cells
Re iew
Ex acellula Vesicles in NAFLD/ALD: F om
Pa hobiology o The apy
Alejand a He nández 1,2, Juan Pablo A ab 1,3 , Daniela Reyes 1, Ainhoa Lapi z 4,
Han Moshage 2, Jesús M. Bañales 4,5,6 and Ma co A ese 1,3,*
1
Depa amen o de Gas oen e ologia, Escuela de Medicina, Pon i icia Uni e sidad Ca olica de Chile. San iago,
Chile 8330077; [email p o ec ed] (A.H.); [email p o ec ed] (J.P.A.); [email p o ec ed] (D.R.)
2Depa men o Gas oen e ology and Hepa ology, Uni e si y o G oningen, Uni e si y Medical Cen e
G oningen, 9713 GZ G oningen, The Ne he lands; [email p o ec ed]
3
Cen o de En ejecimien o y Regene acion (CARE), Depa amen o de Biolog
í
a Celula y Molecula , Facul ad
de Ciencias Biológicas Pon i icia Uni e sidad Ca olica de Chile, San iago 8331010, Chile
4Biodonos ia Heal h Resea ch Ins i u e, Donos ia Uni e si y Hospi al, 20014 San Sebas ian, Spain;
[email p o ec ed] (A.L.); [email p o ec ed]g (J.M.B.)
5Na ional Ins i u e o he S udy o Li e and Gas oin es inal Diseases (CIBERehd), Ca los III Na ional
Ins i u e o Heal h, 28029 Mad id, Spain
6IKERBASQUE, Basque Founda ion o Science, 48013 Bilbao, Spain
*Co espondence: ma [email p o ec ed]; Tel.: +56-2-3543822
Recei ed: 28 Feb ua y 2020; Accep ed: 24 Ma ch 2020; Published: 27 Ma ch 2020
Abs ac :
In ecen yea s, knowledge on he biology and pa hobiology o ex acellula esicles
(EVs) has exploded. EVs a e submic on memb ane-bound s uc u es sec e ed om di e en cell
ypes con aining a wide a ie y o bioac i e molecules (e.g., p o eins, lipids, and nucleic acids
(coding and non-coding RNA) and mi ochond ial DNA). EVs ha e impo an unc ions in cell- o-cell
communica ion and a e ound in a wide a ie y o issues and body luids. Be e delinea ion o
EV s uc u es and ad ances in he isola ion and cha ac e iza ion o hei ca go ha e allowed he
diagnos ic and he apeu ic implica ions o hese pa icles o be explo ed. In he ield o li e diseases,
EVs a e eme ging as key playe s in he pa hogenesis o bo h nonalcoholic li e disease (NAFLD) and
alcoholic li e disease (ALD), he mos p e alen li e diseases wo ldwide, and hei complica ions,
including de elopmen o hepa ocellula ca cinoma. In hese diseases, s essed/damaged hepa ocy es
elease la ge quan i ies o EVs ha con ibu e o he occu ence o in lamma ion, ib ogenesis, and
angiogenesis, which a e key pa hobiological p ocesses in li e disease p og ession. Mo eo e , he
speci ic molecula signa u es o eleased EVs in bio luids ha e allowed EVs o be conside ed as
p omising candida es o se e as disease bioma ke s. Addi ionally, di e en expe imen al s udies
ha e shown ha EVs may ha e po en ial o he apeu ic use as a li e -speci ic deli e y me hod o
di e en agen s, aking ad an age o hei hepa ocellula up ake h ough in e ac ions wi h speci ic
ecep o s. In his e iew, we ocused on he mos ecen indings conce ning he ole o EVs as new
s uc u es media ing au oc ine and pa ac ine in e cellula communica ion in bo h ALD and NAFLD,
as well as hei po en ial use as bioma ke s o disease se e i y and p og ession. Eme ging he apeu ic
applica ions o EVs in hese li e diseases we e also examined, along wi h he po en ial o success ul
ansi ion om bench o clinic.
Keywo ds:
nonalcoholic a y li e disease; a y li e ; alcoholic li e disease; ex acellula esicles;
signaling; exosomes; bioma ke s
Cells 2020,9, 817; doi:10.3390/cells9040817 www.mdpi.com/jou nal/cells
Cells 2020,9, 817 2 o 14
1. In oduc ion
Knowledge o he pa hobiology o ex acellula esicles (EVs) has expanded signi ican ly in he las
decade [
1
,
2
]. Indeed, signi ican ad ances ha e been made in delinea ing he mechanisms o assembly
and elease o EVs, as well as hei subsequen memb ane usion wi h a ge cells [
3
,
4
]. Mo eo e ,
powe ul analy ical echniques ha e made possible he ex ensi e cha ac e iza ion o he ca go o
EVs, which includes my iad molecules including g ow h ac o s, me abolic enzymes, mic oRNAs
and ansc ip ion ac o s, ce ain p o eins, lipids, and me aboli es, among o he s, ha modula e
in e cellula and in e o gan communica ion [
3
,
5
,
6
]. I is o no e ha high- h oughpu da ase s o
esicula componen s a e now a ailable in public da abases, which s ongly suppo s EV esea ch [
7
,
8
].
