erg

SThe

Keywords:MicroautophagyBis(Monoacylglycero) Phosphate (BMP or

thaa desides not only in their protein and RNA contents but also in their lipidic molecules.ids organized in a bilayer membrane and along with the lipid carriers such as fatty

lipoproteins but also by vesicles. The current view of vesicular transport prostaglandin PGE2 participate in tumor immune evasion and promote

sed on the importancephysiologies. Anothermicrovesicles derivedreceiving increasing

Biochimica et Biophysica Acta 1841 (2014) 108120

Contents lists available at ScienceDirect

Biochimica et Bi

j ourna l homepage: www.e llipolytic enzymes such as the different families of phospholipaseA2. Exosome-mediated lipid trafcking is involved in various

interest. Exosomes and microvesicles have been grouped under theterm of Extracellular Vesicles (EV) [6] which are generated frompathophysiologies. Exosomes positive for health are derived from intact viable cells, but their respective functions appear different.glandins, leukotrienes) in their membrane or in their lumen. Exosomallipids interact with receptors on target cell periphery and are thereafterinternalized into endosomes where they concentrate the bioactivelipids that they carry. In addition exosomes are transporters of active

progression over the last ten years and has focuof cell-derived vesicles in cell biology and pathotype of cell-derived vesicle called ectosomes orfrom plasma membrane shedding is alsoof lipids refers mainly to intracellular lipid trafcking [1,2]. However,extracellular nanovesicles termed exosomes are actively secretedfrom the late endosomal compartment of viable cells and trafc be-tween cells [3] transporting lipids (sphingomyelin, cholesterol, lyso-phosphatidylcholine, arachidonic acid and other fatty acids, prosta-

tumor growth. Cholesterol can be removed from endosomes by therelease of exosomes to bypass its accumulation as in the NiemannPickdisease [4], but internalization of exosomes into monocytes triggerscholesterol accumulation into lipid droplets [5].

The number of studies related to exosomes has been in constant Corresponding author at: INSERM-UMR 1037, Cance(CRCT), Team Sterol Metabolism and Therapeutic InnCHU Purpan, Toulouse F-31300, France. Tel.: +33 561 42

E-mail address:michel.record@inserm.fr (M. Record).

1388-1981/$ see front matter 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.bbalip.2013.10.004rier proteins (ABC trans-cid binding proteins) and

enhance DC antigenic capacity. Exosome contents like the fatty aciddocosahexaenoic acid (DHA, 22:6) and lyso-phosphatidylcholine (LPC)can regulate this process. On the contrary exosomes enriched inLipid transport is mediated not only by carporters, phospholipid transfer proteins, fatty aLBPA)Phospholipases (A1, A2, D)Prostaglandin E2Cholesterol epoxide hydrolase (CHEH)Aminoalkyl sterols

1. Introductionas eicosanoids, fatty acids, and cholesterol, and their lipid composition can be modied by in-vitro manipulation.They also contain lipid related enzymes so that they can constitute an autonomous unit of production of variousbioactive lipids. Exosomes can circulate between proximal or distal cells and their fate can be regulated in part bylipidic molecules. Compared to their parental cells, exosomes are enriched in cholesterol and sphingomyelin andtheir accumulation in cells might modulate recipient cell homeostasis. Exosome release from cells appears to be ageneral biological process. They have been reported in all biological uids from which they can be recovered andcan be monitors of specic pathophysiological situations. Thus, the lipid content of circulating exosomes could beuseful biomarkers of lipid related diseases. Since the rst lipid analysis of exosomes ten years ago detailedknowledge of exosomal lipids has accumulated. The role of lipids in exosome fate and bioactivity and how theyconstitute an additional lipid transport system are considered in this review.

2013 Elsevier B.V. All rights reserved.

immunocompetent cells such as dendritic cells (DC) whose exosomesAvailable online 16 October 2013acid binding proteins that they contain, exosomes transport bioactive lipids. Exosomes can vectorize lipids suchAccepted 3 October 2013The bioactivity of exosomes rExosomes display original lipReview

Exosomes as new vesicular lipid transportcommunication and various pathophysiolo

Michel Record , Kevin Carayon, Marc Poirot, SandrineINSERM-UMR 1037, Cancer Research Center of Toulouse (CRCT), Team Sterol Metabolism andInstitut Claudius Regaud, 20-24 Rue du Pont Saint-Pierre, 31052 Toulouse Cedex, FranceUniversit Paul Sabatier Toulouse 3, 118 Route de Narbonne, Toulouse, France

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

Article history:Received 6 June 2013Received in revised form 29 September 2013

Exosomes are nanovesiclesintracellular compartment ofr Research Center of Toulouseovation in Oncology, BP3028,4 648; fax: +33 561 424 631.

ights reserved.s involved in cellcellies

ilvente-Poirotrapeutic Innovation in Oncology, BP3028, CHU Purpan, Toulouse F-31300, France

t have emerged as a new intercellular communication system between anonor cell towards the periphery or an internal compartment of a recipient cell.

ophysica Acta

sev ie r .com/ locate /bba l ipExosome biogenesis occurs inside an intracellular compartment(late endosome/MultiVesicular Bodies [MVB]) by a microautophagyprocess [7] which sorts molecular constituents from various cellcompartments of the parental cells. The lipid composition of exosomesindicates a major sorting of lipidic molecules from the parental cellswith enrichment in sphingomyelin and saturated molecular species of

109M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120phosphatidylcholine and phosphatidylethanolamine [810], whichaccounts for their stability in-vivo. On the contrary, the lipid compositionof microvesicles reects that of the parental plasma membrane [11].Exosome release out of the cells results from a secretion processsubsequent to the displacement of theMVBalongmicrotubules, a processin part controlled by the cholesterol content of the cells. Fusogenic lipidsare then required to allow fusion of theMVBwith the plasmamembraneand secretion of exosomes. The whole process is totally distinct from theplasma membrane shedding which leads to the release of microvesicles.However upon cell stimulation, both exosomes and microvesicles canbe produced as reported in a key study on platelets [12]: thrombinstimulation of platelets triggers the release of 100 nm1 m diametermicrovesicles possessing procoagulant activity whereas plateletexosomes, 40100 nm wide and enriched in the tetraspanin CD63,were unable to bind procoagulantmolecules. Becausemuch knowledgehas been accumulated about exosomes regarding the lipids required fortheir biogenesis, the bioactive lipids they transport, and the in-vitromanipulation of their lipid content, only exosomes will be covered inthis review.

Exosome-mediated intercellular lipid exchange is distinct from thelipoprotein-mediated lipid transport. About 60 g of exosomes isrecovered in 100ml of plasma [13], which is about 500 times less thanthe circulating content of lipoproteins. The molecular organizationbetween the two lipid vehicles is quite different since lipoproteinscontain a core of neutral lipids surrounded by a monolayer of phos-pholipids whereas exosomes are vesicles with a bilayer membrane [8]containing a cytosolic material. In line with this, exosomes carry a setof enzymes related to lipid metabolism [9,14] whereas apolipoproteinshave no intrinsic catalytic activity. In addition cells from the intestineand liver, two organs playing a key role in lipoprotein metabolism,also possess an exosome pathway i.e. they release and internalizeexosomes [15,16]. A connection between lipoproteins and exosomesmay exist since treatment of monocytes with oxidized LDL-immunecomplex leading to the formation of foam cells also triggers the formationof exosomes [17].

Different names for exosomes have been used across the literature inrelation to either their functionality or cells and tissues of origin. It hasbeen emphasized that the more generic term of exosome has broaderutility [6] since it refers to nanovesicles generated inside an intracellularcompartment and released out of viable cells. This release occurs bothupon cell stimulation and constitutively from resting cells such asepithelial and tumor cells. In this latter case, the exosome-associatedprostaglandin E2 participates in the non-recognition of the tumor by theimmune system [18]. Similarly exosomal-PGE2 in intestinal exosomescan anergize Natural Killer T cells in the liver after exosome migrationbetween the two organs [18,19].

The lipids involved in the various steps of exosomes' fate andbioactivity are detailed in this review. The specic endosomalpolyglycerophospholipid called Bis(Monoacylglycero)Phosphate(BMP) [20] plays a role in intralumenal vesicle biogenesis [21] andthis is the primary difference from peripheral microvesicles since BMPis not present in the plasma membrane [22]. Exosomes not only carrybioactive lipids such as eicosanoids but also transport some of theenzymes involved in their metabolism. Several enzymes originatingfrom the endoplasmic reticulum are found in exosomes and cannotbe present in microvesicles (ectosomes) that are derived from theplasma membrane. The accumulation of exosomes in the endosomes ofreceiving cells enables high concentration of bioactive lipids to bereached which can potentially modify the lipid homeostasis of thesecells [9]. Thus exosome trafcking has to be taken into account in generallipid metabolism [23].

