Cha ac e iza ion o blood ma ke s and
hei implica ion in human aging
Ainhoa Albe o Ga i ano
To ob ain a doc o al deg ee in Molecula Biology and Biomedicine
om he Uni e si y o he Basque Coun y
Supe iso s:
Da id O aegui Bicho
Ma ías Sáenz Cues a
Janua y 2020
(cc)2020 AINHOA ALBERRO GARITANO (cc by.nc-nd 4.0)
This hesis has been ca ied ou a he Biodonos ia Heal h Resea ch Ins i u e. Du ing his wo k, I
was suppo ed by a ellowship om he Depa men o Educa ion o he Basque Go e nmen
(PRE_2015_1_0341, 2016-2019), by a ellowship om he Jesús de Gangoi i Ba e a Founda ion
(2016) and by an EMBO Sho -Te m Fellowship (STF_7624, 2018).
Izan zi enei,
di enei,
izango di enei.
Labu pena
Zaha zea de ini zean, gu e go pu zak denbo an zeha jasa en duen aldake a mul zoa dela
esa en dugu, gai asun un zionala en gale a e a bai a gaixo asunekiko e a kanpoko e asoekiko
zau ga i asun handiagoa daka ena.
Bizi kali a ea en hobekun zei e a azken hama kade an medikun zan eman di en au e apen
handiei eske , adinekoen kopu ua naba men igo da. Ba ez bes eko bizi-i xa opena en
hazkun za honen e a jaio za- asa en behe akada en ondo ioz, biz anle ia zen su ge o e a
deso eka uagoa daukagu. Gaine a, bizi-i xa opena igo den a en, osasun su bizi ga en u e
kopu ua ez da hazi e a, be az, luzeago bizi ga a, baina ez hobe o. Ho enbes ez, zaha zea
o oko ean, e a menpeko asuna be eziki, gu e giza ea en e onka bihu u di a, e a ge o e a
baliabide gehiago bide a zen di a haue a a. Gauzak ho ela, espa u hone an bu u zen di en
ike ke a medikuen helbu u nagusia menpeko asuna mu iz ea izan beha da, zaha ze
osasun su ba lo zeko.
Zaha ze osasun sua en bidean, hausko asun kon zep ua ekin egi en dugu lan.
Hausko asuna adina ekin e laziona u ako sind ome medikoa da, e a be e ezauga i nagusiak
e ese ba un zionala en gale a, sis ema isiologikoen egoki ze gai asun mu iz ua e a
zau ga i asuna en a eago zea di a. Hausko asuna hainba ondo io nega ibo ga a zeko
a iskua ekin e laziona u a dago, hala nola, e o ke ak, haus u ak, in ekzioak, desgai asuna,
ospi alizazioa, menpeko asuna e a he io za. Be az, hausko asuna dauka en pe sonen
iden i ikazioa beha ezkoa da neu iak ha u ahal iza eko e a e o kizuneko a azoak saihes en
saia zeko. Baina, o ain a e ezin izan da hausko asuna de ek a zeko me odo e aginko ik
ga a u, e a hainba es e a p oba un zional badauden a en, hauek ez di a gai pe sona
hausko guz iak iden i ika zeko. Gau egun esku aga i dauden esnak osa u e a emai za
hobeak lo zeko asmoz, hausko asuna en bioma ka zaileak au ki zeko ike ke ak bu u zen a i
di a, au e ago ez abaida uko dugun moduan. Bioma ka zaileen bilake a azaldu au e ik,
o dea, zaha ze p ozesua en oina i biologikoa labu uko dugu.
Zaha zea p ozesua unibe sala, p og esiboa e a he e ogeneoa da, e a be az, modu oso
desbe dine an ga a zen da. Zaha zea en ja o i molekula ak e a zelula ak ule zea
biologia en e onka nagusie ako ba da, e a helbu u ho ekin lan uga i egin di a. Ike ke a
ho iei eske , o ain dela u e ba zuk zaha zea en 9 ezauga i nagusiak zehaz u zi en,
ondo engoak di ela ik: ezegonko asun genomikoa, aldake a epigene ikoak, elome oen
labu zea, dis un zio mi okond ialak, zelula amen ago zea, zelulen seneszen zia, zelulen
a eko komunikazio desbe dina, p o eos asia en gale a e a man enugaien hau ema e
18 | Labu pena
oke agoa. Haue a iko ezauga i bakoi za sakonki ike u da komuni a e zien i ikoan, baina,
ja aian esi hone an landu di enak azalduko di ugu labu ki.
Zaha za oa ekin ge a zen den ezegonko asun genomikoa, neu i ba ean, DNA en egi u an
aldake ak me a zea i zo zaio, hala nola mu azioak, delezioak edo DNA en haus u ak ha izpi
ba ean edo bie an. Zelulek badauzka e aka s ho iek kon ola zeko e a konpon zeko
mekanismoak, baina hauek e e adina ekin hu s egi en du ela ikusi da. Bes alde, geneen
adie azpena alda u egi en da zaha zean, e a hau bes e gene ba zuen adie azpena e egula zen
du en ansk ipzio ak o ee an ge a zen bada, e agina a e handiagoa izango da. Gene ez
kodi ika zaileen adie azpena e e alda u egin dai eke, e a ansk ipzio os eko e egulazioan
e agina eduki, mik oRNA molekulen kasuan bezala.
Seneszen ziak zelulen p oli e azioa en gale a daka , ja due a me abolikoa, bide aga i asuna
e a haien be ezko un zio ba zuk man en zen di uz en bi a ean. Zelula kal e u e a po en zialki
a isku suak izan dai ezkeenak seneszen zian sa zea de en sa mekanismoa dela desk iba u
da, hauen p oli e azioa ekidi en bai a. E a be ean, badi udi zelula seneszen eek ja ia u ako
seinaleek ehunen bi so kun zan lagun zen du ela: zelula seneszen een diges ioa e a zelula
amen di e en ziazioa sus a zen du e, zelula be i helduek ehuna be i zen du ela ik. Adinean
au e a egin ahala, o dea, p ozesu honek e e hu s egi eko joe a dauka, zelula seneszen eak
pila u egi en di a e a kal eak e agin di zake e. Lan hone an be eziki immuni a e-sis ema en
seneszen zian (immunoseneszen zian) zen a u ga a, adineko pe sonen zau ga i asunean
e agina bai auka.
Az e u dugun bes e ezauga i ba zelula amen ago zea da. Hau oso lo u a dago
seneszen zia ekin, izan e e, ehunen bi so kun za ezin da osa u zelula ama ik ez badago edo
beha bezala di e en zia zen ez badi a. Zelula amak kieszen zia egoe an man en zen di a e a
beha ezkoa denean baka ik ak iba u, p oli e a u e a di e en zia zen di a. Gaine a, zelula
amek au o-be ikun za gai asuna du e, zelula alabe ako ba zelula ama moduan man endu e a
bes ea baka ik di e en zia zen bada. Baina zelula amen ago zea zelulak sime ikoki bana zen
di enean eman dai eke, edo zaha zea ekin lo u ako a azoak di ela medio kieszen zia
egoe a ik i en e a ak iba zea lo zen ez du enean. Izan e e, kieszen zia ik i e zeko p ozesua
konplexua da e a oso e egula u a dago, e a zenbai egoe ek, hala nola, elome oen labu zeak,
es es oxida zaileak edo a DNAn eman di en kal eek p ozesua e ago zi dezake e.
Azkenik, zaha za oan ema en den zelulen a eko komunikazio desbe dina en ingu uan e e
ike u dugu. Zaha za oko ezauga i hau be eziki ga an zi sua da gu e za bi a azoi enga ik.
Alde ba e ik, zaha ze akoan ema en di en aldake ek in lamazio basal k onikoa ga a ze a
e ama en bai u e, in lammaging izenez ezagu zen dena. In lamazioa sus a zen du en molekula
Labu pena | 19
hauen ja ia zea hainba zelula mo ek bu u zen du e, e a adina ekin ema en den immuni a e-
sis ema en un zionamendu xa a a eago zen du e. Bes alde, zelulaz kanpoko besikulak
(EVak, ingeleseko labu du a ekin) dauzkagu, zelula mo a gehienek ekoiz u e a ja ia zen
di uz en pa ikulak e a zelulen a eko komunikazio ako bide ba di enak. EVak duela 50 u e
baino gehiago au ki u zi en, baina hasie an ez zi zaien ga an zi ik eman e a zelulen “zabo a”
a e a zeko” modu ba zela pen sa u zen. Azken hama kade an hauen un zioak sakonago
az e zen hasi zen, e a o aindik e e, u e o EVen un zio be i en ba desk iba zen da. Hala e e,
zaha zea en ezauga i nagusien a ean zelulen a eko komunikazio desbe dina izenda u
zenean, ez zi en EVak kon uan ha u, e a ho ega ik zaha ze p ozesuan dauka en inplikazioa
e akus en ja ai u beha a daukagu.