No el insigh s in o he biology o EVs show ha hese pa icles egula e c i ical biological unc ions
and may ac as con ibu o s o disease pa hogenesis, and may also se e as disease bioma ke s, wi h
he i ue o he ela i e simplici y o EV isola ion om di e en bio luids [
9
]. In addi ion, EVs a e
gaining in e es om a he apeu ic poin o iew due o hei po en ial as a unique d ug deli e y
sys em [10].
In he ield o hepa ology, EVs ha e ecen ly eme ged as no el playe s in he pa hogenesis and
p og ession o se e al condi ions [
11
–
13
], including he wo mos common li e diseases wo ldwide:
nonalcoholic li e disease (NAFLD) and alcoholic li e disease (ALD) [
14
]. Speci ically, ecen s udies
poin o a signi ican ole o EVs in modula ing inju y, ampli ying in lamma ion, and p omo ing li e
ib osis in bo h NAFLD and ALD [
15
–
17
]. Since in o ma ion on his opic is dynamic and apidly
e ol ing, we aimed o p o ide an up- o-da e o e iew o he cu en knowledge on he ole o EVs in
he con ex o bo h NAFLD and ALD, wi h emphasis on hei po en ial diagnos ic and he apeu ic
impac in hese diseases. We excluded om his e iew da a ega ding EVs in li e cance , since his
has been ecen ly e iewed elsewhe e [18,19].
2. Gene al Concep s o EVs in he Li e : EVs Biogenesis, Sec e ion, and Ca go
De ails on he o ma ion and sec e ion pa hways o EVs ha e been ecen ly e iewed
elsewhe e
[15,20,21]
, and can also be ound in o he con ibu ions o his special issue o Cells [
22
]. Only
basic concep s a e p o ided he ein, as well as in o ma ion on aspec s ha a e o pa icula impo ance
o li e physiology and pa hophysiology.
In gene al, EVs a e classi ied acco ding o size and biogene ic pa hway, such as exosomes,
mic o esicles, and apop o ic bodies [
23
]. Exosomes a e bilaye lipid esicles wi h a diame e o
30–150 nm ha a e de i ed om endosomal mul i esicula bodies (MVBs) [
15
,
23
]. Thei o ma ion
esul s om he in agina ion o he plasma memb ane (ea ly endosome) and he subsequen usion o
endocy ic esicles media ed by he endosomal so ing complex esponsible o anspo (ESCRTs)
and o he componen s (such as ce amides and e aspanins) [
24
]. MVBs can elease he in aluminal
esicles known as exosomes by he usion o MVBs o he plasma memb ane, a p ocess media ed in
pa by Rab GTPases [
25
]. Mic o esicles (MVs) ha e a diame e o 50–1000 nm and o igina e om
he plasma memb ane by budding and ission, ollowed by elease in o he ex acellula space [
15
,
23
].
MVs con ain a subse o cell su ace p o eins depending on he composi ion o he pa en al plasma
memb ane [
26
,
27
]. Apop o ic bodies ha e a diame e o 100–5000 nm, o igina e om he budding o
cell memb anes, and may con ain nuclea ma e ial, which is quickly phagocy osed du ing p og ammed
cell dea h [
15
,
28
]. Unlike exosomes and MVs, he ole o apop o ic bodies is no ela ed o in e cellula
communica ion. The e o e, when s udying EVs unde cellula damage condi ions, apop o ic bodies
a e excluded [29].
The impac o EV esea ch is highligh ed by he exis ence o a dedica ed scien i ic socie y ( he
In e na ional Socie y o Ex acellula Vesicles, ISEV, h ps://www.ise .o g/) ha is cons an ly upda ing
exis en in o ma ion on echnical issues in o de o homogenize he da a being gene a ed in di e en
labo a o ies. To ha end, he ISEV eleased a documen en i led Minimal In o ma ion o S udies
o Ex acellula Vesicles (“MISEV”) Guidelines, o help esea che s o be e isola e and cha ac e ize
hei EV p epa a ions, as well as sugges ing p o ocols o documen speci ic EV-associa ed unc ional
Cells 2020,9, 817 3 o 14
ac i i ies [
29
]. A he p esen ime, MISEV2018 does no p opose molecula ma ke s ha could
speci ically cha ac e ize each EV sub ype. Howe e , unde s anding o exis ing sub ypes and hei
associa ions wi h o he cellula s uc u es is an e ol ing ma e and p og ess is expec ed in his
ega d [
22
]. Cu en ly, he MISEV2018 guidelines sugges h ee ca ego ies o ma ke s o be analyzed in
all EV p epa a ions: Ca ego ies 1 and 2, which demons a e he p esence o EVs, and Ca ego y 3, which
assesses hei pu i y om common con aminan s. The la e ecommends apolipop o eins A1/2 and B
(APOA1/2, APOB), and albumin (ALB) as he bes nega i e ma ke s a ailable a he p esen ime.