This review encompasses the characteristics of exosomes, how lipidsparticipate in their biogenesis, the type of lipids vectorized by exosomesand how exosomal lipids canmodulate the lipid homeostasis of recipientcells. The lipids regulating exosome fate and the involvement of exosomal

lipids in various pathophysiologies are also considered.2. Exosomes: denition and characteristics

Exosomes are nanovesicles (50100 nm) released from viable cells,either constitutively or upon activation of cell secretion, but not fromlysed or apoptotic cells. They are not released by plasma membraneshedding but they are secreted froman intracellular compartment relatedto late endosomes, the MultiVesicular Bodies (MVB) [24,25]. The initialstep in exosome biogenesis is intralumenal vesicle (ILV) formation byinward budding of the MVB membrane. The late endosomes containinginternal vesicles and consequently called MultiVesicular Bodies (MVB)move along microtubules to fuse with the plasma membrane and thenrelease their ILVs which then become exosomes. Stimulation of thissecretion process by a calcium ionophore in basophil leukemia cells(RBL-2H3) levels off at 30min and triggers the release of 13g of proteinas exosomes from 106 cells. Parts of these exosomes are aggregated andcan be quantied by ow cytometry [9]. Measurement of single vesiclesusing the recent technique of Nano-Tracking Analysis [26] indicatedthat 106 RBL-2H3 cells release about 500 106 single exosomes underthe same conditions, i.e. an average of 500 single exosomes per cell.As compared with a previous estimation of 3000 vesicles potentiallyreleasable per cell [3], it means that only one part of the MVB pool inparental cells participates in the exosome secretion. Indeed thesecretion of exosomes is sectorized in the cell, i.e. it occurs only in apart of the whole cell periphery as observed in activated T cells [27]. Inthat study in T cells it was observed that exosomes exited the MVB asaggregates. In addition, aggregated exosomes can be formed by cross-linking of their phosphatidylserine (PS) with the calcium present inbiological uids, since they expose PS in sufcient amount to bind toannexin V [28]. Thereby some of the exosomes can naturally circulateas aggregates.

Because of their endosomal origin, exosomes contain two typicalproteins, Tsg101 and Alix. It is noteworthy that Alix binds the specicendosomal phospholipid Bis(Monoacylglycero)Phosphate (BMP, alsocalled LBPA for Lyso Bis Phosphatidic Acid) by means of a convex surfacein the Bro1 domain corresponding to the sequences 101-KGSLFGGSVK-110 and 232-QYKD-235 which contain two main hydrophobic residuesL104 and F105 [29].

Exosomes are highly enriched in transmembrane proteins termedtetraspanins (from 7 to 124 fold as compared with the parental cells)[30]. Although thesemolecules are also present in the plasmamembraneand can be shed as part of microvesicles, exosomes are characterized bytheir enrichment in some of the tetraspanins such as CD9, CD63, CD37or CD81 [30]. The tetraspanin-enriched microdomains (TEM) and morespecically CD81 play a key role as sorting machinery to target proteinstowards exosomes [31]. Heat-shock proteins (HSP 90/70) are alsoenriched in exosomes and make exosomes distinct from the ectosomesreleased upon plasma membrane shedding [32]. Ectosomes containdistinct sets of proteins [33] and distinct markers such as CD35(complement 3b/4b receptor), GPA (glycophorin A) [32] or CD86(B-lymphocyte activation antigen B7-2), and CD47 (Integrin AssociatedProtein) [34]. Exosomes are also totally distinct from apoptotic micro-particles [12] which bear markers such as CD31 (endothelial cellularadhesion molecule) and CD105 (endoglin, TGF co-receptor) [35,36].Exosomes have a rather high density, ranging from 1.13g/ml for B cellexosomes [25] to 1.19 g/ml for epithelial intestinal cell exosomes [5]and reach a density of 1.21 g/ml when recovered from the plasma[13]. In fact, in-vivo exosomes are found in all biological uids includingurine, plasma, epididymal uid, amniotic liquid, malignant and pleuraleffusions of ascites, bronchoalveolar lavageuid, synovialuid and breastmilk [3]. It isworth noting that exosomes derived from reticulocytes havea density of 1.19 at the early stages of reticulocyte maturation, whichincreases to 1.21 at the late stages [37].

It is important to emphasize that exosomes are released from viablecells. Several studies reporting extracellular vesicle analysis deal withmaterials recovered from experimental conditions in which the state of

the cells has not always been thoroughly characterized. Long term cell

stimulation or cells cultured with low levels of serum can lead to therelease of amixture of vesicles, i.e. those shed from theplasmamembrane(termed ectosomes, microvesicles or microparticles ), exosomes releasedfrom late endosomes (MVB), and apoptotic vesicles, and apoptotic vesi-cles also called apoptotic bodies [38]. The distinction between the varioustypes of vesicles can be ascertained partly from their density [39].Apoptotic vesicles have a higher density than exosomes, ranging from1.18 to 1.28, with specic markers between 1.24 and 1.28 [36]. Thedensity of microvesicles has been reported to be between 1.25 and 1.30[40] and they are heterogeneous in size, ranging from 100 to 1000 nm.Most microvesicles can be pelleted at 10,000 g to 20,000 g [33,41],whereas 10 times higher centrifugation speeds are required to pelletexosomes [42]. However small plasma membrane vesicles with a sizesimilar to exosomes can be recovered at 100,000 g but have a lighterdensity (1.041.07) than exosomes [39].

The comparative density of the vesicles released from viable cellsis depicted in Fig. 1. This gure also shows that choline-containingphospholipids (sphingomyelin and phosphatidylcholine) and theendosomal specic phospholipid Bis(Monoacylglycero)Phosphate(BMP) can help to discriminate the various types of vesicles. Whereasthe content in choline-containing phospholipids is similar betweenmicrovesicules and their parental cells [11], exosomes are highlyenriched in sphingomyelin but not in phosphatidylcholine (Fig. 2).BMP is present in endosomes but not in plasma membrane [45] and istherefore exclusive to exosomes.

Discriminating the various vesicles is important relative to theirfunctional effects. Ectosomes down regulate macrophages and dendriticcells (DC) [46,47] whereas exosomes enhance DC-mediated antigenpresentation [48]. Exosomes can cross the blood-brain barrier at variancewith microparticles as shown by cell imaging in the brain [41]. Thisobservation has been further strengthened by inactivation of the brainprotease BACE by means of bio-engineered exosomes injected into the

Although the various vesicles reported in the literature displaybiological activity, exosomes have long been the subject of considerableinterest andmuch knowledge has been accumulated about them. Indeedthey were described for the rst time by Johnstone and collaborators in1987 studying reticulocyte maturation [24]. Since then, all the cell typesexamined, both normal and pathological, have been shown to secreteexosomes. The observation that launched the interest in exosomes

1

100 nm

> 100 nm Ectosomes(microvesicles)

Exosomes

8 1

0.7 1

10-20 000 x g

ction by lipidic markers. Plasma membrane-derived vesicles (ectosomes) display a large set of39] whereas similar sized exosomes have amuch higher density (1.131.21) due in part to their0,000 g). Large microvesicles have a density N1.25 [40] and are recovered at medium speedsh these vesicles. Values with regard to choline-containing phospholipids i.e. sphingomyelinsf each phospholipid in the vesicles over that in parental cells. SM are enriched in exosomes inaining phospholipid (SM and PC) composition of microvesicles is identical to that of parentalpecic endosomephospholipidBis(Monoacylglycero)Phosphate (BMP) is detected in exosomese plasmamembrane [43,44] and consequently in the plasma-membrane-derivedmicrovesicles

SMCH

OL PS PI

PA CERHx

CERBM

P PE PC0

1

2

3

4

Lipids

Fold

incr

ease

/ pa

rent

cel

l

Fig. 2. Lipid sorting between parental cells and exosomes. The gure represents theratios between each lipid class in exosomes versus that of parental cells. Ratios betweenphospholipids or neutral lipids were calculated from literature data reporting thecomposition of exosomes and parental cells [8,37,62,83]. SM = sphingomyelin;CHOL = free cholesterol; PS = phosphatidylserine; PI = phosphatidylinositol;CER = ceramide; HxCER = hexosylceramide; BMP = Bis(Monoacylglycero)Phosphate;PE=phosphatidylethanolamine; PC=phosphatidylcholine.