Hausko asun sind omea en bioma ka zaileen gaia i be i o eu si a, lan asko egin izan di a
zaha zea en ezauga i o oko ak hausko asuna ekin e laziona zen saia zeko. Ike ke a
hauen hipo esia ze a da: adina ekin aldake a edo p ozesu kal ega iak ge a zen di a, e a
hauek neu i handiago ba e an eman dai ezke hausko asuna edo menpeko asuna pai a zen
du en pe sone an, gai asun un zionala man en zen du ene an (sendoe an) baino. Ildo
ho e a ik, in lamazioa edo es es oxida zailea ekin lo u ako molekulen igoe ak, e a ho monen
edo me abolismoa en e egulazio gale ak hausko asuna ekin izan di zake en lo u ak az e u
di a, adibidez. DNA sekuen zia espezi ikoek, geneen e egulazioak edo geneen adie azpen
aldake ek hausko asuna ga a zeko joe a ekin izan di zake en e lazioak e e ike u izan di a.
Bioma ka zaile hauei e a bes elako ba zuei bu uzko a ikulu zien i iko uga i kale a u di a.
Ho ie ako ba zuek hausko asuna ekin edo hau ga a zeko a iskua ekin ha emanak au ki u
di uz e, baina badaude e e p oposa u ako bioma ka zaileak balioz a u ez di uz en ike ke ak,
e a ho ega ik, gau egun o aindik ez da hausko asuna en iden i ikazioan lagundu dezakeen
bioma ka zaile ik aplika zen p ak ika klinikoan.
Gauzak ho ela, esi honen lehenengo kapi uluan hausko asun bioma ka zaileak au ki zen
saia u ga a. Ho e a ako, adineko pe sonen odol laginak esku a u di ugu, e a hauek sendo e a
hausko aldee an bana u di ugu es desbe dine an lo u ako emai zen a abe a. Lehenik,
in lamazioa en ezauga i di en molekulak neu u di ugu. Hauekin emai za posi iboak lo u
izan di a au eko ike ke a ba zue an, baina badaude hausko asuna ekin e lazio ik au ki u ez
du en lanak e e, e a ho ien an ze a, gu e lagine an ez dugu in lamazio molekulen kon zen azio
handiago ik opa u pe sona hausko e an. Gu e biga en es a egia az e ke a
ansk ip omikoa egi ea izan da. Ho en bidez, pe sona sendoak e a hausko ak be eiz en
di uz en 35 gene iden i ika u ahal izan di ugu. Has eko, gene ho ie ako 3 en adie azpena
neu u dugu koho e zabalago ba e an e a EGR1 genea en gainadie azpena balioz a u dugu
pe sona hausko e an. EGR1 p ozesu zelula ga an zi sue an pa e ha zen duen
20 | Labu pena
ansk ipzio ak o ea da, hala nola mi ogenoekiko e an zunean, p oli e azioan, apop osian e a
hainba zelula mo en di e en ziazioan, e a es imulu desbe dinen ondo ioz ak iba ua izan
dai eke. Gaine a, adineko pe sonen hausko asun egoe a i buel a eman edo mu iz en
saia zeko esku-ha ze ba bu u u da, e a 3 hilabe ez a ike a isikoa egin du e pa aideek. Esku-
ha zea en ondo en, 12 pa aidee a ik 9k EGR1 genea en adie azpena jai si du ela au ki u
dugu. Emai za naba men hauekin, EGR1en adie azpen al ua hausko asun bioma ka zaile
bezala p oposa zen dugu, e a e o kizuneko ike ke e an kon uan ha u beha ekoa dela
de i zogu.
Hausko asun bioma ka zaileen ike ke an, EVen kon zen azio plasma ikoa izan da gu e azken
hu bilke a. Izan e e, zenbai in lamazio p ozesue an, minbizia e a gaixo asun au oimmuneak
esa e ako, EVen kon zen azio al ua au ki u da, e a gu e helbu ua hau adinean ema en den
in lammaging-a ekin e e ge a zen o e den az e zea izan da. Gu e emai zek e aku si du enez,
nahiz e a in lamazio basala adineko pe sone an kon i ma u den, hauek ez du e EV
kon zen azio plasma iko handiago ik, e a ez dago ez a e e sendo e a hausko en a eko
di e en zia ik. Emai za hauek adie az en du enez, be az, ez da EVen kon zen azio igoe a
ema en in lamazio p ozesu guz ie an.
Lan honen biga en kapi uluan EVek zelulen di e en ziazioan du en e agina az e u dugu.
Os eogenesi e a miogenesian zen a u ga a, zaha za oan kal e u ako p ozesuak bai i a e a
os eopo osian e a sa kopenian e agina bai u e, hu enez hu en. Adina ekin ohikoak di en bi
p ozesu hauek ikuspegi desbe dine a ik sakonki az e u izan di a, baina oso gu xik ike u du e
EVen inplikazioa. Gan z ehune ik e a o i ako zelula ama mesenkimalekin bu u u ako gu e
lanak e akus en duenez, plasmako EVek os eogenesi p ozesuan lagun zen du e, e a e agin hau
naba menagoa da heldue a ik esku a u ako EVekin, adineko emaileekin alde a u a. E a
be ean, plasmako EVek miogenesian du en e agina az e zeko p o okolo ba ga a zen saia u
ga a, e a lehen emai zek adie az en du enez, mioblas oen di e en ziazio p ozesua bul za zen
du e EVek, be i o e e adineko pe sone a ik esku a uko EVek e ek u mu iz uagoa du ela ik.
Hi uga en kapi uluan immunoseneszen zia en enomenoa i ja i diogu a e a, e a helduak
(20-49 u e) e a pe sona adindunak (70-104 u e) az e u di ugu. Lehenik, seneszen zia zelula
mailan neu u da, e a ikusi dugu CD8 T lin ozi o seneszen een p opo zioa hazi egi en dela
adina ekin. Zaha za oa en lehen hama kade an be dina ge a zen da CD4 T lin ozi oe an,
baina 90. u e ik go ako pe sone an zelula seneszen e mo a honen kan i a ea mu iz uagoa
dela au ki u dugu. Ondo en, EVek lin ozi o seneszen een ezauga i be dinak e akus en o e
di uz en az e u dugu. Zelula hauen ma ka zaile be dinak EVen min zean au ki u di ugu, baina
ez, o dea, “EV seneszen een” igoe a ik adinak au e a egin ahala. Azkenik, lin ozi oen e a EVen
in i o espe imen uak bu u u di ugu, EVek zelula hauen ak ibazioan e agina du en
Labu pena | 21
ebalua zeko. Emai zek e aku si du e EVek ez dela e an zun immunologiko ik e agi en, e a a e
gehiago, zelulen bide aga i asuna hobe zen du ela. Baina, es imulu immunogeniko ba en
au ean e an zu eko o duan, EVek T lin ozi oen ak ibazioa a eago zen du e. Gaine a,
ak ibazioa a eago zeko gai asun hau ahulagoa da EVak emaile zaha e a ik isola uak badi a.
Ho ela, gu e emai zek adie az en du e, nahiz e a EVen min zeko molekule an
desbe din asunik ez egon, EVek gai asun un zional desbe dinak dauzka ela isola u izan di en
emailea en adina en a abe a.
Tesi honen lauga en e a azken kapi uluan, immunoseneszen zia az e u dugu eskle osi
anizkoi za du en gaixoe an. Izan e e, p oposa u izan da gaixo asun au oimmunee an ema en
den e engabeko immuni a e-sis ema en ak ibazioa dela e a azken honen ago zea eman
dai ekeela, e a ondo ioz, immunoseneszen zia goiz ia a. Hipo esi hau ebalua zeko, eskle osi
anizkoi za du en pe sonen e a heldu osasun suen laginak alde a u di ugu. Eskle osi
anizkoi za dauka en gaixoek, a amendu immunomodula zaileak ha zen e a e emisioan
egon a en, pe sona osasun suek baino in lamazio ma ka zaile kon zen azio al uagoak
dauzka ela au ki u dugu. Honek in lamazio basala dauka ela adie az en du, in lammaging-
a ekin e laziona u a egon li ekeena. Bes alde, T lin ozi oen analisian, eskle osi anizkoi za
dauka en gaixoek zelula seneszen e gehiago ik ez du ela ikusi dugu, baina gaixo hauen T
lin ozi oek e an zun desbe dina dauka e in i o jaso ako es imulu ba en au ean, ak ibazio
mu iz uagoa e akus en du ela ik pe sona osasun suen zelulekin alde a u a.
