other lipids are described to be involved in biogenesis of micr-ovesicles [72,73]. It has been reported that increase in cytosoliccalcium leads to exposure of phosphatidylserine residues, whichin turn causes budding from the plasma membrane [74]. Severalreports suggest the involvement of the ESRCT complex indicatingthat microvesicle budding might be similar to exosome buddinginto MVBs [75]. Actin-based motors are also proposed to beinvolved in the detachment of the microvesicle bud from theplasma membrane [76]. Apoptotic bodies, big vesicles with a diam-eter range between 0.8 and 5 lm, are released from cellular break-down of dying cells [10]. These EVs are usually engulfed byphagocytic cells and quickly removed before the contents can bespilled out to the surrounding cells and cause damage. Studiesshowing their role in intercellular communication suggest thatthey can escape phagocyte ingestion and target neighboring cells;however, the molecular mechanism of apoptotic body targeting isstill unknown [63].

Interactions with target cells

EVs originate from their parent cells, and travel in extracellularspace until they interact with their target cells. The literature con-tains an increasing number of studies verifying the specificity of EVtarget interaction and subsequent biological effect. [77,78]. Eventhough the molecular mechanism of EV targeting is not yet welldescribed, it is thought to mainly occur via adhesion (peripheral)and heat shock proteins that interact with the receptors locatedon the target cell surface [79–82]. Phosphatidylserine lipids alsoparticipate in target recognition via interaction with their recep-tors on the target cell [83]. Upon interaction with the target cellsEVs can dock at the plasma membrane and exert their biologicalfunction via signal transduction or fuse with the cell membraneand transfer their bioactive cargo [8]. EV membrane fluidity andpH of the environment greatly affects the efficiency of EV fusion.High cholesterol, sphingomyelin, and saturated fatty acid contentcontribute to EV rigidity, and prevent fusion at neutral pH. Theacidity of the tumor microenvironment increases the efficiency oftumor EV fusion by the target stromal cells [84]. EVs can also beinternalized by endocytosis, broken down in lysosomes, and subse-quently release their cargo into the lumen of their targets. EV tar-geting can be monitored via different methods that rely on imagingtechniques and fluorescence dyes. Docking can be detected by tar-geting cell surface proteins with antibodies, while fusion is com-monly detected using self-quenching lipophilic dyes (e.g.R18)[59]. Thanks to their tendency to form aggregates in vitro,EVs internalized by endocytosis can be easily observed by

42 J. Morhayim et al. / Archives of Biochemistry and Biophysics 561 (2014) 38–45

membrane intercalating dyes, such as PKH dyes (Fig. 3). EV aggre-gation is usually explained by the formation of bridges betweenthe anionic phospholipids due to the calcium content of culturemedia; however, we cannot rule out that aggregation may beimportant for EV propagation similar to viruses [85].

Biological functions

Only a few decades ago it was commonly accepted that intercel-lular communication was solely mediated via receptors and smallsoluble molecules. Vesicular structures secreted to the extracellu-lar space were mainly considered to be cell artefacts as a way ofdisposing cellular waste. During the ‘80s, several studies attributedmore functions to EVs in antigen-presenting, antitumor activityand immune response [86–89]. Today, EVs have been shown tobe involved in many biological functions, such as development,immunity, inflammation, coagulation, cardiovascular functionand stem cell biology, as well as cancer and chemotherapyresistance.

EVs are important regulators of coagulation via the exchange ofactive cargo that promotes vessel formation, angiogenesis, clottingand platelet aggregation [8,89,90]. EVs regulate coagulation bytransferring cargo with either anticoagulant activity (e.g. activatedprotein C) or procoagulant activity (e.g. arachidonic acid) betweenactivated and resting platelets [91–93]. Neuronal EVs are known toplay a regulatory role in myelin formation, neuron outgrowth andneuronal survival [94–96]. Neuronal EVs are also essential in recy-cling receptors and removing potential pathological proteins fromthe central nervous system [95]. EV production is up-regulatedduring inflammatory response [5]. Elevated ATP levels caused bycellular injury triggers macrophage EV secretion, which in turnhave the capacity to activate other macrophages [97]. EVs alsohave essential implications in immune response as they arereleased both by infected cells and immune cells. Hepatitis Cinfected cells release EVs that contain viral RNA that is transferredto target plasmacytoid dendritic cells, and consequently activateimmune response [98]. Ramakrishnaiah and colleagues showedthat hepatitis C virus RNA could also evade the immune systemby transmitting the infection between human hepatocytes [99].Recently studies showed that EVs have crucial implications in theregulation of stem cell fate. Ratajczak and colleagues showed thatembryonic stem cells stimulate the pluripotency of hematopoieticprogenitors via transferring Wnt3 protein [100]. EV released fromapoptotic T cells induced differentiation of leukemic cells towardsmegakaryocytes via the associated hedgehog morphogen [101].EVs isolated from the peripheral circulation of pregnant women

(A)

Cou

nt

FITC

(B)

Fig. 3. EV uptake by HEK 293 cells. (A) Flow cytometry histogram shows increasing fluPKH67-labeled SV-HFO EVs. EV concentrations: red-1X; blue-2X; orange-5X; light greenHEK 293 cells that were treated with the highest dose (40X) of PKH67-labeled SV-HFO EVEVs. Scale bar: 8 lm.

contained different cargo in different gestation phases, suggestinga role for EVs in morphogenesis during vertebrate development[102]. Several reports showed that EVs carry developmental pro-teins such as b-catenin, Wnt proteins and Sonic Hedgehog, furthersupporting their involvement during development [103].