110 M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120tails of mice [49].

1.1 1.151.05

SM 1 1.5-2.

BMP 10PC 1 0.6-0

100-120 000 Xg

Fig. 1. Respective density of extracellular vesicles recovered from viable cells and their distinsizes, from 100nm to 1m. Plasmamembrane-derived 100nm vesicles have a density b1.1 [high protein content[9]. Those vesicles are recovered at high speed centrifugation (10012centrifugation (1020,000 g). In addition to their density, some lipidic markers distingui(SM) and phosphatidylcholines (PC) in the gure express the ratio between the amount othe range of 1.53 fold [8,10] whereas PC are decreased (see also Fig. 2). The choline-contcells [11], and the ratios betweenmicrovesicles andcells have beenassigned a value of 1. The s[8,82] but not enriched comparedwith the parental cells (ratio=1), whereas it is absent in th

(ratio=0).was their enrichment in antigen-presenting molecules [50] and their

.2 1.31.25 density

111M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120involvement in the immune response [51,52], but the characterizationof exosomes as vectors of mRNA and microRNA [53,54] has boostedthe eld. Finally an additional interest has arisen from the observationthat exosomes carry bioactive lipids such as prostaglandins [9] andleukotrienes [14] which trigger cell-to-cell signaling.

3. Lipids required for exosome biogenesis and release

The rationale for studying exosomal lipids is not only becausethey carry bioactive lipids involved in inammation and immunitybut also that lipid second messengers such as phosphatidic acid,diglycerides, and ceramides are involved in their biogenesis. It isworth noting that the packaging of miRNA into exosomes requiresthe neutral sphingomyelinase (nSMase2) which generates the lipidmediator ceramide. This suggests a relationship between exosome lipidcomposition and their functionality. Because of their high cholesterolcontent and their accumulation in recipient endosomes, exosomes canbe implicated in cholesterol-related storage disease and they canmodifythe lipid homeostasis of target cells [5,55,56].

3.1. Exosome biogenesis: a microautophagy process involving various lipids

The precursors of exosomes, i.e. intralumenal vesicles (ILV) aregenerated from the limiting membrane of late endosomes by a micro-autophagy process which requires the Endosomal Sorting ComplexRequired for Transport (ESCRT) I and III, the sorting protein vps4 andthe constitutive heat-shock protein Hsc-70 [7]. Indeed the ESCRTmachinery is transiently recruited to the cytosolic side of the endosomalmembrane for sorting of selected proteins to the ILV [57]. The ESCRTmachinery is constituted of three separate protein complexes calledESCRT-I, ESCRT-II and ESCRT-III. Two typical exosomal proteins areTsg101 which belongs to the ESCRT-I protein complex, and Alix whichis associated with the ESCRT-III complex [3].

Proteins of the ESCRT machinery interact with various lipids or lipid-related enzymes. Vps4 interacts with an oxysterol binding protein Oshp7[58] making a link with cholesterol metabolism. Genome-scale screensfor genetic interactions that affect the Golgi/endosome/vacuole sortingunveil a key role for lipids in general, and more specically for sterolsand fatty acids [59].

The constitutive heat shock protein Hsc-70 specically requiresphosphatidylserine (PS) from the endosomal membrane to partici-pate in the microautophagy process generating exosomes. TheESCRT-III associated protein Alix binds the polyglycerophospholipidBMP (BisMonoacylglyceroPhosphate) [21], specically enriched in endo-somes and lysosomes [45]. BMP can potentially be formed by the phos-pholipase D (PLD) transphosphatidylation reaction [60] and the PLD2has been shown to participate in exosome production [61].

Beside the phospholipids PS and BMP, the neutral lipid ceramide hasbeen shown to trigger an exosome biogenesis pathway independent ofthe ESCRT machinery [62]. Cholesterol enhances secretion of otillin-2positive exosomes [4]. In addition, lipid transporters such as ABCA3,that transport phosphatidylcholines, have been involved in exosomeproduction [63].

Therefore not only BMP, cholesterol, oxysterols, and ceramides,but also phosphatidic acid and ATP binding cassette-type transportersparticipate in ILV/exosome biogenesis. Details of the involvement ofthese lipids are developed below.

3.2. BMP (also called LBPA)

BMP (BisMonoacylglyceroPhosphate) is a polyglycerophospholipidwhose location in cells is restricted to MVB and lysosomes and whichis required for MVB formation [64] and subsequently for ILV biogenesis[65,66]. BMP binds Alix, a protein that is part of the ESCRT machinery.Inactivating Alix triggers a large decrease in both the MVB and BMP

contents of cells [21]. BMP also binds the chaperon protein Hsp70,which is present in exosomes, with high afnity: the point mutationTrp90Phe almost completely abolishes the lipid binding [67]. Formationof intra-endosomal ILVs can be mimicked by adding BMP to largeliposomes provided that there is a 2 unit pH difference betweenthe inside (pH5.5) and the outside (pH7.4) of the liposome [21]. AddingBMP in a phospholipid mixture representative of the endosome mem-brane composition increases the molecular area of the lipid moleculesat acidic pH but not at neutral pH [3,60] thus generating an excessof lipids in the inner leaet of the liposome membrane which requiresan inward budding of vesicles to reequilibrate the two layers of themembrane. Inward budding of a 60 nm vesicle will eliminate twice asmany lipid molecules from the parent membrane inner leaet ascompared to the outer leaet because of the vesicle curvature. Indeed a60nmwide vesicle contains two thirds of its lipids in the external leaet[8].

The canonical pathway for BMP biosynthesis starts from phos-phatidylglycerol (PG) [68,69]. PG is biosynthesized in themitochondria[70], or by the transphosphatidylation activity of the plasmamembranephospholipase D2 (PLD2) in the presence of exogenous glycerol [71].In both cases PG has to be addressed to the late endosome (MVB)membrane which is the site of BMP biosynthesis. A transbilayertransport mechanism is required to bring the PG molecule insidethe MVB lumen to reach the enzymes involved in its transformationinto BMP. Indeed a PG-specic and MAFP-sensitive phospholipaseA2 operating at acidic pH has been ascribed to the formation oflyso-phosphatidylglycerol (LPG) as the rst step of BMP biosynthesis[72]. This PLA2 could be the PLA2 GXIV [73,74], although it is notestablished whether this enzyme is sensitive to MAFP. The secondstep of the intra-endosomal BMP biosynthesis has been assigned to anacid acyltransferase converting LPG into BMP [75].

Another possible BMP biosynthesis pathway could be located on thecytoplasmic side of the endosome, involving the sequential action of thesecreted sPLA2IIA which not only hydrolyses PG in-vitro into LPG butalso possesses intracellular activity [76], followed by acylation of LPGinto BMP by the polyglycerophospholipid acyltransferase ALCAT1[77].However this pool of BMP could be degraded by the phospholipase A1activity of the Pancreatic Lipase Related Protein 2 (PLRP2) locatedoutside the MVB and which hydrolyses BMP [60], unless this BMP istranslocated to the inner leaet of the endosome membrane. Thisinner leaet has been shown to be enriched in BMP by immunoelectronmicroscopy [45] using an anti-BMP antibody. The enrichment in BMP ofthis leaet participates in ILV biogenesis by ssion of the vesicle fromthe endosome membrane [64], provided that BMP accounts locally forabout one-third of the phospholipids [43].

3.3. Phosphatidic acid

It has been shown that the formation of phospholipase D2-mediatedphosphatidic acid (PA) enhances exosome production from the leukemiccell line RBL-2H3 [61]. PA resulting from the action of diglyceride kinasealso increases exosomes following T cell activation [78]. PA activates themammalian Target of Rapamycin (mTOR) complex [79] and consistentinhibition of mTOR by rapamycin decreases exosome production [63].

3.4. Ceramides

It has been demonstrated that in Oli-neu cells (mouse oligoden-droglial cell line, myelinating cells of the central nervous system) theproteolipid PLP (a major component of myelin) is released in associationwith exosomes. PLP is segregated into distinct subdomains on the MVBmembrane whose transfer into exosomes is independent of the ESCRTmachinery but requires the sphingolipid ceramide. Ceramides providean alternative pathway for sorting cargo into MVB that seems to dependon sphingomyelin (SM)/cholesterol enriched microdomains [62].