Buka zeko, labu pen modu a, dok o e za esi honek zaha ze p ozesua en hainba alde di i
bu uzko ezagu za au e a zen lagundu duela esan dezakegu. Hausko asun bioma ka zaileak
ike u di ugu, EGR1 genea en adie azpena e o kizunean kon uan ha u beha eko ma ka zaile
bezala p oposa uz. Plasma ik isola u ako EVen ezauga iak e e az e u di ugu, os eogenesia
e a miogenesia bezalako zelulen di e en ziazio p ozesue an lagundu dezake ela e aku siz,
bai a T lin ozi oen ak ibazioa sus a zen du ela es imulu immunogenikoen au ean, e a EVen
un zio hauek zaha zea ekin ahuldu egi en di ela. Emai za hauek guz iek e akus en digu enez,
EVek pape ga an zi suak be e zen di uz e hainba p ozesue an, e a ain za ha u beha ko
li a eke zaha zea en ezauga i nagusie an. Azkenik, eskle osi anizkoi za en ingu uan
bu u u ako espe imen uek adie az en digu e gaixo asun hau dauka en pe sonek in lamazio
k onikoa badauka ela, e a immunoseneszen zia goiz ia a eskle osi anizkoi zean e a bes elako
gaixo asun au oimmunee an ga a zen o e den ike zen ja ai u beha dugula.
Table o con en s
Abb e ia ions......................................................................................................................................................... 31
INTRODUCTION ......................................................................................................................................................... 33
1. Aging ..................................................................................................................................................................... 35
1.1. F ail y and o he ele an age- ela ed heal h concep s .......................................................... 37
1.1.1. F ail y and obus ness .................................................................................................................. 37
1.1.2. F ail y scales ..................................................................................................................................... 38
1.1.3. Resilience ........................................................................................................................................... 40
1.1.4. In insic capaci y ............................................................................................................................ 41
1.1.5. Heal hy aging, success ul aging and ela ed concep s..................................................... 42
1.2. The biology o aging ............................................................................................................................... 43
1.2.1. Genomic ins abili y and gene exp ession ............................................................................. 45
1.2.2. Telome e a i ion and epigene ic al e a ions ................................................................... 46
1.2.3. Loss o p o eos asis ....................................................................................................................... 49
1.2.4. De egula ed nu ien sensing .................................................................................................... 50
1.2.5. Mi ochond ial dys unc ion ......................................................................................................... 51
1.2.6. Cellula senescence ........................................................................................................................ 52
1.2.6.1. Immunosenescence ...................................................................................................................... 53
1.2.7. S em cell exhaus ion ...................................................................................................................... 55
1.2.8. Al e ed in e cellula communica ion ..................................................................................... 58
1.2.8.1 In lammaging ................................................................................................................................. 59
1.3. Molecula bioma ke s o ail y ........................................................................................................ 60
2. Ex acellula esicles ..................................................................................................................................... 66
2.1. Biological cha ac e is ics o ex acellula esicles .................................................................... 66
2.2. Isola ion and cha ac e iza ion o ex acellula esicles ......................................................... 68
2.3. Ex acellula esicles in physiological and pa hological p ocesses ................................... 71
2.4. Po en ial clinical applica ions o ex acellula esicles........................................................... 73
3. Mul iple scle osis ............................................................................................................................................ 76
3.1. E iopa hology o mul iple scle osis ................................................................................................. 76
3.2. Mul iple scle osis and p ema u e aging ........................................................................................ 78
JUSTIFICATION .......................................................................................................................................................... 81
HYPOTHESIS AND OBJECTIVES .......................................................................................................................... 85
CHAPTER ONE Bioma ke s o ail y ................................................................................................................ 89
In lamma ion ......................................................................................................................................................... 95
T ansc ip omics ................................................................................................................................................ 103
Ex acellula esicles ....................................................................................................................................... 113
CHAPTER TWO In luence o ex acellula esicles and age on cell di e en ia ion ................... 119
Os eogenesis ....................................................................................................................................................... 123
Myogenesis .......................................................................................................................................................... 133
CHAPTER THREE Immunosenescence and he ole o ex acellula esicles .............................. 143
CHAPTER FOUR Mul iple scle osis and p ema u e aging..................................................................... 163
GENERAL DISCUSSION, PERSONAL OPINION AND FUTURE PERSPECTIVE .............................. 179
CONCLUSIONS ......................................................................................................................................................... 187
PUBLICATIONS ....................................................................................................................................................... 191
REFERENCES ........................................................................................................................................................... 197
| 31
Abb e ia ions
7-AAD 7-aminoac inomycin D
ADL Ac i i ies o daily li ing
ASC Adipose issue-de i ed
s em cells
BBB Blood-b ain ba ie
BSA Bo ine se um albumin
BDNF B ain-de i ed neu o ophic
ac o
ci cRNA ci cula RNA
CNS Cen al ne ous sys em
CRP C- eac i e p o ein
CSHA Canadian S udy o Heal h
and Aging
DHEAS Dehyd oepiand os e one
sul a e
EM Elec on mic oscopy
EV Ex acellula esicle
FMO Fluo escence minus one
GFST Ge on opole F ail y
Sc eening Tool
GS Gai speed
HC Heal hy con ol
HSC Hema opoie ic s em cell
HSP Hea shock p o ein
IGF-1 Insulin-like g ow h ac o -1
IIS Insulin and insulin-like
g ow h ac o 1
IL In e leukin
MD Myogenic di e en ia ion
MHC Majo his ocompa ibili y
complex
miRNA mic oRNA
MS Mul iple scle osis
MSC Mesenchymal s em cell
m DNA mi ochond ial DNA
MuSC Muscle s em cell
NSC Neu al s em cell
NTA Nanopa icle acking
analysis
OD Os eogenic di e en ia ion
PBMC Pe iphe al blood
mononuclea cell
PCA P incipal Componen
Analysis
PHA Phy ohemagglu inin
PRP Pla ele - ich plasma
ROC Recei e Ope a ing
Cha ac e is ic
ROS Reac i e oxygen species
RT-qPCR Quan i a i e eal- ime PCR
SASP Senescence-associa ed
sec e o y pheno ype
sncRNA small non-coding RNA
SNP Single nucleo ide
polymo phism
SPPB Sho Physical Pe o mance
Ba e y
TAC T ansc ip ome Analysis
Console
TFF Tangen ial low il a ion
TFI Tilbu g F ail y Indica o
TNF-α Tumou nec osis ac o
alpha
TUG Timed up-and-go
UPS Ubiqui in-p o easome
sys em
INTRODUCTION
In oduc ion | 35
1. Aging
The p ocess o ge ing old a ec s each o us and ou socie y as a whole. Aging is de ined as he
accumula ion o ime- ela ed modi ica ions ha lead o dec eased unc ional capaci y, as well
as inc eased suscep ibili y and ulne abili y o disease o ex e nal insul s [1]. I is a uni e sal,
complex and he e ogeneous p ocess. The p esen wo k is ocused on biological aspec s o
aging, bu we conside we should i s ou line some gene al cha ac e is ics o be e
unde s and he implica ions and mo i a ions o ou esea ch.
Rega ding demog aphics, a d ama ic change in he p opo ions o young and elde people is
obse ed in mos o he coun ies. On one side, he imp o emen s in social, medical and
economic condi ions ha e esul ed in educed mo ali y, be e quali y o li e and consequen
inc ease in li e expec ancy. On he o he side, he educed bi h a e con ibu es o he o e all
inc ease o he popula ion age. In Figu e 1 he popula ion py amids o he Eu opean Union in
2003 and 2018 a e depic ed, showing a clea d op o he inhabi an s younge han 44 yea s
and a ise o he ones o e 45 yea s.
Figu e 1. Popula ion py amids o he 28 membe s a es o he Eu opean Union in he yea s 2003 and
2018. The pe cen age o people o all age anges up o 40-44 yea s has dec eased, while all he age anges
o e 45-40 yea s ha e inc eased, demons a ing he aging o he popula ion. Sou ce: Eu os a .