All EVs anspo a a ie y o bioac i e molecules including cy oplasmic p o eins, lipids, speci ic
lipid- a -in e ac ing p o eins, messenge RNA (mRNA), mic oRNA (miRNA), ibosomal RNA ( RNA),
ans e RNA ( RNA), noncoding RNAs (ncRNAs), DNA, mi ochond ial DNA (m DNA), and
me aboli es [
24
,
30
]. Lipidomic analysis has shown ha EVs, independen ly o hei biogenesis,
con ain a mul i ude o lipids such as choles e ol, sphingomyelin, ce amide, sa u a ed a y acids,
phospha idylcholine, phospha idyle hanolamine, and phospha idylse ine [
4
,
23
,
29
,
30
]. In addi ion,
p o eomic analysis has shown ha EVs con ain di e en ypes o p o eins, such as hea shock p o eins
(Hsp70 and Hsp90), e aspanins (CD9, CD63, CD81, CD82), endosomal so ing complex p o eins
equi ed o anspo (Alix and Tsg101), ecep o s including epide mal g ow h ac o ecep o (EGFR),
memb ane a icking p o eins (GTPases, Flo illin and Annexins), cy oskele al p o eins ( ubulin and
ac in), and cy osolic p o eins [
5
,
26
]. I is wo h no ing ha he ca go o EVs a ies depending no only
on hei cellula o igin, bu also on he condi ion unde which hey a e eleased (i.e., physiological s.
pa hological).
In he li e , bo h pa enchymal (hepa ocy es) and non-pa enchymal cells (e.g., hepa ic s ella e
cells, endo helial, cholangiocy es, Kup e cells, and li e endo helial cells) ha e been ound o elease
EVs in bo h physiological and pa hological s a es [
20
]. Howe e , in o ma ion on a ge cell epe oi e,
ecep o s, o o he speci ic ac ions is s ill limi ed and incomple e. I has been shown ha heal hy
hepa ocy es p oduce limi ed numbe s o exosomes con aining p o eins po en ially ele an o cell
su i al, g ow h, and p oli e a ion [
31
], whe eas s essed hepa ocy es boos exosome elease [
32
] and
en ich hei con en in speci ic p o eins, lipids, and mic oRNAs ha modula e he ansc ip ional
p og am o neighbo ing hepa ocy es and non-pa enchymal cells, hus modula ing in lamma ion and
ib osis, which a e c i ical o he p og ession o li e diseases (Figu e 1). In e es ingly, ecen e idence
sugges s ha EVs om a -laden hepa ocy es can also signal o o he o gans such as adipose issue,
in luencing adipogenesis and issue emodeling [33].
Cells 2020,9, 817 4 o 14
Cells 2020, 9, x FOR PEER REVIEW 4 o 14
Figu e 1. Ex acellula esicles (EVs) can be eleased by hepa ocy es upon lipo oxic o alcohol-induced
inju y. EV ca goes include a mul i ude o molecules ha can ac on a ge cells, e oking in lamma o y and
ib ogenic e en s and p omo ing neoplas ic ans o ma ion, hus con ibu ing o he p og ession o bo h
alcoholic li e disease (ALD) and nonalcoholic li e disease (NAFLD) o hei in lamma o y and mo e
agg essi e o ms alcoholic s ea ohepa i is (ASH) and nonalcoholic s ea ohepa i is (NASH), espec i ely
as well as o hepa ocellula ca cinoma (HCC de elopmen . EVs hold p omise o bo h diagnosis (i.e. as
bioma ke s o disease se e i y o o diagnosis o HCC) and he apeu ic pu poses (i.e. he apeu ic use o
mesenchymal s em cell [MSC]-de i ed EVs, d ug o miRNAs li e a ge ed he apies and an i- umo
accines).
Figu e 1.
Ex acellula esicles (EVs) can be eleased by hepa ocy es upon lipo oxic o alcohol-induced
inju y. EV ca goes include a mul i ude o molecules ha can ac on a ge cells, e oking in lamma o y
and ib ogenic e en s and p omo ing neoplas ic ans o ma ion, hus con ibu ing o he p og ession
o bo h alcoholic li e disease (ALD) and nonalcoholic li e disease (NAFLD) o hei in lamma o y
and mo e agg essi e o ms alcoholic s ea ohepa i is (ASH) and nonalcoholic s ea ohepa i is (NASH),
espec i ely as well as o hepa ocellula ca cinoma (HCC de elopmen . EVs hold p omise o bo h
diagnosis (i.e., as bioma ke s o disease se e i y o o diagnosis o HCC) and he apeu ic pu poses (i.e.,
he apeu ic use o mesenchymal s em cell [MSC]-de i ed EVs, d ug o miRNAs li e a ge ed he apies
and an i- umo accines).