It is noteworthy that exosomes from oligodendroglial cells are

highly enriched in ceramide and hexosylceramides [62] (Fig. 2). Also

112 M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120exosomes/prostasomes from PC3 (human prostate carcinoma) cells areenriched in both types of ceramides [10]. This is consistent with the roleof the neutral sphingomyelinase in the biogenesis of ILVs [62].

Ceramides generated by the neutral sphingomyelinase 2 (nSMase2)play a key role by their involvement in the generation of the exosomeswhich package miRNA [80] and allow those miRNA to circulate in bodyuids by protecting them from degradation by the circulating RNases.This ceramide-mediated exosome biogenesis is independent of theESCRT protein sortingmachinery [80] and emphasizes a key role of lipidsin exosome biogenesis. Indeed the ceramide content of the membraneregulates the cellular distribution of the tetraspanin CD81 [81], anessential tetraspanin for exosome biogenesis [31] and function [82].

3.5. Cholesterol

ILVs accumulate cholesterol during their biogenesis from the endo-some (MVB) membrane, and subsequently exosomes are enriched incholesterol as compared to the parental cells [37,83]. ESCRT complexesinduce lateral lipid phase separation generating ordered membranemicrodomains provided that cholesterol accounts for more than 10%of the membrane lipids [84]. The cholesterol content of the membranealso regulates cellular trafcking of the tetraspanin CD82 to endosomes[85] which is thereafter detected in exosomes [9]. Cholesterol can beloaded into endosomes and on ILVs by means of specic transporterssuch as ABCG1 which has been observed in late endosomes [86]. TheBMP content and the presence of the BMP-interacting protein Alix arecritical for the MVB cholesterol content: accumulation of cholesterolinto endosomes of BHK cells by means of U18666A is decreased whenBMP increases [87]. However, in oligodendroglial cells, accumulationof endosomal cholesterol by U18666A treatment favors exosomesecretion, which requires the cholesterol binding protein otillin [4].A similar observationwas reported in NPC1 cells [4]. Therefore exosomesaccount for a mechanism to bypass endosomal cholesterol accumulationand which depends upon the amount of endosomal BMP, which alsomight favor exosome secretion [64].

3.6. Oxysterol binding protein interaction with Vps4 and endosomemovement

The vesicular protein sorting 4 (Vps4), which is an AAA-ATPasepresent in the MVB-exosome protein sorting machinery ESCRT-III,directly interacts with an oxysterol binding protein (OSBP) [58]. TheseOSBP proteins are part of the molecular mechanisms involved inmembrane contact between intracellular secretory vesicles and theplasma membrane. OSBP might play a role in the membrane fusionbetween MVBs and the plasma membrane to allow the release of ILVs/exosomes outside the cells [88]. In addition an OSBP protein, namelyORP1L, controls the endosome movement along microtubules, sinceORP1L is part of the endosome displacement machinery and sensesthe cholesterol content. In the presence of high endosomal cholesterolconcentrations any endosome movement is blocked [89].

4. Exosomes as new vesicular lipid transporters

4.1. Exosome membrane lipid composition and organization

Exosome lipid composition differs substantially from that of theparental cells as shown by the ratios of each individual class of phos-pholipids between exosomes and cells (Fig. 2). Lipid sorting leads toan enrichment in some of the exosome phospholipids (sphingomyelin,phosphatidylserine, phosphatidylinositol, phosphatidic acids) andneutrallipids (free cholesterol, ceramides) as compared to the parental cells.Instead the phosphatidylcholine content is decreased since its ratio islowered to 0.6 in exosomes over parental cells (Fig. 2), except inreticulocytes [37]. The endosomal phospholipid Bis(Monoacylglycero)

Phosphate (BMP) is present in exosomes but not enriched compared toparent cells (ratio of 1, Fig. 2). Changes in the ratios between choline-containing phospholipids indicate an enrichment in sphingomyelinat the expense of phosphatidylcholine (Fig. 2) that is a lipidic signatureof exosomes and reveals active phospholipid sorting during exosomebiogenesis from theMVB limitingmembrane. Indeed the lipid composi-tion of the MVB membrane from BHK cells [90] shows 15 times morephosphatidylcholine than sphingomyelin. Vesicles derived from prostatePC3 cells grown for three days in the absence of serum [10] and relatedto exosomes/prostasomes [91] have a phospholipid pattern similarto that reported in Fig. 2. They are also enriched in hexosylceramidesand glycosphingolipids (Gb3, GD1, GM1-3) [10]. Exosomes from immu-nocompetent cells have been reported to contain gangliosides with asialyllactose moiety [92,93].

Exosomes arehighly enriched inphosphatidylserine (PS) as comparedwith the parental cells [21]. Although exosomes have the same trans-membrane orientation as the plasma membrane of parental cells,exosomal PS appears randomly distributed between the twomembraneleaets [94], whereas PS is retained in the inner leaet of the cell plasmamembrane [95]. This can be subsequent to the presence of a calcium-dependent phospholipid scramblase in exosomes [9] together withthe lack of an organized cytoskeleton even though actin is detected.Phosphatidylethanolamine is consistently randomly distributed betweenthe two exosome membrane leaets [8]. Because of the membranecurvature, a 60 nm diameter exosome contains two thirds of its phos-pholipids in the outer leaet [8]. This leads to an apparent enrichmentin PS in the external exosomal membrane. Indeed exosomes bind to PSreceptors and are stained by annexin-V [28,5]. However, as ectosomesand apoptotic bodies also express PS on their surface [96] this stainingcannot be use to discriminate between these vesicles.

As shown by probes sensing the membrane uidity such asdiphenylhexatriene, the membrane of exosomes is rigid [8] and hasbeen characterized as an ordered Lo lipid phase [37]. This certainlyaccounts for the long-lasting presence of exosomes in biological uids,and the lack of attack by lipolytic enzymes in the circulation. It has beenshown that exosomes can remain for up to two weeks in the lymphnodes [97]. This membrane rigidity is subsequent not only to the highamounts of disaturated PC and PE [8], but also to the enrichment bothin sphingomyelin and cholesterol. Consistently, detergent-resistantmem-branes can be isolated from the exosomes. [98].

4.2. Transport of bioactive lipids and related enzymes by exosomes

Lipids and related enzymes found in exosomes are reported inTable 1 and detailed below.

4.3. Exosomes as fatty acid transporters

It has been shown that exosomes carry a whole set of fatty acids,which could be produced in the exosome itself or loaded intoexosomes during their biogenesis in the parental cell [9]. Thusexosomes appear as a reservoir of a large panel of free fatty acidsaccumulated in their membranes, or bound to potential proteincarriers located in their lumen. Exosomes carry mostly saturatedfatty acids, but monounsaturated and polyunsaturated ones are alsopresent. Among the unsaturated species, arachidonic acid is one of themost represented. However, fatty acid concentrations in exosomesovercome the capacity of the exosome itself to generate fatty acidfrom its own phospholipids, indicating that a part of exosome fattyacids originates from the parent cell [3,9]. As compared with fatty acidtransporters such as serum albumin, lipoproteins or fatty acid bindingproteins (FABP), exosomes appear as an additional mechanism totransport fatty acids across the plasma membrane by means ofendocytosis.

It is noteworthy that exosomes can carry several arachidonic-derivedlipid mediators at the same time, and since they are targeted to either

neighboring or distant cells, they vectorize bioactive lipids very efciently.

2):taseLTBate

s

113M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120Table 1Lipid related enzymes and bioactive lipids in exosomes. Enzymes: phospholipases A2 (PLAphospholipase D2 (PLD2); diglyceride kinase (DGK); PTEN is a phosphoinositide phosphaacid (DHA); phosphatidic acid (PA); lysophosphatidylcholine (LPC); leukotrienes (LTA4,[10,98]. Phosphatidylinositol 3,4,5-triphosphate (PIP3), Phosphatidylinositol 4,5-bisphosph

Enzymes Lipids Exosomes from

Saturated FAMonounsaturated FAPolyunsaturated FA (AA, DHA)

Rat mast cells

cPLA2, iPLA2 Arachidonic acid and LPC Rat mast cellsHuman mast cells

sPLA2 IIA Rat mast cellssPLA2 V Rat mast cellsPLD2 PA Rat mast cellsDGK PA LymphocytesLTA4 hydrolase Human MDM

Human MDDCLTA4, LTB4, LTC4 Human MDM

Human MDDCLTC4 synthase LTC4 Human MDM

Human MDDCCOX 1,2 PGE2

15d-PGJ2Rat mast cells

PGE synthase PGE2 Human mast cells

nSMase 2 (in parental cell) Ceramide; Hexosylceramide OligodendrocytesCholesterol accumulation(in parental cell)

OligodendrocytesSkin broblastB lymphoma cell line

PIP3 phosphatase (PTEN) FibroblastsEpitheliumGlioblastoma4.4. Exosomes carry functional lipid-related enzymes

As shown in Table 1, exosomes are a unique cell compartment sincethey carry at the same time the three classes of phospholipase A2, thecalcium-dependent cPLA2, the calcium independent iPLA2, and thesecreted sPLA2. They also carry some of the enzymes involved in theeicosanoidmetabolism [6,28]. Among the phospholipases, phospholipaseD2 is targeted to exosomes and is involved at the cellular level inexosome release.