“Popula ion s uc u e and ageing”. A ailable a : h ps://ec.eu opa.eu/eu os a /
36 | In oduc ion
Mo eo e , he cu en pic u e will con inue e ol ing and he popula ion is p ojec ed o age
no ably mo e. In Figu e 2 he p esen popula ion s uc u e and u u e p ojec ions a e shown.
In e es ingly, we can obse e ha he p opo ion o people aged mo e han 80 yea s is
expec ed o inc ease om 5.6% o 14.6% in he yea 2100.
Figu e 2. Popula ion s uc u e by majo age g oups o he 28 membe s a es o he Eu opean Union in
he yea 2018 and he p ojec ions o he nex decades. The p opo ion o child en (0-14 yea s) will ha e
only a mino dec ease, while a ma ked dec ease o he adul s (15-64 yea s) and inc ease o elde s (65-
79 yea s and 80+ yea s) is expec ed. Sou ce: Eu os a . “Popula ion s uc u e and ageing”. A ailable a :
h ps://ec.eu opa.eu/eu os a /
Howe e , he epo s om he Eu opean Union also show ha li e expec ancy a bi h is
inc easing, while he heal hy li e yea s a bi h (also called disabili y- ee li e expec ancy) is no
ising. Fo ins ance, in 2016 li e expec ancy a bi h was 83.6 yea s o women and 78.2 yea s
o men and heal hy li e yea s we e 64.2 and 63.5 yea s espec i ely. This indica es ha a
woman bo n in 2016 will li e he app oxima ely 77% o he li e wi hou disabili y, while i
would be an 81% o his li e o a man (h ps://ec.eu opa.eu/eu os a /). Fu he mo e, hese
pe cen ages would con inue o decline as long as li e expec ancy inc eases, bu no educ ion o
disabili y is achie ed.
In oduc ion | 37
In ligh o he commen ed demog aphic changes and disabili y da a, he impo ance o aking
app op ia e and e ec i e ac ions becomes e iden . Mo eo e , as s a ed by he Wo ld Heal h
O ganiza ion, he aging o ou socie ies is one o he majo challenges o he 21s Cen u y [2],
as i eaches no only sani a y bu also many socioeconomic aspec s. In consequence, decisions
coo dina ed by expe s o di e en ields should be aken, aiming o achie e he well-being and
heal hy aging o he popula ion while main aining inancial sus ainabili y.
One o he key ac ions o ace he aging challenge is esea ch. Resea ch on he unde lying
mechanisms, he age-associa ed diseases and loss o unc ions, he in e en ions and he
ou comes a e essen ial o be e unde s and he aging p ocess and o be able o implemen
inno a i e ea men s and/o in e en ions. Besides, he p ima y objec i e o biomedical
esea ch on aging should no be ocused on he ex ension o li e, i should aim o imp o e he
quali y o li e o he elde ly, educing disabili y and p olonging heal hy aging.
1.1. F ail y and o he ele an age- ela ed heal h concep s
Many wo ks ha e p e iously s udied he loss o unc ions associa ed wi h aging. No ably, he e
ha e been di e en app oaches o in es iga e he age- ela ed dys unc ions, and consequen ly,
mul iple e ms ha e also been p oposed. In he nex lines, he main concep s a e p esen ed,
and hei p incipal cha ac e is ics explained.
1.1.1. F ail y and obus ness
F ail y is a common age- ela ed medical synd ome, cha ac e ized by a educed unc ional
ese e, impai ed adap i e capaci y ac oss mul iple physiological sys ems and inc eased
ulne abili y o s esso s [3]. The accen ua ed ulne abili y esul s in high isk o nega i e
ou comes, such as alls, ac u es, in ec ions, disabili y, hospi aliza ion, dependency and dea h
[4]. F ail y synd ome has been widely s udied o decades, bu s ill, no consensus has been
eached on i s de ini ion and iden i ica ion ools.
Rega ding he concep o ail y, a wo k by Rod íguez-Mañas and colleagues ga he ed he
de ini ions o expe s in he ield and p esen ed a lis o accep ed s a emen s ha de ine ail y
[5]. This lis included aspec s o physical pe o mance, nu i ional s a us, men al heal h, and
cogni ion. Howe e , hey concluded ha , e en i some concep s o ail y a e widely ag eed,
he e is no consensus on an ope a ional de ini ion o ail y.
Despi e he lack o a comple e de ini ion, as men ioned be o e, ail y implies a educed
unc ional capaci y o an indi idual ha esul s in an inc eased isk o de eloping dependence.
The opposi e si ua ion o ail y is mos o he imes e med obus ness. An elde is classi ied
as obus when he /his unc ional capaci y is conse ed, and besides, pheno ypic s abili y is
44 | In oduc ion
A ew yea s ago, in 2013, a no ewo hy classi ica ion o he cellula and molecula aspec s o
aging was p oposed by López-O ín and collabo a o s [33]. They dis inguished 9 hallma ks o
aging, and u he ca ego ized hem as p ima y, an agonis ic and in eg a i e hallma ks (Figu e
6). I is impo an o men ion ha he au ho s p oposed his ca ego iza ion because he
hallma ks a e igh ly in e connec ed and hey canno be unde s ood indi idually.
In he nex sec ions, he main cha ac e is ics o he p oposed hallma ks o aging a e explained,
wi h a special ocus on he 4 hallma ks ha ha e been in es iga ed in his hesis. Besides, we
complemen ed hese sec ions wi h in o ma ion ha was no included in he o iginal
desc ip ion o he hallma ks, bu ha we conside ele an bo h in he con ex o his wo k and
o he gene al unde s anding o he biology o aging.
Finally, we should bea in mind ha hanks o new indings he knowledge abou aging is
apidly e ol ing. The hallma ks p oposed in 2013 ep esen ed he cu en s a e o he a , bu
p obably, modi ica ions such as he inclusion o new ca ego ies o u he explana ions on hei
connexions and implica ions will be made in he nea u u e.
Figu e 6. The hallma ks o aging and hei in e connec ions. Genomic ins abili y, elome e a i ion,
epigene ic al e a ions and loss o p o eos asis a e conside ed he p ima y hallma ks, he p ima y causes
o cellula damage. De egula ed nu ien sensing, mi ochond ial dys unc ion and cellula senescence a e
pa o compensa o y o an agonis ic esponses o he damage. These esponses a e p oposed o ini ially
mi iga e he damage, bu e en ually, hey become dele e ious hemsel es. S em cell exhaus ion and
al e ed in e cellula communica ion compose he in eg a i e hallma ks, hey a e he end esul o he
p e ious wo g oups and a e ul ima ely esponsible o he unc ional decline associa ed wi h aging. The
black a ows indica e he hallma ks ha ha e been in es iga ed in he p esen wo k. Adap ed om [33].
In oduc ion | 45
1.2.1. Genomic ins abili y and gene exp ession
The e is ex ensi e e idence showing ha genomic damage accompanies aging. The
accumula ion o DNA damage is caused by bo h endogenous and exogenous h ea s, such as
eac i e oxygen species (ROS) and ul a iole adia ion espec i ely [34]. Examples o DNA
damage include mu a ions, single- and double-s and b eaks o in e s and c osslinks [33].
The cell has mul iple mechanisms o genome main enance and e o epai , which illus a e
he impo ance o genome s abili y. Whe he he accumula ion o DNA damage is a cause o a
consequence o aging was long deba ed, bu he desc ip ion o genome ins abili y in diseases
o accele a ed aging demons a ed he causali y, as ex ensi ely e iewed by Niede nho e e
al. [35]. Indeed, in he same publica ion, hey also e iewed he cu en da a showing ha
mu a ions inc ease and DNA epai capaci y dec eases wi h age.
Apa om genomic ins abili y, bu di ec ly ela ed o i , gene exp ession modi ica ions ha e a
ele an ole in aging. I has been widely shown ha he exp ession o p o ein-coding genes
changes wi h age [36–38]. Impo an ly, ansc ip ion ac o s and ela ed signalling pa hways
ha e also been ound o a ec cell senescence and aging. The insulin and insulin-like g ow h
ac o 1 (IIS) pa hway is he bes s udied one, and i would be commen ed in he sec ion o
de egula ed nu ien sensing, along wi h o he ele an sys ems. The in e connec ions
be ween he p oposed hallma ks o aging become e iden , as he modi ica ion o ansc ip s
leads o de egula ed nu ien sensing.