Cells 2020,9, 817 5 o 14
3. EVs in Li e Pa hobiology o The apy
3.1. EVs and Li e In lamma ion
Hepa ocellula damage de e mines he elease o a numbe o signals in o he ex acellula
en i onmen ha can con ibu e o issue in lamma ion [
34
]. Some o hese signals (collec i ely e med
damage-associa ed molecula pa e ns (DAMPs)) a e packaged in EVs and signal be ween hepa ocy es
and non-pa enchymal cells such as li e - esiden mac ophages (Kup e cells, KCs) [
35
]. Indeed, EVs
may e oke he syn hesis and elease o p oin lamma o y cy okines such as p o-in e leukin (IL)-1
β
and IL-6 [
36
] by KCs, hus con ibu ing o local in lamma ion [
37
]. EVs eleased om hepa ocy es can
also p omo e he ec ui men o addi ional immune cells (e.g., p oin lamma o y monocy e-de i ed
mac ophages) in o he li e , main aining and ampli ying in lamma ion. EVs can also signal o
endo helial cells and can con ibu e o ascula in lamma ion [
38
]. Fu he mo e, o add complexi y,
EVs can also be sec e ed om o he cells and in luence li e in lamma ion. In his ega d, some
e idence sugges s ha pla ele -de i ed EVs may ha e p oin lamma o y e ec s in he li e , bu his
needs u he con i ma ion [
39
,
40
]. Finally, i has also been shown ha EVs a e eleased om monocy ic
cells and induce pola iza ion owa ds he an i-in lamma o y M2 pheno ype o neighbo ing nai e
monocy es by deli e ing ca go miR-27a, hus con ibu ing o esolu ion o in lamma ion [
41
]. O he
examples o modula ion o immune esponse ha e been shown in s udies o hepa i is B and C [
42
,
43
],
which can pa icipa e in exace ba ing li e inju y when bo h i al in ec ion and o he inju ing agen s
(i.e., alcohol) a e a play.
Collec i ely, hese indings sugges ha EVs eleased om inju ed hepa ocy es ha e an impo an
ole in modula ing he in lamma o y esponse du ing li e damage h ough in e cellula communica ion
be ween di e en cell ypes, wi h po en ial con ibu ions om o he cell-de i ed EVs [
35
]. Speci ic
mechanisms in ol ed in NAFLD and ALD a e e iewed below.
3.2. EVs and Li e Fib osis
Pe sis en li e ib ogenesis and he de elopmen o ci hosis a e esponsible o he li e - ela ed
mo bidi y and mo ali y associa ed wi h ch onic li e diseases [
44
]. Ac i a ion o ans-di e en ia ion
o HSCs esul ing in insoluble collagen deposi ion and dis o ion o he no mal mac o- and
mic o-ana omical s uc u e o he li e is he majo d i e o li e ib ogenesis [
45
]. The ole o
pa ac ine signals o igina ing om inju ed epi helial cells (hepa ocy es) ha can di ec ly o indi ec ly
induce HSC ac i a ion has been ecognized in ecen yea s [
46
]. In his ega d, EVs seem o play a
ole, as shown by p e ious epo ed s udies. Lipid-induced hepa ocy e-de i ed EVs seem o egula e
HSC ac i a ion by shu ling speci ic mic oRNAs (e.g., miR-128-3p) ha supp ess PPAR-
γ
exp ession in
HSCs, leading o a ma ked inc ease o p o ib ogenic gene exp ession [
47
]. O he au ho s ha e shown
ha in e naliza ion o endo helial-de i ed exosomes by HSCs enhances cell mig a ion in a p ocess
media ed by sphingosine 1-phospha e (S1P) [
48
]. Addi ionally, in e cellula communica ion be ween
quiescen and ac i a ed HSCs ia exosomes can also modula e ib osis. In his ega d, a ole o shu led
mic oRNA 214 (miR-214) in egula ing he exp ession o alpha-smoo h muscle ac in and collagen
in ac i a ed HSCs has been demons a ed [
49
,
50
]. Mo eo e , EVs de i ed om non- esiden cells
such as pla ele s o g anulocy es ha e been shown o inc ease angiogenesis and o ha e p ocoagulan
p ope ies, hus p omo ing ib ogenesis [
51
,
52
]. These indings sugges ha EVs appea o be key
modula o s in ib osis, as signals om bo h pa enchymal and non-pa enchymal li e cells can ei he
d i e o slow down HSC ac i a ion. In addi ion, ci cula ing EVs may be used as a bioma ke o
hepa ic ib osis and ha e po en ial implica ions o he de elopmen o no el an i- ib o ic a ge s [
53
],
as e iewed in he ollowing sec ions.
Cells 2020,9, 817 6 o 14
3.3. EVs as Bioma ke s in Li e Diseases
Dynamic changes o EV gene a ion in pa hological condi ions and he accessibili y o measu e
and analyze hem in biological samples (e.g., blood, u ine, bile and o he bio luids) make EVs good
candida es o be used as disease bioma ke s. The ela i e accessibili y o es ing and he abili y o
pe o m epea ed measu emen s o e ime could acili a e ea ly diagnosis, disease moni o ing, and
he de elopmen o pe sonalized medicine. Fu he mo e, e inemen o echniques allowing isola ion
and in-dep h cha ac e iza ion o EV ca goes could lead o iden i ica ion o disease-speci ic molecula
signa u es o p o iles, p o iding ample oppo uni ies o EVs o be used as sui able, non-in asi e
bioma ke s [54].