Remarkably all these enzymes are catalytically active in the exosomes.In addition, the total PLA2 activity is enhanced upon incubation ofexosomes with GTP [9]. This observation was unexpected since no directinteraction between PLA2s and G proteins has been reported in anin-vitro system, at variance with PLDs that are directly activated bymonomeric GTPases. Almost all family members of the RasGTPasesuperfamily are present within exosomes. Activation of PLA2 byGTP suggests a specic mode of regulation of PLA2s in the exosomevesicle. In line with this, the exosomal LTC4 synthase involved inleukotriene biosynthesis appears to be activated during exosomeformation since it displays 5-fold more activity in exosomes than inparental cells [14].

Compared with parental cells, exosomes are enriched in phosphatidicacid [61] which can originate from PLD or DG kinase activity present inthe exosomes [102]. Also exosomes/prostasomes contain about twice asmuch PA than the parental cells [10].

Altogether these observations show that exosomes are autonomousunits of active enzymes related to lipid metabolism.

4.5. Exosomes as shuttles of eicosanoid precursors involved in the trans-cellular metabolism of eicosanoids?

Eicosanoids are bioactive lipids derived from arachidonic acid andinclude prostaglandins and leukotrienes. Leukotrienes are involved incytosolic (cPLA2); calcium-independent (iPLA2); secreted group IIA and V (sPLA2 IIA; V);converting PIP3 to PIP2. Lipids: fatty acid (FA): arachidonic acid (AA), docosahexaenoic4, LTC4); prostaglandins (PGE2, 15d-PGJ2). Exosomes also contain a set of gangliosides(PIP2).

Parent cell model Functional effects References

RBL-2H3 Fatty acid carrier Subra, C [9]

RBL-2H3.MC9,HCM1

Fatty acid and LPC carriers Subra, C ; Valadi, H [9]

RBL-2H3 Prostaglandin biosynthesis Subra, C [9]RBL-2H3 Prostaglandin biosynthesis Subra, C [9]RBL-2H3 Increase exosomes Laulagnier, K [61]Jurkat Decrease exosomes Alonso, R [99]MDM Leukotriene biosynthesis Esser, J [14]MDDCMDM Granulocyte migration Esser, J [14]MDDCMDM Leukotriene Esser, J [14]MDDC ChemotaxisRBL-2H3 Immunosuppression

PPAR ligandSubra, C [9]

MC9,HCM1

Inammation Valadi, H [53]

Oli-neu Increase exosomes Trajkovic, K [62]Oli-neu Increase exosomes Strauss, K [4]NPC1/Su-DHL-4 Decrease exosomes Aung, T [63]Balm-3OCI-Ly1MEF Phosphatase activity in target cells.

Tumor suppressor.Putz, U [100]

HEK 293TU87MG Gabriel, K [101]various pathophysiologies such as inammatory asthma, atherosclerosisand cancer [103]. Evidence for a transcellular biosynthesis processas a regulatory mechanism to control the biological effects of theselipid mediators has been characterized in several cell models [104].Leukotriene A4 (LTA4) is a key intermediate in the generation ofeither LTB4 by the action of the LTA4 hydrolase or LTC4 by the actionof the LTC4 synthase [105]. More than 50% of LTA4 produced byneutrophils is exported from the cells [106], and since the half-lifeof LTA4 in a buffer is about 5 s [107], some protective mechanismmust exist to avoid its degradation during intercellular transfer.Although albumin has been shown to stabilize LTA4 [107], exosomescould play that role by harvesting LTA4 from the parental cell andtransporting the compound for the long periods of time required forbiosynthesis of downstream leukotrienes such as LTB4 which lasts for1 h, or for LTE4, a cysteinyl leukotriene whose synthesis peaks around15 min [105]. Both LTB4 and LCT4 have been found in exosomesfrom antigen-presenting cells such as macrophages or dendritic cellsfollowing differentiation with TGF or IL4 respectively [14]. Thoseexosomes displayed a higher capacity for the conversion of LTA4 intoLTB4 and LTC4 than the parental cells. Remarkably, exosomes carriedthe LTA4 hydrolase and the LTC4 synthase (see Table 1). The LTA4hydrolase activity is 7-fold higher than in parental cells, and consistently5 times more LTC4 was recovered in exosomes [14]. Exosomesfrom these antigen-presenting cells are functional and trigger poly-morphonuclear leukocyte migration.

A similar observation has been reported for prostaglandins. Exosomesderived from the mast-cell line RBL-2H3 are about 10-fold enriched inPGE2 and 15d-PGJ2 as compared with the parental cells upon cellchallenge with a calcium ionophore [9]. These exosomes carried theprecursor arachidonic acid but its exosomal concentration overcamethe capacity of exosomes to generate this fatty acid from their ownphospholipids, suggesting that exosomes also harvest fatty acids andpossibly prostaglandins from the parental cells. Both cyclooxygenases

TxB2 N 6-keto-PGF1a [119]. It is worth noting that internalization ofexosomes by recipient cells enables the prostaglandin 15d-PGJ2 to bypass

114 M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120COX-1 and COX-2 [9] and PGE synthase [53] are present in exosomes;they also contain secreted sPLA2 IIA and V which have been reported tobe involved in transcellular PGE2 biosynthesis [108,109].

Therefore exosomes appear both as transporters and autonomousproduction units of eicosanoids able to participate in their transcellularmetabolisms. As reported from RBL-2H3 cells [110], several subpopu-lations of exosomes could be involved in the process of lipidmetabolismcommunication, bearing different sets of enzymes and eicosanoidprecursors.

5. Accumulation of exosomal lipids in target cells

Once released, exosomes are either washed away in the circulationand constitute potential biomarkers or can interact with proximalor distal cells and be internalized. Indeed, exosomes transfer materialbetween cells and their interactionwith target cells operates via severalmechanisms involving receptor-mediated endocytosis, phagocytosis,non-receptor mediated endocytosis (macropinocytosis), and, morerarely, membrane fusion.

5.1. Lipid receptors involved in exosome fate

Exosome recognition by target cells involves lipid receptors such asTIM for PS and potentially the G protein coupled receptor (GPCR) calledG2A for LPC. Although direct binding of LPC on G2A is controversial,LPC-mediated effects such as lymphocyte chemotaxis is abolishedwhen G2A is inactivated [111]. This lysophospholipid has been shownto be produced not only by the calcium independent iPLA2, but also bythe cPLA2 from parental cells or by exosome on-board phospholipases.

The fate of exosomes depends uponwhich type of cells both exosomalPS and LPC phospholipids interact with by means of their respectivespecic receptors. PS bind TIM-1 and TIM-4 receptors [112]. It hasbeen proposed that PS on exosomes can bind separately to these tworeceptors, forming a bridge between cells which could bring closerimmunocompetent cells such as antigen presenting cells and Tcells, therefore facilitating antigen presentation [113]. Also PS receptorsmediate exosome endocytosis in immunocompetent cells withsubsequent enhancement of antigen presentation.

Althoughmechanisms have not been characterized at themolecularlevel, exosomal LPC could attract T lymphocytes to the lymph nodes andparticipate in the immune response. Immunocompetent exosomesaccumulate in the lymph nodes [97]. LPC is a chemoattractant forlymphocytes [114] and induces the maturation of dendritic cells [115].

The interaction of exosomal PS and LPC with cells undergoingapoptosis triggers exosome elimination. Exosomal LPC binds IgM-typeimmunoglobulins which also recognize apoptotic cells. This allowsexosome interaction with apoptotic cells with subsequent eliminationof both of them [116]. Also the PS receptor TIM-4 is involved in theengulfment of apoptotic cells and in this way can mediate thephagocytosis of exosomes by macrophages [112].