Besides, no only he p o ein-coding ansc ip s a e a ec ed by aging. Se e al wo ks ha e
in es iga ed he pos - ansc ip ional egula ion o gene exp ession by mic oRNAs (miRNAs)
and o he small non-coding RNAs (sncRNAs). Di e en ially exp essed miRNAs ha e been
ound in a ious o gans as well as in he ci cula o y sys em o bo h humans and model animals
[39,40]. We also s udied he exp ession o sncRNAs in human leukocy es wi h age and
iden i ied a subse o 69 sncRNAs ha g adually inc ease o dec ease. In e es ingly, we
epo ed an accele a ed change in sncRNA exp ession be ween 47-54 yea s, sugges ing ha a
his age ele an gene exp ession modi ica ions occu [41]. No ably, wo ks by Bo ás and
colleagues ecen ly showed ha bo h mRNA and miRNA exp ession a e di e en when
oc ogena ians and cen ena ians a e compa ed, and mo eo e , he da a om cen ena ians a e
mo e simila o he ones ob ained om adul s [26,27]. Fu he mo e, a longi udinal s udy
pe o med by Smi h-Vikos e al. e alua ed he exp ession o se um miRNAs in 16 subjec s and
iden i ied di e en ially exp essed miRNAs be ween he sho -li ed and long-li ed subg oups
[42]. The e is s ill a long way o ge o unde s and he unc ion o all sncRNAs in aging, bu he
a ailable da a highligh hei implica ion in he p ocess and hei po en ial use as bioma ke s
o age- ela ed modi ica ions.
46 | In oduc ion
In addi ion, in he las yea s, a new playe has en e ed he game: ci cula RNA (ci cRNA).
ci cRNAs a e co alen ly closed ansc ip s o med h ough an RNA back-splicing e en and
cha ac e ized by he p esence o a back-splicing junc ion ha makes hem dis inguishable om
hei linea coun e pa s [43]. Al hough he unc ion o mos o he ci cRNAs emains
unknown, i has been ound ha hey can ac as miRNA sponges and ha hey a e also in ol ed
in gene exp ession egula ion, as ci cRNAs can egula e he ansc ip ion o hei pa en al
genes. Mo eo e , ibosome p o iling s udies ha e ecen ly shown ha ci cRNAs can be
ansla ed bo h in i o and in i o, which challenge he s e eo ypic iew o ci cRNAs as non-
coding RNAs [44]. Wi h ega d o aging, se e al s udies ha e in es iga ed hese molecules and
di e en ial exp ession o a la ge numbe o ci cRNAs du ing aging has been ound in a wide
ange o o ganisms. In humans, o ins ance, hey ha e been p oposed o play a ole in
Alzheime ’s disease and immunosenescence [45]. E en i he e a e s ill ew epo s on he
unc ions o ci cRNAs, his is an eme ging ield ha will con inue de eloping and ci cRNAs ha e
o be conside ed as ano he piece o he complex puzzle o aging.
Finally, i is wo h men ioning ha se e al in es iga ions ha e been ca ied ou in he las
decades o y o ind gene ic a ian s ela ed o heal hy aging. S udies conduc ed on
excep ionally long-li ed indi iduals and genome-wide associa ion s udies e ealed many
candida e loci and single nucleo ide polymo phisms (SNPs) ha could be linked o heal hy
aging and longe i y. Howe e , he e a e con o e sial esul s and mos o he candida es
iden i ied in some wo ks ha e no been con i med in o he s udies. These di e ences could be
due o di e en s udy designs, pheno ype de ini ions and in e -e hnic cha ac e is ics, as well
as by he e ec s o epigene ics, en i onmen al ac o s and li es yle di e ences [46].
1.2.2. Telome e a i ion and epigene ic al e a ions
As men ioned be o e, al e a ions in he sequence o genomic DNA a e common in aging, bu
he e a e o he ele an modi ica ions ha a ec he s uc u e and ansc ip ion o DNA, which
include elome e sho ening (o a i ion) and epigene ic al e a ions (Figu e 7).
The sho ening o elome es is caused by he incomple e eplica ion o he e minal ends o
genomic DNA. The eplica ion o ch omosomes is conduc ed by eplica i e DNA polyme ases
ha lack he capaci y o eplica e comple ely he ends o DNA molecules. This unc ion is
ca ied ou by a specialized DNA polyme ase called elome ase. Howe e , mos soma ic cells
do no exp ess elome ase, leading o a p og essi e and cumula i e loss o elome es in each
eplica ion cycle, and consequen ly, wi h inc easing age [47].
Telome es a e implica ed in essen ial biological unc ions: hey p o ec ch omosomes om
ecombina ion, end- o-end usion, and ecogni ion as damaged DNA, con ibu e o he
In oduc ion | 47
unc ional o ganiza ion o ch omosomes wi hin he nucleus, pa icipa e in he egula ion o
gene exp ession, and se e as molecula clocks ha con ol he eplica i e capaci y o human
cells and hei en y in o eplica i e senescence [48]. De ec s in elome ase, elome es o
shel e in ( he p o ein complex ha p o ec s elome es), a e linked o di e se p oblems,
including pulmona y ib osis, p ema u e aging and cance [49]. Besides, i has been ound ha
elome ase-de icien mice exhibi p ema u e aging, which can be e e ed by gene ically
eac i a ing elome ase [50].
Figu e 7. Schema ic ep esen a ion o age- ela ed modi ica ions in DNA s uc u e. Aging a ec s DNA
o ganiza ion a he ch omosome le el ( elome e a i ion) and a he ch oma in le el (diso ganized
he e och oma in). In addi ion, epigene ic al e a ions, such as me hyla ions o deace yla ions can a ec
bo h DNA sequences and his ones. Adap ed om [51].
In humans, he elome e leng h o leukocy es has been widely s udied, and i has been
p oposed ha longe leukocy e elome es a e linked o longe i y [52]. Howe e , se e al
au ho s ha e in es iga ed he a e o leukocy e elome e a i ion and ound ha elome e
leng h is highly a iable a bi h, and besides, hei sho ening is e y high du ing he i s yea s
o li e, while i slows down conside ably du ing adul hood [53,54]. Mo eo e , a publica ion ha
e alua ed he anking o leukocy e elome e leng h o ou longi udinal s udies demons a ed
ha , despi e he in e indi idual di e ences in elome e a i ion pe yea , mos subjec s
main ain hei classi ica ion wi h espec o hei age-ma ched pai s, meaning ha he ones
ha ha e sho e elome es a he age o 30 a e he ones ha ha e sho e elome es one
decade la e [55]. These esul s indica e ha elome e leng h is mos ly p ede e mined and
en i onmen al o li es yle changes ha e only mino e ec s on elome e a i ion. In
consequence, he measu emen o elome e leng h ea ly in li e is use ul o he iden i ica ion o
elome opa hies and as an age- ela ed isk ac o , while i s u ili y o in e en ion moni o ing
in he elde ly is no p omising.
48 | In oduc ion
On he o he hand, he age-associa ed epigene ic al e a ions ha e been ound o be pa ially
e e sible. The bes desc ibed epigene ic modi ica ion ha occu s du ing aging a e
me hyla ions, his one modi ica ions and ch oma in emodelling [33].
Rega ding DNA me hyla ion, only a small ac ion o he CpG si es ha e shown age- ela ed
modi ica ions (a ound 2%), bu his ac ion ep esen s be ween 2 and 3 million cy osines in
he genome, deno ing he complexi y o he sys em. In addi ion, bo h hypo- and
hype me hyla ion o he CpG si es happen wi h age, and he modi ica ions can occu in ce ain
issues o cell ypes, o e en a ec only one pa o a speci ic cell popula ion, adding u he
complexi y [56]. Despi e his, obus ma hema ical me hods ha e been de eloped and he e
a e eliable algo i hms ha in e p e he me hyla ion pa e n o selec ed CpG si es o an
indi idual and p edic ch onological age wi h high accu acy [57]. These ools a e called
epigene ic clocks, and ecen publica ions indica e ha hey could also be use ul o he
de ec ion o accele a ed epigene ic aging ela ed o se e al p oblems o diseases, including
cance , Alzheime ’s disease, ail y and he p edic ion o mo ali y isk [56].
Besides, modi ica ions in his ones a ec he o ganiza ion o he DNA, as well as gene
ansc ip ion. His ones can also be me hyla ed, bu he bes s udied cha ac e is ic o hese
s uc u es is he deace yla ion pe o med mainly by si uins. Si uins a e a amily o NAD-
dependen enzymes able o pos - ansla ionally deace yla e his ones, which is associa ed wi h
ansc ip ion ep ession. The inc eased exp ession o si uins has been ela ed o longe i y in
humans and model o ganisms, and mo eo e , o e exp ession s udies in hese animal models
esul ed in elonga ed li espan and heal hie aging, while he down egula ions o si uins
inc eased senescence and accele a ed aging [58].