EVs also have many implications in cellular pathologies, such asdiabetes, coagulopathies, inflammation, infection, autoimmunedisease and cancer, being of particular interest in the field [104–108]. Both tumor cells and the surrounding non-malignant cellsin the tumor microenvironment produce EVs that contribute topre-metastatic niche formation, immunosuppression, angiogene-sis, invasion, metastasis and tumor progression [109–113]. TumorEVs generally carry oncogenes and pro-metastatic cargo consistingof metalloproteinases, tetraspanins, plasminogens, integrins, heathshock proteins, growth factors, etc. that promote extracellularmatrix degradation, invasion and consequent tumor progression[54]. Several reports showed the role of EVs in priming metastaticniches for enhanced metastatic propensity of the target cells viathe exchange of pro-angiogenic factors and chemo-attractants[114]. Hood and colleagues reported that melanoma EVs primethe sentinel lymph nodes for melanoma metastasis [115], whilePeinado and colleagues reported that melanoma EVs stimulatethe bone marrow cells to support metastasis and tumor growthvia receptor tyrosine kinases [116]. Interestingly, EVs shed bytumor cells inhibit immune surveillance and promote chemother-apy resistance by expulsion of therapeutic drugs from tumor cellsvia exchange of ABC transporters and drug metabolizing enzymes[117–119].

Extracellular vesicles and bone

Over the past few years, researchers have shown increasinginterest in delineating the role of EVs in bone biology. The workof Anderson and Bonucci not only pioneered the development ofEV field but also contributed to study the regulation of bone matrixdevelopment, mainly via matrix vesicles secreted by osteoblasts.Matrix vesicles with diameters between 30 and 300 nm originatefrom the plasma membrane of mineralizing osteoblasts, and areinvolved in bone matrix mineralization via hydroxyapatite deposi-tion [120]. Matrix vesicles are also released by cartilage duringendochondral calcification. Proteome profiles of matrix vesiclesobtained from bone and cartilage of different species revealed alarge number of shared proteins, such as phosphatases, annexinsand ion channels, that contribute to the understanding of mineralformation [121,122]. Studies with hypophosphatasia patientsshowed that the defects in proper mineral crystal propagation

orescence intensity shift of HEK 293 cells treated with different concentrations of-10X; dark green-20X; pink-40X. (1X: 0.25% v/v) (B) Confocal microscopy image ofs shows EV uptake. Analyses were performed after overnight treatment with labeled

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from matrix vesicles led to improper or complete failure of bonecalcification [123]. In vitro manipulation of osteoblast mineraliza-tion further supported the parallels between matrix vesicleproduction and mineralization. Inhibition of mineralization byActivin A triggered reduced expression of matrix vesicle markersimplying deficient or altered matrix vesicles production [124],while vitamin D treatment successfully increased matrix vesiclesecretion from osteoblasts leading to higher mineralization rates[125]. Osteocytes also secrete EVs with possible roles in regulationof mineral deposition via communication with osteoblasts andosteoclasts [126,127]. On-going studies have reported preliminaryresults showing the enrichment of osteocyte EVs with receptoractivator of nuclear factor kappa-B ligand, of which the vesicularlevels are elevated upon parathyroid hormone treatment, sugges-tive of a role in promoting osteoclast formation (BoneKEy reports).Recent studies with osteoclasts reported the importance of themiRNAs in osteoclastogenesis [128,129]. Current studies are focus-ing on elucidating whether miRNAs are transported via EVssecreted by osteoclasts.

Bone forms a complex microenvironment that hosts a greatdiversity of cells, such as hematopoietic stem cells, fat cells, MSCs,endothelial cells, cartilage and nerves. Keeping the constant bal-ance of bone homeostasis as well as the other physiological eventsthat occur within the bone microenvironment requires the forma-tion of intricate intercellular communication networks betweenthe resident cells. MSC–EVs regulate inflammation by inhibitingauto-reactive lymphocyte proliferation and promote secretion ofthe anti-inflammatory cytokines IL-10 and TGF-b [130]. Ekstromand colleagues demonstrated that monocyte EVs stimulate osteo-genic differentiation of regenerative MSCs during bone injury atthe site of titanium implants [131]. CD34+ bone marrow progeni-tors stimulate angiogenesis via miRNA exchange [132]. In cancer,the favorable bone microenvironment attracts distant bone-metas-tasizing tumors. Prostate tumor cells release EVs that educate thebone cells to act as a pre-metastatic niche [57]. On the other hand,studies show contradictory roles of MSC–EVs in tumor microenvi-ronment. Several groups reported that MSC–EVs favored tumorgrowth and angiogenesis, while others showed the inhibition ofthese processes [133–135].