Although not specic for exosome capture, Siglec-1 (Sialic acid-binding Ig-like lectin-1; CD 169) is a receptor expressed in mature DCthat allows exosome internalization by recognition of the sialyllactosemotif of exosome gangliosides [93].

5.2. Exosome trafcking concentrates bioactive lipids in endosomes oftarget cells

Exosomes have been shown to be internalized by a variety of cells.When added to rat basophil leukemia cells, exosomes stay on the cellperiphery for about 15 min and then are internalized in resting or inactivated cells [9]. Uptake is receptor-mediated and involves both thescavenger receptor CD36 [23] and the Hsp90 receptor CD91 [117].Another aspect of exosome internalization in cells is the concentrationof exogenous lipid mediators into recipient MVB-endosomes [9]. Rat

basophil leukemia cells contain an average of 30 MVB per cell, with anthe plasmamembrane and potentially reach the nuclear receptor PPAR.Data on how the 15d-PGJ2 could cross the plasma membrane has not, sofar, been clearly documented.

5.3. Lipids involved in the fusion between exosomes and the recipient lateendosome membrane

Because of the high rigidity of the exosome membrane at neutralpH, which is decreased in an acidic environment [8], fusion of anexosome with another membrane is more likely to occur at the acidicpH of the endosome rather than at the neutral pH environment of theplasma membrane, unless the cell periphery is in acidic conditionssuch as with tumors [120]. Indirect evidence of fusion betweenexosomes accumulated into recipient endosomes and the endosomemembrane arises from the observations that (1) exosomal mRNAs aretranslated into proteins in target cells [53] and that (2) a cycling processof endosomal vesicle (ILV) formation and return to the endosomemembrane called back-fusion has been reported [29,121]. The back-fusion requires BMP, and how lipids can participate in exosome fusionwith the endosome membrane is considered in this part of the review.

Exosomes transport not only lipids, but also lipolytic enzymes, such asphospholipases A2 and D, COX, and leukotriene-related enzymes(Table 1). How these enzymes participate to the lipid metabolism of thetarget cells remains to be investigated. We postulated that the presenceof an active PLD in exosomes that accumulated inside recipient lateendosomes might generate fusogenic molecules such as PA or BMP(LBPA) which would trigger the fusion of the exosome membrane withthat of the recipient endosome, allowing the release of the exosomecontents into the cytosol.

As previously mentioned, immunolabeling of BMP in the endosomecompartment showed an enrichment of this lipid in the inner leaetof the endosome membrane [45], and in the ILVs [43,122]. Howeverbiochemical BMPdeterminations on ILV-derived exosomes failed to showaverage diameter of 600 nm. Calculation of the total volume of theseMVB gives a value which is 500 times lower than the correspondingvolume of the whole cell, considering an average cell diameter of 15 M.Thus internalization of exosomes into endosomes of target cell allowsthe lipid mediators they carry to concentrate into a specic cellularcompartment with a resulting concentration 500 times higher than ifthey hadbeen targeted to thewhole cell. This process enablesmicromolarconcentrations of prostaglandins to be reached, which is in the range oftheir biological effects [9].

As compared to a single free molecule of a lipid mediator that wouldbe released out of a cell, and dispersed in the intercellular mediumbefore reaching target cells, exosomes vectorize numerous moleculesfrom a donor cell into a small compartment of a recipient cell. Thereforeexosomes appear as an efcient vectorization device allowing inter-nalization of prostaglandins such as the 15d-PGJ2 whose target is thenuclear receptor PPAR. It is worth noting that PGE2, whose regulartarget is a peripheral GPCR, will also be internalized in this way. Afunctional role of intracellular PGE2 remains hypothetical. Only a fewreports emphasize a possible activity of intracellular PGE2 to induce aBax-dependent apoptosis in a Bax-expressing subset of glioblastomatumors [118]. In these experiments on glioblastomas, PGE2 wasmicroinjected, while PGE2-containing exosomes provide a natural wayto supply intracellular PGE2.

Exosome exchanges between cells can also affect the cellular lipidhomeostasis [94] by accumulating sphingomyelin and cholesterol intothe endosomes of target cells [5].

On thewhole, exosomes appear as a newmechanismof prostaglandintransport complementary to the transmembrane prostaglandin trans-porter (PGT) whose efciency is PGE1 PGE2 (or PGD2) PGF2a Nany enrichment in BMP [8,83,110], suggesting that some remodeling

of the lipid composition might occur between the stages of the intra-endosomal ILVs and exosomes.

Therefore exosomesmust recover a way to generate fusogenic lipidsonce in the recipient endosomes in order to fuse with the endosomalmembrane (Fig. 3).

The PLD hydrolysis product, phosphatidic acid (PA), can triggermembrane fusion in the presence of calcium [123] and exosomes havebeen shown to be enriched in PA (Fig. 2). PA rst allows the removal ofthe water molecules present on the polar head-group of phospholipids,subsequently facilitating lipid interdigitation between membranes.Precisely, an ARF6-activatable PLD [9] has been found on exosomes, andthis PLD is also active in acidic conditions comparable to the intra-endosomal pH [124].

Another candidate for triggering membrane fusion in acidic condi-tions is BMP, providing it accounts for an appropriate concentration atthe contact between membranes (about 30% of total phospholipids[43]). Although the canonical pathway of BMP biosynthesis derivesfrom lyso-phosphatidylglycerol (LPG), this pathway accounts for onlyhalf of the cellular BMP content [68]. Another possible pathway involvesthe transphosphatidylation activity of PLD between PA and DG or MG(Fig. 3). A transphosphatidylation reaction between PA and DG hasbeen already reported in signaling downstream of the bradykininreceptor and leads to the formation of BisDiacylglyceroPhosphate (BDP,previously called bisphosphatidic acid) and degradative products [125].Indeed PLD activity is often associated with that of the phosphatidatephosphatase (PAP1) that converts PA into DG, which is then possiblyconverted into monoglycerides (MG) by a lipase acting at acidic pHconditions, the lysosomal acid lipase (LAL). MG and DG bearing primary

since the isomerase required to transform the precursor sn3,sn1 LPGinto sn1,sn1 BMP has never been characterized, and the sn-3stereoconguration of phospholipids started with bacteria throughevolution. Provided that a transphosphatidylation reaction could occurbetween PA andMG in cells and directly generate hemi-BMP (previouslycalled semi-LBPA), this compound could be a source of BMP (Fig. 3).Indeed it has been shown that the Pancreatic Lipase-Related Protein 2(PLRP2), whose activity is not stereospecic, can hydrolyze hemi-BMP[60]. Because BMP is acquiring increasing interest regarding ILV/exosometrafcking [128] or cholesterol clearance [129,130] its metabolism has tobe thoroughly reconsidered.

Although less documented, the cPLA2 can play a role in membranefusion [131], and the cPLA2 is also present in exosomes [9].

5.4. Distribution of endosomal-accumulated lipids to other cell compartments

Once fusion between internalized exosomes and the recipientendosomal membrane has occurred the components present within theexosome could be released and dispatched to other cell compartmentssuch as cytosol, endoplasmic reticulum and nucleus. Since exosomalmRNA and miRNA can reach the translation machinery of the recipientcell [53], one can conceive that exosomal lipid molecules can beaddressed to various cell compartments using soluble carrier proteinspresent in the exosome lumen and in the cell cytosol. The fatty acidsarachidonic acid and docosahexaenoic acid (DHA) reach calculatedconcentrations in recipient endosomes of 4mM and 1mM respectively[9]. Exosomal arachidonic acid could be addressed to the cell prosta-glandin biosynthesismachinery in the endoplasmic reticulumand supply

AG

B

em

LA

em

twee6, Pis(Mhosogen

115M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120alcohol moieties can be involved in a transphosphatidylation reactionwith PA mediated by the PLD. Both PLD2 and PAP1 have been reportedin exosomes [9]. BDP displays a large molecular area [60] which mightperturbate the membrane organization suggesting that BDP should notaccumulate in membranes. As a result of PLD stereospecic trans-phosphatidylation, BDP generated by PLD activity will contain a mixtureof sn3,sn3 and sn3,sn1 congurations [126]. This BMPwould differ fromthat with the unexpected sn1,sn1 stereoconguration reported in BHKcells [127]. How cells can make this stereoconguration is not clear