EVs ha e been s udied as po en ial bioma ke s o li e inju y in he se ings o ALD, NAFLD,
d ug-induced li e disease, and cholangiopa hies [
13
,
27
,
55
,
56
], as well as being conside ed diagnos ic
ools o li e cance (e.g., hepa ocellula ca cinoma and cholangioca cinoma) [
19
,
56
]. In his
ega d, de e mina ion o he numbe o ci cula ing EVs and nucleic-acid based, lipid-based, and
p o ein-based diagnos ics ha e been applied o measu e EVs en iched in li e -de i ed DNA,
mic oRNAs, lipids, o p o eins [
13
,
27
]. Howe e , be o e implemen ing EV-based bioma ke s in
clinical se ings, s anda diza ion o sample p ocessing (e.g., collec ion, anspo a ion, s o age, and
handling) and assay sys ems is needed, as well as la ge eplica i e s udies o allow EV molecula
signa u es o be conclusi ely linked o speci ic li e diseases. Recen ad ances ela ed o NAFLD and
ALD a e e iewed below.
3.4. The apeu ic Po en ial o EVs
F om a he apeu ic pe spec i e, EVs, ei he unmodi ied o enginee ed, can be u ilized o
he apeu ic pu poses [
57
]. Wi h ega d o li e diseases, e o s ha e been ocused on wo majo a eas:
(a) he use o EVs as deli e y ehicles o d ugs o he li e [
58
] and (b) he use o EVs hemsel es as
he apeu ic agen s o s imula e li e egene a ion, modula e in lamma ion, educe li e ib osis, o
hal hepa oca cinogenesis [
59
,
60
]. The o me app oach in ol es he use o di e en echniques o
load EVs wi h a desi ed ca go (e.g., miRNA, siRNA, chemo he apeu ic agen s) o ac as a “T ojan
ho se” o a ge cells. Theo e ically, he memb ane p ope ies o EVs allow o gan- and cell-speci ic
deli e y, immune-e asion, and a ge ing o dis inc in acellula a icking pa hways. Mo eo e , EVs
could be modi ied o imp o e li e -speci ic a ge ing h ough complex chemical me hods (e.g., click
chemis y) o al e hei su ace o a o a ge ing, o loading hem wi h desi ed he apeu ic agen s
using a a ie y o echniques (e.g., elec opo a ion, saponin-induced po e o ma ion, hypo onic dialysis,
and o he s) [
60
]. La ge EVs (e.g., mic o esicles) could be mo e sui able o he apeu ic pu poses, as
hey may allow be e d ug loading and biodis ibu ion [
61
]. In spi e o hese ad an ages, a numbe o
challenges ela ed o he manu ac u ing o EVs (i.e., p oduc ion, coa ing, loading, e c.) s ill need o be
sol ed be o e con olled clinical s udies can be ca ied ou [62,63].
Rega ding he use o EVs as he apeu ic agen s, mos o he a ailable e idence has been gene a ed
using mesenchymal s em cell (MSC)-de i ed EVs ob ained om human umbilical co ds o human
emb yos ha ha e been es ed in nume ous p eclinical li e disease models (e.g., ca bon e achlo ide,
hioace amide, D-galac osamine/TNF-
α
-induced le hal hepa ic ailu e, and bile duc liga ion) wi h
p omising esul s [
11
,
53
]. Da a o NAFLD and ALD a e mo e limi ed and a e e iewed in he
co esponding sec ions below.
A hi d, and s ill nascen , app oach ela ed o EV-based he apy is based on he concep ha
in e e ing wi h EV sec e ion o up ake may a enua e ha m ul e ec s on a ge cells [
11
,
64
]. In
his ega d, se e al pha macological agen s a e being explo ed ha ha e been shown o inhibi EV
a icking, modi y lipid me abolism, o dec ease EV sec e ion. Howe e , he complexi y o EV
biogenesis poses signi ican challenges o he de elopmen o speci ic agen s able o selec i ely block
EV p oduc ion (see Re e ence [64] o an in-dep h e iew o his opic).
Cells 2020,9, 817 7 o 14
4. EVs in NAFLD and ALD
4.1. EVs in NAFLD
A numbe o s udies ha e demons a ed a ole o EVs in bo h he pa hogenesis and p og ession o
NAFLD [
14
,
15
]. T igge ing o in lamma ion and ib osis de elopmen a e key o p og ession om
isola ed s ea osis (also e e ed as NAFL o non-NASH a y li e ) o nonalcoholic s ea ohepa i is
(NASH), which is hallma ked by he p esence o hepa ocy e ballooning as a e lec ion o ongoing li e
inju y and dea h [
36
]. Da a om di e en die -induced animal models o NASH ha e shown ha EV
concen a ion inc eases wi h disease p og ession in a ime-dependen manne [
17
,
65
,
66
]. This seems o
be a esponse o he accumula ion o oxic lipids and hei downs eam media o s in he li e , which
inc ease he capaci y o hepa ocy es o o m and elease di e en ypes o EVs [
37
,
65
–
67
].