The o ganiza ion o his ones in luences in a mo e gene al iew, he packaging o ch oma in
in o he e och oma in and euch oma in. This o ganiza ion is coo dina ed by si uins and many
o he DNA- and his one- modi ying enzymes. The p ope assembly o his ones ensu es a
packaged he e och oma in, which is, howe e , pa ially los and edis ibu ed wi h aging [33].
In e es ingly, he age-associa ed ch oma in emodelling deeply in luences he ansc ip ion o
mul iple genes, as he coding sequence o gene ally ep essed genes can hen be accessible o
he binding o ansc ip ion ac o s, o he o he way a ound. Indeed, as explained in he
p e ious sec ion, he gene exp ession modi ica ions ha occu wi h aging a e di e se, and
ch oma in eo ganiza ion is jus one o he changes ha a ec i . In e es ingly, epigene ic
modi ica ions ha e been ound o play a ole in in lammaging and immunosenescence [51,59],
wo o he p oblems linked o aging ha will be add essed la e in his wo k.
In oduc ion | 49
Finally, i has o be men ioned ha , in con as o elome e a i ion, epigene ic changes a e
e e sible. Fo ins ance, se e al in es iga ions ha e demons a ed ha calo ic es ic ion
a ec s DNA me hyla ion, a enua ing some age- ela ed CpG modi ica ions and showing
deaccele a ed epigene ic aging in mice. Besides, he mTOR inhibi o apamycin also a ec s
me hyla ion and educe epigene ic age in ea ed mice [56]. Simila ly, o he s udies e alua ing
calo ic es ic ion epo ed si uin media ed slowe aging and ex ended li espan [60]. Again,
compounds ha mimic he posi i e e ec o calo ic es ic ion ega ding si uin modula ion
a e being es ed, such as es e a ol and cu cumin [58]. In summa y, he da a om animal
models indica e ha epigene ic modi ica ions a e p omising a ge s, and nu i ional o
pha macological in e en ions could po en ially be applied o a enua e age- ela ed changes in
humans.
1.2.3. Loss o p o eos asis
P o eos asis is de ined as he p ope con ol o p o eins, including hei biogenesis, olding,
a icking, unc ion and deg ada ion. All he men ioned s eps a e essen ial o main aining he
co ec unc ioning o each cell and he o ganism as a whole. The e o e, he p ocesses
implica ed in p o eos asis a e igh ly egula ed, bu hey can su e modi ica ions ha lead o
mis unc ioning du ing aging [61]. Fo ins ance, he p e iously desc ibed genomic ins abili y o
epigene ic al e a ions ha e a di ec impac on p o ein biosyn hesis, as coding sequences can
be inaccessible, damaged o inapp op ia ely copied.
Besides, e en i he biogenesis is comple ed, many p o eins equi e a speci ic olding o be
unc ional. Chape ones a e a class o hea shock p o eins (HSPs) implica ed in p o ein olding
and s abiliza ion. The HSPs, and speci ically chape ones, ha e been widely s udied in he ield
o aging. Indeed, chape ones a e in ol ed in he esponse mechanisms agains s esso s, and
ha e been shown o ail in elde s [62]. Fu he mo e, many s udies ha e been conduc ed in
model o ganisms, and epo s om wo ms, lies o mice among o he s ha e demons a ed he
accele a ed aging in chape one de icien animals, while hei o e exp ession elonga ed
li espan and educed he accumula ion o p o ein agg ega es [62].
The deg ada ion o p o eins is ano he impo an s ep o main ain p o eos asis. In ac ,
mis olded, agg ega ed o non- unc ional p o eins mus be emo ed om he sys em o p e en
he accumula ion o oxici y. The e a e wo majo pa hways o p o ein deg ada ion: he
ubiqui in-p o easome sys em (UPS) and au ophagy. Bo h pa hways ha e hund eds o di e en
componen s, including chape ones, and his complexi y demons a es he in es men o he
cell on p ope p o ein deg ada ion [61]. Howe e , he unc ion o UPS and au ophagy declines
wi h age, and despi e he de ec o one o he pa hways can be pa ially compensa ed by he
o he , many cell ypes accumula e de ec i e p o eins [63]. The p o ein deg ada ion, in
50 | In oduc ion
combina ion wi h he p e iously men ioned de ec s in biosyn hesis and olding, a e
esponsible o he age- ela ed loss o p o eos asis. Indeed, he de ici s o each s ep con ibu e
o he inal ailu e o he sys em.
1.2.4. De egula ed nu ien sensing
As p esen ed be o e, he hallma k desc ibing de egula ed nu ien sensing in aging is igh ly
connec ed o he gene exp ession modi ica ions. In his sense, he gene ic polymo phisms o
mu a ions ha educe he unc ion o he IIS pa hway ha e been associa ed wi h longe i y.
These include he g ow h ho mone, insulin-like g ow h ac o -1 (IGF-1) ecep o , o
downs eam e ec o s such as AKT, mTOR and FOXO [64–66]. Howe e , he componen s o he
IIS pa hway a e mul iple and i s egula ion and in e connec ions wi h o he signalling
pa hways a e complex. Indeed, apa om being ela ed o longe i y, educed le els o he IIS
componen s a e also epo ed du ing no mal aging and in animal models o p ema u e aging
[67]. This could seem con adic o y, bu i has been p oposed ha depending on he du a ion
and ex en o down egula ion he elici ed esul s could be bene icial o dele e ious. Thus, he
cons i u i ely dec eased IIS unc ioning implies lowe cell g ow h and me abolism, and
consequen ly educed a es o cellula damage, while acu e dec eases o ex emely low le els
lead o p ema u e aging [33]. This p ocess is compa able o o he de ensi e esponses ha can
become dele e ious when no p ope ly con olled, as in he case o in lamma o y esponses.
O he nu ien sensing sys ems igh ly connec ed o IIS and also associa ed wi h aging include
mTOR and si uins. The mTOR kinases a e implica ed in anabolic me abolism and he gene ic
as well as pha macologic a enua ion wi h apamycin o his sys em ha e been linked o
inc eased longe i y in dis inc animal models [68,69]. On he o he hand, as desc ibed in he
sec ion o epigene ic al e a ions, si uins a e enzymes implica ed in he o ganiza ion o
his ones and hey play a ole in he age-associa ed ansc ip ional egula ion. No ably, si uins
also espond o nu ien a ailabili y, and hey ge ac i a ed unde nu ien sca ci y. We
p e iously commen ed he ela ion be ween he ele a ed exp ession o si uins and longe i y,
and he posi i e esul s ob ained wi h calo ic es ic ion o ea men s wi h es e a ol o
cu cumin and associa ed wi h si uins [58,60]. Simila ly, apa om he pha macological
in e en ions, he bene icial esul s o calo ic es ic ion ha e also been ound o be media ed,
a leas in pa , by he educ ion o mTOR ac i i y. This link has been demons a ed in animal
models unde calo ic es ic ion, in which he down egula ion o dele ion o mTOR genes
p e en ed he o he wise obse ed ex ension o li espan [70].
In oduc ion | 51
1.2.5. Mi ochond ial dys unc ion
The wellbeing o mi ochond ia is essen ial o he app op ia e unc ioning o cells. Despi e his,
he e a e hund eds o housands o mi ochond ia in a single cell ( he numbe depends on he
o ganism, issue and cell ype), and hus, single o small numbe s o mu a ions o de ici s can
be managed by a cell and main ain p ope unc ioning [71]. Howe e , as we age, he inc eased
damage, educed espi a o y chain unc ioning, imbalanced usion and ission, and de ec i e
clea ance o mi ochond ia (mi ophagy) con ibu e o cellula and o ganismal aging [72]. In he
las yea s, he po en ially bene icial e ec s o mild de iciencies in mi ochond ia ha e been
p oposed. Indeed, he elici ed mi ochond ial de ensi e esponse and he low ene gy s a e ha e
been ound o induce bene icial compensa o y esponses, and e en o ex end li espan in model
o ganisms [33]. This could seem pa adoxical, bu i is in line wi h o he age- ela ed
cha ac e is ics, such as cell senescence o in lamma ion, ha could be bene icial when
e ec i ely con olled, bu de imen al when main ained o abno mally inc eased.