PC PA DG

BDG H

PAP1PLD2

PLD2

?

scramblase

PLD2

MVB peripheral m

Arf6GTP

GTPexosome

Ca++

Fig. 3. Putative pathways for fusogenic lipids (PA and BMP) triggeringmembrane fusion beexosomal membrane are depicted based on the enzymes present in exosomes (PLD2, Arftrigger the formation of Bis(Diacylglycero)Phosphate (BDP) further hydrolysed to hemi-Btransphosphatidylation between PA and MG and converted in BMP by the PLRP2 lipase/pgroup-associated water layer. PA is fusogenic in presence of calcium [123] and BMP is fus

lysophospholipide phosphatase 1); LAL: lysosomal acid lipase; PLRP2: Pancreatic Lipase-Relatemore substrate for cellular prostaglandin biosynthesis. Exosomal DHAcould be targeted to another lipid metabolism pathway related to sterolmetabolism and located also in the endoplasmic reticulum. Indeed DHAis a competitive inhibitor of the cholesterol epoxide hydrolase (CHEH)[132] which catalyzes the hydrolysis of cholesterol 5,6 epoxide intocholestane-triol. Inhibiting the CHEH is clinically relevant since increasedactivity of this enzyme is observed in cancer development and thera-peutic resistance, whereas various anti-tumor compounds are inhibitorsof CHEHat therapeutic doses [133]. In that respect a newaminoalkylsterol

rf6TP

GTP

MP

i-BMP

MGL

PLRP2

scramblase

exosome

pH 5.5

pH 7.4brane

n exosomes and the recipient endosome innermembrane. BMP biosynthesis pathways onAP, scramblase [6]) and those inside endosomes (LAL). These enzymatic activities mightonoacylglycero)Phosphate (hemi-BMP). Hemi-BMP could also be generated directly bypholipase A1. MG=monoglycerides; DG= diglycerides. Green line: phospholipid headic at acidic pH [43]. PLD2: phospholipase D2; PAP1: phosphatidate phosphatase 1 (LPP1,

d Protein 2. Color code: red: PA; green: DG; blue: MG; brown: hemi-BMP; yellow: BMP.

116 M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120named Dendrogenin A, naturally present in healthy tissues but not intumor ones, has recently been characterized as a potent inhibitor of theCHEH and displays anti-tumor activity [134]. DHA has been shown toadd a benecial effect to breast cancer chemotherapy [135] and anexosome-mediated DHA supply could participate in this process.Exosomes contain fatty acid binding proteins (FABP) in their lumen [9]which can transport DHA from the exosomes to other locations.

FABP also binds arachidonic acid (AA) and the FABPAA complexreleased from exosomes can bind to the PPAR receptor located in thecytosol with subsequent targeting of the PPAR to the nucleus where itwill regulate the transcription of specic sets of genes. A specic agonistof PPAR is 15d-PGJ2 and exosomal 15d-PGJ2-driven accumulationinside recipient endosomes enables concentrations of around 50 M tobe attained in these endosomes [9]. This is an efcient concentration tomediate PPAR-dependent effects such as tumor reduction, whichsuggests that exosomal 15d-PGJ2 can bind to the PPAR receptor.

6. Vectorization of bioactive lipids by ex-vivo manipulation ofexosomes

6.1. Engineering of the lipid phase of natural exosomes

Manipulating the lipid phase of natural exosomes to vectorizebioactive lipids appears as an alternative strategy to the use of classicalliposomes made only of phospholipids/cholesterol.

Exosomes feature a typical lipid bilayer membrane [8] and someamphiphilic molecules can be inserted into this lipid phase duringin-vitro manipulations. Those manipulations open the possibility tosupplement exosomes with appropriated lipidic molecules. LPCpenetrates easily into lipid bilayers and has been shown to triggerlymphocyte chemotaxis and dendritic cell differentiation. LPC-enrichedexosomes might boost the immune response specically in the lymphnodes in which they have been shown to stay for a long period of time[97]. Another interesting lipidic manipulation of exosomes has been toenrich them with curcumin [136], a yellow pigment used abundantly inIndian food. Curcumin is a lipidic molecule with a polyphenolic structureand which shows anti-oxidant and anti-inammatory properties, andit also inhibits tumor growth. Whereas free curcumin is ineffective in awhole organism because of its poor solubility in biological uids,exosome-vehiculated curcumin has proven to be very efcient towardstumors or inammation related-diseases in the brain [136]. In thatrespect intranasal injection in mice of curcumin-enriched exosomesallowed these vesicles to rapidly reach the brain and to protect againstLPS-induced brain inammation, or to inhibit brain tumor growth[132]. This work showed for the rst time that exosomes are able tocross the blood-brain barrier. Exosome-mediated transport throughthe blood-brain barrier has been further assessed by systemic injectionin mouse tails of exosomes bio-engineered with an siRNA directedagainst the brain protease BACE. Expression of BACE, which is involvedin the Alzheimer disease, was inhibited by this exosome-based treat-ment [49].

6.2. Engineering of exoliposomes

While the use of natural exosomes for medical purposes isrestricted to autologous treatments, liposomes mimicking exosomes(exoliposomes) would overcome that problem. Exoliposomes can bedened as liposomeswith the same size and lipid composition as naturalexosomes. Liposomes have been considered for a long time as a tool tovectorize molecules of interest, but liposome compositions reportedacross the literature have been adapted more or less faithfully from thecompositions of biological membranes. Exosomes have provided theopportunity to elaborate liposomes with a lipid composition of a naturalbioactive nanovesicle (Fig. 2). Interestingly those exoliposomes ex-hibited a similar membrane uidity as natural exosomes [8], indicating

that exoliposomes could mimic natural exosomes. Recently it wasobserved that exoliposomes loaded with antigenic peptides stimulatedIL-2 secretion from in-vitro cultured lymphocytes similarly to classicalliposomes (M.R. unpublished data), suggesting that encapsulated antigensare more efcient than a free form whatever is the lipid composition ofthe vesicular carrier.

To optimize their biodistribution, exoliposomes could be coated withMHCI/peptide complexes and selective ligands for adhesion to T cells inorder to target the immune system [137]. However such engineeringappears tedious.

Although not strictly related to exosomes since they display distinctsize and density and lacked typical exosome markers, nanovesiclesreleased by pancreatic tumor SOJ-6 cells trigger tumor cell death [138].Liposomes made from the lipid composition of those nanovesicles areable to kill the tumor cells. Their efcacy appears to rely on the amountof raft as shown by the ratio of cholesterol and sphingomyelin overglycerophospholipids [138].

In summary the lipidic composition of the natural exosomes providesa way to dene the optimal lipid composition of exoliposomes. In thatrespect the lipidomic analysis of exosomes/prostasomes brings insightsin this domain [10].

Thereby manipulating the lipidic composition of exoliposomes mightlead to production of bioactive liposomal vesicles, provided that theirbiodistribution is adequate in the body as compared with naturalexosomes. This is of interest because of the involvement of exosomes inseveral pathologies.

7. Pathophysiological role of lipid exosomes

7.1. Exosomal lipids in tumor growth and resistance

Although PGE2 is secreted via a plasma membrane transporter[139,140], it has been shown that exosomes vehiculate this prostaglandin[9,18]. PGE2 triggers a coordinated immunosuppression allowingtumor development [141]. Exosomes derived from murine mammaryadenocarcinoma cell lines (TS/A; 4 T-1) and the respective tumorscontain PGE2whose amount is correlatedwith the induction ofMyeloidDerived Suppressor Cells (MDSC) [18]. These cells down-regulate T cellfunctions in-vivo. Inactivating exosomal PGE2 with a specic antibodypartly reverses MDSC proliferation and tumor growth [18]. Recentlya cell death-induced tumor repopulation pathway including caspase3/calcium independent phospholipase A2/PGE2 has been proposedas a mechanism for tumor recurrence following radiotherapy [142].Precisely all the components of this pathway (caspase3-activatediPLA2, arachidonic acid, PGE2) have been found in exosomes [6]. Thusthe balance between the immunosuppressive PGE2 and the antitumor15d-PGJ2 as reported above [6] in tumor exosomes appears as a keypoint for their outcome in tumor development.

Exosomes have been shown to carry leukotrienes and to contributeto granulocyte recruitment indicating a role in inammatory diseases[14]. Leukotrienes in exosomes (Table 1) might also participate intumor development since LTB4 and LTC4 receptors are highly expressedin some cancers [141].