In i o
ea men o hepa ocy es wi h non-es e i ied a y acids e okes he elease o EVs con aining nume ous
molecules including C-X-C mo i ligand 10 (CXCL10), sphingosine-1-phospha e (S1P), mi ochond ial
DNA (m DNA), mic o-RNAs, ce amides, and umo nec osis ac o - ela ed apop osis-inducing ligand
(TRAIL) (15). These molecules may ampli y in lamma ion h ough mul iple mechanisms such as
mac ophage ac i a ion and monocy e chemo axis, as well as in lammasome ac i a ion and modula ion
o he NF-
κ
B pa hway in a ge cells [
67
,
68
]. As men ioned ea lie , EVs may be eleased by di e en
mechanisms including a caspase-3-dependen mechanism [
66
] o ac i a ion o dea h ecep o 5 (DR5)
in hepa ocy es. Hepa ocy e-de i ed EVs a e able o induce exp ession o p oin lamma o y cy okines
and p omo e M1 pola iza ion o hepa ic mac ophages [
37
,
69
]. CXCL10-bea ing EVs can also se e as
chemo ac ic s imuli o mac ophages, as shown ecen ly [
67
]. Mo eo e , EVs eleased om hepa ocy es
can con ibu e o hepa ic ec ui men o monocy e-de i ed mac ophages by p omo ing monocy e
adhesion ia in eg in
β
1 (ITG
β
1)-dependen mechanisms, as shown in mu ine NASH [
70
]. Addi ional
s imuli can also s imula e EV elease om hepa ocy es. In his ega d, i has been obse ed ha
hypoxia de e mines ha a -laden hepa ocy es elease EVs able o signal KCs, e oking p oin lamma o y
pheno ypes in hese cells, a phenomenon ha may explain he agg a a ing e ec o obs uc i e sleep
apnea synd ome on NAFLD [
71
]. Thus, i seems clea ha lipo oxic inju y o hepa ocy es de e mines
EV elease, p omo ing in lamma ion h ough ac i a ion and ec ui men o mac ophages [
14
], wi h clea
implica ions o he igge ing o in lamma ion in NAFLD/NASH [
36
]. In addi ion, hepa ocy e-de i ed
EVs may p omo e HSC ac i a ion [
47
,
72
] in expe imen al models o NAFLD/NASH. In e es ingly,
bo h mouse and human HSCs elease EVs ha a ge hepa ocy es and HSCs hemsel es [
49
,
50
,
73
].
Collec i ely, hese da a implica e EVs as one aspec o he cellula e en s igge ing hepa ic ib ogenesis,
a key p ocess in NAFLD p og ession [
74
]. In addi ion o signaling o mac ophages and HSCs, EVs
may ac on endo helial cells [
38
,
66
], p omo ing ascula in lamma ion wi h po en ial implica ions o
NAFLD- ela ed a he oscle osis. In his ega d, i has been shown ha EVs ca ying miR-1 as ca go
media e p oin lamma o y e ec s in endo helial cells in mice ia down egula ion o KLF4 and ac i a ion
o he NF-
κ
B pa hway [
38
]. Finally, o he o gan-de i ed EVs (e.g., isce al-adipose- issue-de i ed
exosomes) can con ibu e o NAFLD pa hogenesis and p og ession and in luence ib ogenic pa hways
in bo h hepa ocy es and HSCs [
75
], unde sco ing he ole o o he insulin-sensi i e o gans in NAFLD.
S udies ca ied ou in mouse models o NASH ha e shown ha o al ci cula ing EVs and
pa icula ly hepa ocy e-de i ed EVs a e ele a ed ea ly in he disease p ocess, while o he cell-de i ed
EVs (e.g., mac ophage- and neu ophil-de i ed EVs) appea in he ci cula ion la e , likely e lec ing
he ongoing in lamma o y p ocess [
65
,
76
]. P o eomic p o iling o ci cula ing EVs in expe imen al
NAFLD has been demons a ed o allow di e en ia ion be ween NAFLD s. con ol animals [
17
].
These indings unde sco e he po en ial o EVs as minimally in asi e bioma ke s o NAFLD [
77
],
which a e u gen ly needed o clinical ials and in clinical se ings. Since ci cula ing EVs (mainly
exosomes) a e also inc eased in human NAFLD and ha e been ound by some au ho s o co ela e
wi h disease his ological ea u es [
78
], EV analysis in se um in ol ing quan i a i e and quali a i e
de e mina ions (including cell su ace ma ke assessmen and measu emen o di e en ca goes (e.g.,
p o eins, lipids, and mic oRNAs)) is now a ocus o in ense esea ch. Se e al s udies ha e been
Cells 2020,9, 817 8 o 14
published in his ega d, showing ha he numbe o CD14+and CD16+EVs is in e sely associa ed
wi h he se e i y o NAFLD- ela ed li e ib osis, while also inc easing he diagnos ic capabili y o
he enhanced li e ib osis sco e (LFS) in pa ien s wi h NAFLD (AUC: 0.948 and 0.967 o CD14+and
CD16+EVs, espec i ely, s. 0.915 o LFS alone) [
79
]). O he e o s include de ec ion o ci cula ing
EVs con aining C16:0 ce amide- and S1P-en iched lipid species, which p og essi ely inc ease in
he plasma o obese pa ien s wi h simple s ea osis and in NASH pa ien s wi h ea ly ib osis [
65
].