Mi ochond ial dys unc ion is closely ela ed o o he cha ac e is ics o aging. The case o
genomic ins abili y becomes e iden , as mu a ions in mi ochond ial DNA (m DNA) a e one o
he main causes o he se e e impai men on ene gy con e sion. E en i only a ound 1% o he
mi ochond ial p o eome is encoded by m DNA, hese include c i ical componen s o he
oxida i e phospho yla ion complexes [71]. And besides, apa om he mu a ions o dele ions
in m DNA, he accumula ion o changes in nuclea sequences, also a ec he componen s and
dynamics o mi ochond ia. In addi ion o he de ec s in m DNA ha accumula e du ing he
o ganismal li e, i has been demons a ed ha single SNPs and mi ochond ial haplog oups can
in luence he aging p ocess. Fo ins ance, SNPs in mi ochond ial uncoupling p o ein genes ha e
been ela ed o heal hy aging [73], he indi iduals wi h mi ochond ial H haplog oup showed
dis inc mi ochond ial dynamics [74,75] and he D4, D5 and J haplog oups ha e been
associa ed wi h longe i y. Mo eo e , s udies pe o med in mice ha e shown ha he m DNA
haplo ype p o oundly in luences mi ochond ial p o eos asis and unc ion, as well as ROS
gene a ion, insulin signalling and elome e leng h, esul ing in di e ences in he aging p ocess
and median li espan be ween conplas ic s ains [76].
Wi h ega d o senescence, he issue o whe he mi ochond ial dys unc ion is causa i e o jus
pa o he consequences o he en ance o a cell in senescence is s ill deba ed. Howe e , he
bidi ec ional link be ween senescence and mi ochond ia could be oo simple o explain he
complex unde lying p ocesses, and his in e play could be bes ou lined as a icious ci cle,
in ol ing a numbe o eedback loops be ween he playe s. In spi e o he igge ing
mechanisms, i has been widely desc ibed ha senescen cells ha e mi ochond ial de ici s bo h
in genome and p o eome main enance, and consequen ly p esen dynamic changes and
52 | In oduc ion
dys unc ions [77]. On he o he hand, he age-associa ed p oblems in mi ochond ia can also
a ec s em cells. The accumula ion o soma ic m DNA mu a ions al e s em cell homeos asis
and can induce de ec s such as imbalanced biogenesis, abno mal mi ophagy o inc eased ROS
p oduc ion, which can e en ually accele a e s em cell senescence [78,79].
The e is also a igh connexion be ween mi ochond ial de ici s and he ch onic in lamma o y
s a e (in lammaging) wi h aging. The de ec i e o dys unc ional mi ochond ia can elease
molecules ha p omo e he ac i a ion o he immune sys em, including m DNA and ROS among
o he s. The mi ochond ial componen s can boos he immune esponse h ough di e en
pa hways, as he ac i a ion o he in lammasome, he ecogni ion by he cy osolic senso o
dsDNA cyclic GMP-AMP syn hase, o he ac i a ion o immune cells wi h sec e ed me aboli es
like succina e, as comp ehensi ely e iewed by Jang and collabo a o s [72]. Rema kably, he
con ibu ion o mi ochond ial componen o in lammaging can esul in u he inju y, as in he
case o in lammasome and caspase-1 media ed mi ochond ial damage [80].
1.2.6. Cellula senescence
Mo e han hal a cen u y ago, he senescence o human cells was desc ibed in in i o cul u es
o ib oblas s [81]. Hay lick and Moo ehead ound ha a e a ce ain numbe o passages
cul u ed cells lose p oli e a i e capaci y. Besides, i was long desc ibed ha cellula senescence
happens also in i o [82]. Fu he mo e, hanks o all he in es iga ions ha ha e been
conduc ed, nowadays we know ha apa om he eplica i e a es , senescen cells show
many o he ea u es dis inc om non-senescen cells. Some o he ea u es associa ed wi h
senescen cells, and commonly used as senescence bioma ke s, include he inc eased ac i i y
o lysosomal -galac osidase, as well as he ele a ed exp ession o p53 and p16INK4a [83,84].
In e es ingly, senescence can be induced as a con olled mechanism o p e en he
p oli e a ion o damaged cells, be o e hey lose he eplica i e con ol and de elop umo igenic
ea u es. Simila ly, he en ance o cells in o senescence can be igge ed by elome e a i ion,
accumula ion o DNA damage, o ROS, which induce he ac i a ion o he DNA damage esponse
mechanism and lead o senescence mainly h ough he p53 pa hway [85,86]. Thus, hese
p ocesses a e linked o he men ioned cha ac e is ic exp ession o p53 and p16INK4a by
senescen cells. Indeed, hey a e umou supp esso p o eins, pa o complex signalling
pa hways ha espond o he exp ession o oncogenes, and consequen ly induce cellula
senescence o apop osis [87]. This indica es he p o ec i e ole o p e en he o ma ion o
umou s, e en i con ibu ing o he accumula ion o senescen cells. In consequence, he
posi i e o nega i e impac o senescen cells is s ill discussed.
In oduc ion | 53
The induc ion o senescence in damaged and po en ially haza dous cells is undoub edly
bene icial. In addi ion, senescence has been p oposed o igge issue enewal, bu in con as ,
his p ocess may no be e icien ly comple ed in aged issues o pa hological con ex s, esul ing
in he accumula ion o senescen cells (Figu e 8) [86]. The e o e, inc easing e idence indica es
ha bo h p o-senescen and an isenescen he apies can be a ou able. Fo example, in cance ,
du ing ac i e issue epai and e en o p e en age- ela ed damage, con olled p o-senescen
he apies could be bene icial, limi ing p oli e a ion, accumula ion o de ec i e cells and ib osis
[88]. Con e sely, an isenescen he apies may help o elimina e he al eady accumula ed
senescen cells and o eco e issue unc ion in aged indi iduals [89].
Figu e 8. P oposed model o senescence. Senescence ini ia es a issue emodelling p ocess by ec ui ing
immune cells h ough he senescence-associa ed sec e o y pheno ype (SASP, explained in he sec ion
1.2.8). Mac ophages clea he senescen cells, and p ogeni o cells egene a e he damaged issue. This
sequence is impai ed unde pe sis en damage, pa hological s a es o aging. In hese cases, senescen
cells a e no e icien ly clea ed, he issue is no ully egene a ed, and i s unc ionali y diminishes.
Resolu ion o he damage in hese cases in ol es a ib o ic sca wi h senescen cells, in lamma o y cells
and ib o ic issue. Adap ed om [86].
Mo eo e , e en i some molecula ea u es –like eplica ion a es o ele a ed -galac osidase
ac i i y– a e ep oduced in mos senescen cells, senescence a ec s in a di e en manne o
dis inc animals, indi iduals, issues and cell ypes [89–91]. In his wo k, we ha e ocused on
he senescence o he human immune sys em, and consequen ly, in he nex lines he
al e a ions o his sys em a e p esen ed.
1.2.6.1. Immunosenescence
The e m immunosenescence is used o e e o all he changes ha occu o he immune
sys em du ing aging leading o i s dys unc ion. Howe e , immunosenescence is no only caused
60 | In oduc ion
media o s such as umou nec osis ac o alpha (TNF-) and C- eac i e p o ein (CRP) ha e also
been in es iga ed by many au ho s, and hei concen a ion ha e also been ound o be
ele a ed in elde s [143–145].
Figu e 11. The bene i s o main aining balance. The impo ance o keeping a unc ional immune sys em
and a balanced in lamma o y s a e has been shown be ela ed wi h heal hy aging and longe i y. On he
o he hand, when he p oin lamma o y molecules accumula e and he balance is los du ing aging,
ch onic low-g ade in lamma ion o in lammaging de elops.
Howe e , wi h ega d o he oles o cy okines, we should always bea in main he complexi y
o hei unc ions. Ac ually, molecules such as he men ioned IL-6 and TNF- ha e
p oin lamma o y bu also an i-in lamma o y unc ions [146–150]. Thei e ec depends on he
su ounding en i onmen , on he memb ane molecules exp essed in ecep o cells and on he
signalling cascaded hey elici . Thus, e en i IL-6 and TNF- a e in mos o he cases indica o s
o in lammaging among he old indi iduals, in some cases he e could be o he unde lying
p ocesses and he p esence o hese molecules could be bene icial [95].
1.3. Molecula bioma ke s o ail y
When de ining ail y and he main cha ac e is ics o he people a ec ed by his synd ome
(in oduc ion sec ion 1.1), we commen ed abou he complexi y o i s iden i ica ion. Due o he
lack o consensus on an ope a ional de ini ion o ail y, many di e en es s a e employed
nowadays a p ima y ca e se ices. The physical, cogni i e o psychological cha ac e is ics ha
he ail y scales measu e, a e conside ed unc ional bioma ke s [151,152]. In his sense, he
aim o molecula bioma ke s is o complemen he al eady applied es s o help he
iden i ica ion o ail indi iduals.