Interestingly, exosome secretion involving the phosphatidylcholinetransporter ABCA3 has been implicated in the resistance to B lymphomatreatment with rituximab, an antibody that targets the B-lymphocyteantigen CD20. ABCA3 is over-expressed in B lymphomas and triggersthe clearance of the antitumor drug by an enhanced exosome release,thus leading to some resistance to this therapy [63]. ABCA3 has beencharacterized mainly as a phosphatidylcholine transporter participatinginmultilamellar body formation in pneumocytes [143] but it is expressedin all major types of malignant lymphohematopoietic diseases [144] andis localized in endosomes [145],which are the site of exosomebiogenesis.This suggests that an enrichment in phosphatidylcholine of the exosomemembrane would favor a clearance pathway rather than an intercellularcommunication function of exosomes. In this respect exosomes

from reticulocytes which clear uneeded proteins during reticulocyte

even though it is now possible to recover exosomes from minimalvolumes of various biological uids. Two lipidic molecules, LPC and PS

117M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120maturation into erythrocytes have a much higher content of phos-phatidylcholine as compared with the typical exosome composition([37] and Fig. 2). Subsequently these exosomes can exhibit high levelsof LPC which is an eat-me signal for exosome elimination by apoptoticcells [116].

7.2. Exosomal cholesterol in cholesterol-related diseases

Exosomes derived from T cells interact with the PS receptor onmonocytes and alleviate these cells of cholesterol which accumulatesinto lipid droplets, suggesting that such exosomes could play a role inatherosclerosis development [5]. On the contrary, the role of exosomesin cholesterol clearance has been unveiled by the defect in the NPC-1cholesterol transporter in NiemannPick disease. It is an autosomaI-recessive lysosomal disorder with abnormal cholesterol accumulationin the late endosomal and lysosomal compartments, with subsequentneurodegeneration and dysmyelination. It has been proposed thatexosomal release of cholesterol could partially bypass the lysosomalcholesterol accumulation which is deleterious in this disease [4].Interestingly, the capacity of endosomes in cholesterol storage iscontrolled by their BMP content [87]. Therefore the lipidic compositionof exosomes and their secretion can play a role in cellular cholesteroltrafcking.

Cholesterol also becomes an important parameter in cancer devel-opment [146]. The quantitative effect of cholesterol clearance fromtumor cells by exosomes can be estimated from the following parameters.The cholesterol/phospholipid ratio is 14mol% in cells [8] but increasesto 30 mol% in exosomes [37] consistent with the average two-foldenrichment in exosomal cholesterol as compared to parental cells(Fig. 2). 109 rat basophil leukemia cells (RBL-2H3 cell) which contain24 mol phospholipids i.e. 3.4 mol cholesterol (24 0.14 = 3.4) [8]release an amount of exosomes which contain 1.7 mol phospholipidsand 0.5 mol cholesterol (1.7 0.3 = 0.5) [8,37] in 24 h. Thus theclearance of exosome-associated cholesterol in 24 h can account forabout 15% (0.5/3.4) of the cellular cholesterol, therefore being a non-negligible process.

Endosomal cholesterol appears to regulate exosome production.Treatment of cells from the central nervous system with the cationicamphiphile U18666A that accumulates cholesterol into endosomesincreases by two-fold the amount of exosomes containing thecholesterol-binding protein otillin [4]. Similarly, broblasts fromNiemannPick C patients accumulate cholesterol into endosomesbecause of the mutation on the NPC1 transporter activity and show anenhanced release of exosomes [4]. On the contrary, B-cell lymphomawhich originally has high production of exosomes decreases thisproduction upon treatment with U18666A [63]. This opposite observa-tions emphasize that the role of cholesterol in exosome release mightdepend upon the cell type considered and their own regulation of choles-terol metabolism. Besides acting on the NPC1 transporter, U18666Amodies the activity of several enzymes of cholesterol metabolism[44]. In addition, otillin2-bearing exosomes might be a subpopula-tion of exosomes, whereas the effect of U18666A on lymphomawas checked on total exosomes [63]. These lymphoma cells alsofeature an over-expression of the phosphatidylcholine transporterABCA3 [63]. Altogether these observations suggest that the cholesterol/phosphatidylcholine content of parental cells might regulate exosomeproduction.

8. Regulation of circulating exosomes in a whole organism

Release of exosomes appears as a general feature of all cells andtissues and requires a tight regulation of their amount at the level of awhole organism. The highest amount of exosomes produced in thebody derives from reticulocyte maturation since about 2 1011

reticulocytes are differentiated every day into erythrocytes by releasing

proteins via exosomes. Therefore an efcient clearingmechanismmustappear as key regulators of the circulating exosomes.

9. Conclusion

Since the pioneer studies demonstrating their involvement in theimmune response and reticulocyte maturation, exosomes have emergedas a new communication systembetween cells and arewidespread in thebody. They represent an example of natural nanovesicles actively se-creted by viable cells, therefore linking cell secretion with intercellularsignaling. As compared to soluble agonists, they carry multiple signalingmolecules together, and since they are addressed to specic target cells,exosomes constitute a mechanism of vectorized signaling.

In this context, exosomes vectorize bioactive lipids, and appear asanother biological system for transporting a variety of lipids, such asfatty acids, prostaglandins and cholesterol. Exosome trafcking canmodulate endogenous cell lipid metabolism by supplying additionalsubstrate for eicosanoid biosynthesis and eicosanoids themselves. Sinceexosomes can be released and taken up by cells, it means exosomescan cycle between proximal cells as those present in the tumor micro-environment and modify their lipid metabolism. The secretion ofexosomes has been quantitatively underestimated and further experi-ments are required to assess the role of these bioactive vesicles in theoverall cell metabolism.

Because of exosome involvement in various pathophysiologies,it appears essential to control their production. For that purpose,addressing therapeutic molecules to endosomes via the endocytic trackcould be a strategy to enhance the production of immunocompetentexosomes, or to decrease the formation of tumor exosomes. Ex-vivomodication of the lipid composition of exosomes could allow a betterway to address lipidic therapeutic molecules than the use of liposomes.Alternatively, it will be worth developing pharmacological compoundsable to modify the molecular composition of exosomes during theiroperate in the body. So far only the interaction of exosomes withcirculating apoptotic cells has beenwell characterized as an eliminationprocess. The lyso-lipid LPC present on exosome surface binds circulatingimmunoglobulins IgM which in turn bind apoptotic cells allowing theelimination of exosomes together with apoptotic cells [116].

Exosomes are enriched in LPC as compared with the parental cells[8]. This content in LPCmight originate not only from the phospholipaseactivities borne by exosomes but also from the parental cells. It hasbeen observed that the phospholipase iPLA2 in parental reticulo-cytes is activated by reactive oxygen species (ROS) produced by the15-lipoxygenase during exosome production [116].

On the other hand, the LPC content in immunocompetent dendriticcell-derived exosomes can favor their migration to the lymph nodesthat appear to be protected homing sites since exosomes can stay therefor a long period [97]. In addition exosomal LPC in the lymph nodescan participate in the immune response since LPC bound to anotherparticulate carrier (oxidized lipoproteins) has been shown to induceDC maturation [147].

In summary LPC can provoke opposing fates for exosomes dependingupon the organ in which they are located. Similarly, exosomal PS willdetermine exosome elimination when interacting with phagocyteswhereas it can favor interaction between immunocompetent cells [113].

So far the impact of exosome trafcking in the physiology of thewhole human body requires further investigations, but lipids exposedon the exosome periphery appear to play a key role in exosome fateand bioactivity.

In addition lipids of circulating exosomes canbeused as biomarkers oflipid-related pathologies, in a complement to protein and miRNA bio-markers [148,149]. In this context, the regulation of the amount ofcirculating exosomes is critical for the detection of exosomal biomarkers,biogenesis in order to improve their benecial effect in health [3]. New

[24] R.M. Johnstone, M. Adam, J.R. Hammond, L. Orr, C. Turbide, Vesicle formationduring reticulocyte maturation. Association of plasma membrane activities with

118 M. Record et al. / Biochimica et Biophysica Acta 1841 (2014) 108120released vesicles (exosomes), J. Biol. Chem. 262 (1987) 94129420.[25] A. Bobrie, M. Colombo, G. Raposo, C. Thery, Exosome secretion: molecular

mechanisms and roles in immune responses, Trafc 12 (2011) 16591668.lipidic molecules, such as aminoalkylsterols [150,151] appear of interestin this pharmacological approach.

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