Un o una ely, he diagnos ic accu acy o hese de e mina ions emains incomple ely explo ed in he
ield o NAFLD/NASH, and igo ous alida ion o his app oach is needed [
13
,
77
]. Mo eo e , signi ican
challenges emain ega ding he isola ion, ep oducibili y, and de ini ion o no mal con ols [13,80].
The apeu ic e o s in ol ing EVs in he ield o NAFLD/NASH a e nascen . A emp s o hal
in lamma ion and ib osis in oden models o NAFLD/NASH using EVs as he apeu ic agen s ha e
been published ecen ly. EVs ob ained om amnion-de i ed MSCs (AMSCs) we e used o ea a s
wi h ei he NASH o li e ib osis induced by he hepa ic oxican CCL4. AMSC-EVs we e gi en
in a enously in one o wo doses, and amelio a ion o in lamma ion and ib ogenesis was obse ed [
81
].
Mo e ecen ly, human li e s em cell (HLSC)-de i ed EVs ha e been used o ea mice wi h die -induced
s ea ohepa i is [
82
]. The au ho s ound ha EV-HLSC ea men signi ican ly down egula ed hepa ic
p o ib o ic and p oin lamma o y gene exp ession and amelio a ed he his ological abno mali ies
in mice wi h NASH. P o eomic analysis o EV-HLSCs showed ha hei ca go included a ious
an i-in lamma o y p o eins such as In e leukin-10, which may ha e con ibu ed o he obse ed
bene icial e ec s. In addi ion, al hough ye no explo ed in he ield o NAFLD, he use o MSC-de i ed
EVs o a es ib ogenesis [
53
,
83
] holds p omise o ea his condi ion, as ib osis is one o he mos
impo an de e minan s o su i al. These esul s unde sco e he concep ha EVs can be exploi ed o
he apy in NAFLD/NASH.
4.2. EVs in ALD
Recen s udies ha e ocused on he ole o EVs in ALD [
11
,
14
,
84
,
85
]. Bo h hepa ocy e- and
monocy e-de i ed EVs ha e been pos ula ed o egula e mac ophage di e en ia ion, he eby p omo ing
in lamma ion in alcoholic hepa i is (AH) [
24
,
41
,
55
,
72
,
86
]. Se e al molecules ha e been p oposed o
be esponsible o EV-media ed cell- o-cell signaling, including miRNAs (in pa icula , miR-122 and
miR-155) [
41
,
85
,
87
–
92
]. Also, he CD40 ligand was p oposed as an EV ca go ha could p omo e
mac ophage ac i a ion
in i o
and
in i o
in expe imen al models o AH [
85
]. Ano he s udy
in ol ing a mouse model o ALD using gas ic in usion o e hanol, ound ha ci cula ing EVs eleased
by hepa ocy es con ain a mic oRNA ba code (le 7 , miR-29a, and miR-340), which was speci ic o
alcohol- ela ed li e inju y [16].
F om a clinical poin o iew, he e is cu en ly no bioma ke able o assess he ea ly s ages o
ALD. EVs ha e been shown o co ela e wi h he diagnosis and p ognosis o alcoholic hepa i is [
93
].
Addi ionally, EVs ha e been p oposed as po en ial bioma ke s o di e en ia e mild and se e e o ms
o ALD, and hei ca go, such as sphingolipids, could be used o disc imina e be ween di e en li e
disease e iologies [
93
]. Ea ly iden i ica ion o hese subjec s migh lead o imely in e en ion in he
disease p ocess. A p esen , he diagnosis o AH elies on a his o y o alcohol consump ion in a
compa ible clinical scena io. A s udy conduc ed in auma pa ien s showed ha wo mic oRNAs
(miR-122 and le 7 ) we e inc eased only in he ci cula ing EVs ob ained om alcohol d inke s wi h
e idence o li e inju y [
94
]. This sugges ha hese mic oRNAs could se e o de ec “a - isk”
popula ions o ALD. Wi h ega d o AH, he mos se e e o m o he disease, a p esen ime he e a e
no bioma ke s which pe mi he ea ly diagnosis o AH, o sc eening es s ha can an icipa e hose
a isk o mo e se e e mani es a ions o AH. Iden i ica ion o his g oup o pa ien s is pa icula ly
impo an since he e is no e ec i e he apy o se e e AH, and e o s o a oid i could be implemen ed.