Applications in diagnostics and therapy

In the last two decades, significant advances have been made inthe characterization of EV content and function leading to theirimplications in health and disease. Better understanding the roleof EVs in bone remodeling and bone microenvironment may pro-vide insights into the complexity of diverse physiological and path-ophysiological events. Furthermore, their clinical implementationsmay open the doors for translational medicine, both in diagnosticsand therapy.

Biomarkers and diagnosisPathological EVs contain molecular disease signatures, and

hence function as excellent molecular biomarkers to be used fordiagnosis and prognosis. Particularly, EVs in body fluids can be eas-ily collected making them optimal non-invasive biomarkers todetect renal disease, obesity and diabetes, and cancer [136–140].EVs are considered to be promising biomarkers for early diagnosisbecause they are already released at early stages of the disease andthey are very stable and easily detectable. EV-derived miRNAs rep-resent one of the most attractive cancer biomarkers as they pro-vide a miRNA pattern that is different in benign and malignantforms, correlating with different stage of tumors [141]. Altered lev-els of circulating miRNAs are also proposed to be biomarkers forbone cancers, such as osteosarcoma [142]. However, the involve-ment of EVs is still not clear. Early diagnosis of metabolic bone

diseases is essential for subsequent effective treatment. Currently,the conventional diagnostics techniques rely on the detection ofbone turnover biomarkers in patient urine and blood [143,144].Interestingly, osteoprotegerin containing EVs were detected in ele-vated levels in the urine of patients with chronic kidney diseasesuggestive of a role in preventing vascular calcification [145].Investigating the roles of bone EVs in metabolic bone diseasesand inappropriate biological mineral formation is promising toprovide novel avenues for early diagnosis.

Regenerative medicine, vaccines, and drug deliveryStem cell-derived EVs have been of increasing interest for their

potential use in cell-free therapies in regenerative medicineexploiting their physiological functions mimicking their parentalcells. They have been shown to improve cellular function in dam-aged organs by preventing apoptosis and promoting proliferationand angiogenesis via the horizontal transfer of pro-regenerativefactors [146–148]. Several studies reported the potential of MSC–EVs as therapeutic agents for tendon repair [149] and rheumaticdiseases based on their anti-fibrotic, anti-apoptotic, anti-inflam-matory and pro-regenerative properties [150]. Furthermore,MSC–EVs have been shown to be involved in repairing invertebraldisc degeneration via transfer of their membrane components tonucleus pulposus cells [151]. These findings stress their potentialin bone regenerative therapy.

Thanks to their immunosuppressive properties, EVs have beenproposed as vaccines for immunotherapy [152,153]. Particularly,EVs secreted by tumors have been shown to contain tumor anti-gens that could have an antitumor activity on other tumors[154]. Remarkably, clinical trials with cancer patients showed thatmelanoma EVs added to autologous dendritic cells in vitro stimu-lated T cells that induced antitumor effects when injected intothe patient [155,156]. EVs have been increasingly seen as physio-logical drug delivery agents [157]. As opposed to synthetic alterna-tives, such as liposomes and nanoparticles, EVs occur naturally,exhibit a long half-life and an intrinsic homing ability, and aremore efficiently tolerated by the recipient cells. Several reportsdemonstrated the possibility to load EVs with therapeutic mole-cules via in vivo and in vitro manipulations [158]. Wood and col-leagues showed that they could use electroporation techniquesto load EVs with siRNA that was delivered to target cells resultingin gene regulation [159]. Human MSC–EVs are shown to be prom-ising for drug delivery. Chai Lai and colleagues manipulated MSCsto produce infinite numbers of EVs from a single clone [160]. Sev-eral groups are focusing on developing biomimetic drug deliveryvehicles inspired by EVs to effectively deliver the therapeutics tothe target site [161,162].

Conclusions

EVs are now recognized as biologically significant structureswith important roles in cell-to-cell communication. Advancementin the field produced tremendous information that gives insightsinto the biological role of EVs in many aspects of physiologicaland pathophysiological events. Due to their signature contentand biocompatibility, EVs represent a potential source of diagnos-tic tools and therapeutic agents. As well, their use might overcomelimitations and risks associated with cell-therapy approaches. Fur-ther characterization of the EV-mediated modes of communicationin the bone microenvironment will not only provide insights intothe complexity of bone development and maintenance but alsoto other biological processes that require the bone microenviron-ment. We anticipate that future studies on bone-EV biogenesis,uptake and content will unravel biomarkers, and help us develop

44 J. Morhayim et al. / Archives of Biochemistry and Biophysics 561 (2014) 38–45

tailored drug delivery vehicles to treat a broad variety of patholog-ical conditions.

Acknowledgments

We acknowledge the support from the INTERBONE researchprogram. We thank Hector Hugo Perez Garza for help with atomicforce microscopy imaging, and Gert-Jan Kremers for confocalimaging.

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