Se e al clinical p ognos ic ma ke s ha e been p oposed, bu he a iables used e lec he se e i y
o li e disease. These indices (e.g., he Madd ey disc iminan unc ion, model o end-s age li e
disease (MELD), and he Lille sco e) a e based on he non-speci ic biochemical assessmen o li e and
Cells 2020,9, 817 9 o 14
enal unc ion and ely hea ily on se um o al bili ubin, p o h ombin ime, and c ea inine [
95
]. The
eliance on bili ubin limi s hei diagnos ic u ili y, as speci ici y is con ounded by hype bili ubinemia
in co-exis en choles a ic li e diseases such as d ug-induced li e disease, o choles a ic hepa i is such
as p ima y scle osing cholangi is and p ima y bilia y cholangi is. None o hese p ognos ic sco ing
me hods u ilize a pa hophysiologically alida ed bioma ke ha e lec s he unde lying molecula
and signaling mechanisms o he disease. Fu he mo e, he in lamma o y esponse is p edic i e o
mo ali y bu is no aken in o accoun in ma hema ical models. They can iden i y subjec s a highes
mo ali y isk wi h an a ea unde he ecei e ope a ing cu e (AUROC) o unde 0.8; ideal su i al
models should ha e an AUROC >0.8 [
96
]. The eason hese sco es a e impe ec may be ha hey
a e no based on he pa hophysiological mechanisms ha media e li e inju y. Li e biopsy, he gold
s anda d o diagnosis o AH, emains unde u ilized due o he concu en coagulopa hy, which g ea ly
inc eases he isk o biopsy- ela ed complica ions [
97
]. Li e -biopsy-based hepa ic his ology sco e has
an AUROC o 0.73 in p edic ing 90 day mo ali y [
96
]. In his ega d, he use o EVs as bioma ke in AH
is p omising. Thus, i was ecen ly epo ed ha he o al numbe o EVs was signi ican ly inc eased
in pa ien s wi h AH and ha wo mic oRNAs (e.g., miRNA-192 and miRNA-30a) we e signi ican ly
inc eased in he plasma o subjec s wi h AH [
86
]. Addi ionally, a e y ecen s udy showed ha
ci cula ing EVs ca ying a sphingolipid ca go migh be help ul o he diagnosis o AH, and may allow
dynamic isk p o iling o hese pa ien s. [
98
]. Mos ecen ly, EVs ha e been used as a su oga e ma ke
o imp o emen in clinical ials o pa ien s wi h AH [
99
]. Fu he s udies will be equi ed o alida e
EVs as a bioma ke o AH diagnosis and/o p ognosis.
5. Concluding Rema ks and Fu u e Pe spec i es
The ield o EVs in a y li e diseases is apidly e ol ing as he impo an unc ions o hese
pa icles in cell- o-cell communica ion and in he pa hogenesis o bo h ALD and NAFLD, he
mos p e alen li e diseases wo ldwide, a e un eiled. The elease o la ge quan i ies o EVs
by s essed/damaged hepa ocy es con ibu es o in lamma ion, ib ogenesis, and angiogenesis, ueling
li e disease p og ession and p o ides oppo uni ies o in e en ion. The iden i ica ion o speci ic
molecula signa u es o eleased EVs is p omising in he sea ch o disease-speci ic bioma ke s,
al hough mo e da a a e needed o alida e hese ma ke s in la ge coho s and in a igo ous manne .
EVs may ha e po en ial o he apeu ic use, bu his ield is s ill nascen . Mo e esea ch is needed o a
success ul ansi ion o cu en EV knowledge om he bench o he clinic.
Au ho Con ibu ions:
Concep ualiza ion and w i ing—o iginal d a p epa a ion, A.H., J.M.B. and M.A.;
C i ically e iewing and con ibu ing o he inal e sion o he e iew J.P.A., D.R., A.L. and H.M. W i ing— inal
e sion o he manusc ip , A.H. and M.A. All au ho s app o ed he inal e sion o he manusc ip o submission.
Funding:
This a icle was pa ially suppo ed by he Chilean go e nmen h ough he “Fondo Nacional de
Desa ollo Cien
í
ico y Tecnol
ó
gico” (FONDECYT 1191145 o M.A. and 1200227 o J.P.A.) and he Comisi
ó
n
Nacional de In es igaci
ó
n Cien
í
ica y Tecnol
ó
gica (g an CONICYT PIA/Basal PFB12, Basal Cen e o Excellence
in Science and Technology o M.A.). Suppo om he “Vice ec o
í
a de In es igaci
ó
n de la Pon i icia Uni e sidad
Ca
ó
lica” (g an -in-aid o suppo sho esea ch isi s ab oad, PEBI1913) is also acknowledged. J.M.B is
unded by Spanish Ca los III Heal h Ins i u e (ISCIII) [FIS PI15/01132, PI18/01075 and Miguel Se e P og am
CON14/00129) co inanced by “Fondo Eu opeo de Desa ollo Regional” (FEDER), CIBERehd (ISCIII), “Dipu aci
ó
n
Fo al Gipuzkoa” (DFG15/010, DFG16/004), BIOEF (Basque Founda ion o Inno a ion and Heal h Resea ch:
EiTB Ma a oia BIO15/CA/016/BD), Depa men o Heal h o he Basque Coun y (2017111010), Euskadi RIS3
(2016222001, 2017222014, 2018222029, 2019222054), La Caixa Scien i ic Founda ion (HR17-00601), “Fundaci
ó
n
Cien
í
ica de la Asociaci
ó
n Española Con a el C
á
nce ” (AECC Scien i ic Founda ion, Ra e Cance s g an 2017).
M.A. and J.M.B. a e unded by he Eu opean Ho izon 2020 p og am (ESCALON p ojec : SEP-210503876). A.L. is
unded by he by he Basque Go e nmen (PRE_2017_1_0345).
Acknowledgmen s:
We a e g a e ul o Nicolas T ian a ilo, o his help in designing he igu e accompanying o
his a icle.
Con lic s o In e es : Au ho s decla e ha he e is no con lic o in e es ega ding he publica ion o his pape .