In oduc ion | 61
Many e o s a e being made o y o iden i y and alida e molecula bioma ke s o ail y, and
e en i some ha e been p oposed, o ou knowledge none o hem a e applied in he clinic. In
he sea ch o ail y bioma ke s, se e al s udies ha e in es iga ed molecules implica ed in he
biology o aging and, he e o e, included in he hallma ks o aging, aiming o ind di e ences
be ween heal hy aging and ail y. In he nex , lines we will commen some o he bioma ke s
ha ha e been sugges ed o be implica ed. A g aphical ep esen a ion o he biological
cha ac e is ics and bioma ke s o ail y is shown in Figu e 12.
Figu e 12. Schema ic ep esen a ion o he main biological p ocesses, hei in e connec ions, he
implica ed molecules and hei ela ion o ail y synd ome [153].
The cu en knowledge poin s, o ins ance, o a ole o oxida i e s ess in he de elopmen o
ail y. In his sense, mos wo ks ha e shown inc eases in oxida i e damage indica o s, while
educed le els in an ioxidan mic onu ien s, in ail indi iduals, which ga e ise o he
ecen ly p oposed ee adical heo y o ail y [154]. This heo y sugges s a change in he ocus
o oxida i e s ess, as di e se s udies showed ha oxida i e damage does no co ela e wi h
ch onological age, bu a he wi h ail y.
One o he oxida i e s ess bioma ke s o ail y is he ele a ed le els o ci cula ing p o ein
ca bonyls, a well-es ablished indica o o p o ein oxida i e damage. Fo example, high p o ein
ca bonyla ion co ela ed o poo g ip s eng h, pa icula ly among olde women [155].
Besides, low le els o ci cula ing an ioxidan s like i amin E ha e been ound o be associa ed
wi h ail y [156,157]. Howe e , o he au ho s did no ind di e ences be ween i amin E
le els and ail y [158], and he ac ha i amin le els can be easily al e ed wi h die o du ing
62 | In oduc ion
disease, complica es e en mo e he in e p e a ion o esul s. Ano he i amin ha has been
s udied in many diseases and p ocesses, including ail y, is i amin D. Lowe le els o his
ho mone ha e been linked o ail y, bu he no mal anges o i amin D a e highly a iable
depending on he geog aphical a ea and among seasons, and he e ec s o long- e m
supplemen a ion wi h i amin D a e s ill con o e sial [3,159,160].
The le els o o he ho mones ha e also been in es iga ed as po en ial bioma ke s. Mos o he
endoc ine ma ke s p oposed in he con ex o ail y a e hose ela ed o he decline in muscle
mass and unc ion. Du ing aging, he e is a p og essi e swi ch om anabolic o ca abolic
me abolism ha a ec s muscle p o eos asis, which has been ela ed o a ia ions in ce ain
ho mone le els. Indeed, dehyd oepiand os e one sul a e (DHEAS) is an impo an egula o o
muscle mass and s eng h ha dec eases wi h age and i has been ela ed o sa copenia [161].
Besides, DHEAS s imula es he p oduc ion o IGF-1, which is equi ed o muscle egene a ion.
Some publica ions ha e epo ed lowe le els o DHEAS in ail subjec s and imp o ed physical
unc ion wi h DHEAS supplemen a ion and exe cise [162,163]. In con as , o he au ho s did
no ind signi ican co ela ions be ween ail y and he le els o es os e one, DHEAS o IGF-1
indi idually, while he accumula ion o mul iple anabolic de iciencies was a good p edic o i
ail y [164].
Ano he cha ac e is ic gene ally associa ed wi h aging and widely in es iga ed in ail y is
me abolic imbalance, and specially glucose and insulin dys egula ion. Ele a ed basal le els o
glucose and insulin, insulin esis ance and abno mal insulin-glucose dynamics ha e been
ela ed o highe a es o baseline ail y and g ea e odds o ail y onse [153,165–167].
No ably, i should be men ioned ha e en i mos o he wo ks ound some al e a ions, no all
o hem ob ained he same esul s. Fo example, he basal le els o glucose we e epo ed o be
ele a ed in ail subjec in some wo ks, while hey we e no signi ican ly di e en om non-
ails in o he publica ions. In addi ion, as epidemiological s udies indica e ha diabe es is a
isk ac o o de eloping ail y, and some ail elde s wi hou diabe es ha e ele a ed le els o
glucose, he ques ion o whe he his imbalance could be a cause o a consequence o ail y
emains open.
The link be ween ch onic in lamma ion and ail y has been ex ensi ely in es iga ed. The
concen a ion o in lamma o y media o s in ci cula ion has been measu ed in many di e en
coho s aiming o es whe he p oin lamma o y molecules a e specially inc eased in ail
indi iduals when compa ed o obus s. Ce ainly, an ele a ed concen a ion o IL-6, TNF- and
CRP, among o he s, ha e been epo ed in mos o he coho s in ail elde s [144,168–172],
bu he e a e also some s udies ha did no ind signi ican di e ences be ween obus and
ail indi iduals [159,173]. Mo eo e , hese h ee molecules a e inc eased in a as ange o
In oduc ion | 63
in lamma o y o in ec ious condi ions, so hey could no be used as a single measu e, and
should be combined wi h o he bioma ke s ha p o ide in o ma ion abou addi ional
a iables ela ed o ail y, such as muscle loss o bone degene a ion [174].
Also, he coagula ion ac i i y is ela ed o in lamma ion. Indeed, hype coagulabili y bo h
e lec s and con ibu es o enhanced in lamma ion [153]. Hype coagulabili y is gene ally
obse ed du ing aging, and ele a ed le els o coagula ion ma ke s, such as ib inogen, ac o
VIII, D-dime and issue plasminogen ac i a o ha e also been linked o highe a es o ail y
[165,173,175]. Howe e , simila o he p e iously men ioned bioma ke s ha ha e been
p oposed o ail y, no all he au ho s ob ained he same esul s. Fo ins ance, ele a ed
ib inogen was ela ed o a highe isk o ail y in women and men by Wals on and
collabo a o s [165], while i was only associa ed wi h ail y isk in women by Gale and co-
wo ke s [175], and he s udy pe o med only in women by Reine e al. ound no associa ions
be ween ib inogen and he isk o inciden ail y [173].
Ano he ema kable sou ce o ail y bioma ke s is linked o b ain changes. This o gan is
ma kedly a ec ed by aging and indeed, he incidence o many b ain diseases inc eases no ably
in elde s. A s udy by Buchman and collabo a o s ollowed nea ly 800 aged people and showed
ha ail y p og esses wi h age, and an accele a ed decline was epo ed in he pa icipan s
ha we e ound o ha e b ain pa hologies in he pos mo em e alua ion [176]. In an a emp
o iden i y easily measu able b ain bioma ke s, educed ce ebella g ey ma e olume
assessed by magne ic esonance imaging ha e been ound in ail elde s when compa ed o
obus s [177]. Simila ly, he neu op o ec i e b ain-de i ed neu o ophic ac o (BDNF), which
p o ec s adul neu ons om dea h du ing s ess and p omo es he de elopmen o imma u e
neu ons, can be measu ed in plasma, and dec eased le els o his p o ein we e linked o highe
a es o ail y in women. Mo eo e , a physical in e en ion ele a ed he concen a ion o
BDNF bo h in obus and p e- ail pa icipan s, sugges ing i s implica ion in he
pa hophysiology o ail y [178].
In a di e en app oach, al e a ions a he gene ic le el a e e alua ed. This is he case o
elome e leng h, epigene ic changes and gene exp ession modi ica ions o e en
pos ansc ip ional egula ion. Wi h ega d o elome e leng h, many au ho s ha e
in es iga ed whe he he e a e associa ions be ween sho e elome s and ail y synd ome. A
sys ema ic e iew and me a-analysis ha was published ecen ly, iden i ied 155 publica ions
on his opic [179]. In e es ingly, hey selec ed 9 s udies ha measu ed elome e leng h in
leukocy es and concluded ha , in acco dance wi h p e ious epo s, elome e leng h migh no
be a meaning ul bioma ke o ail y. In ac , hey epo ed no signi ican di e ences in 8 o he
selec ed s udies and ound only sho e elome es in he s udy ha was pe o med in Hispanic
64 | In oduc ion
indi iduals. As discussed in one o he wo ks ha in es iga ed he associa ion be ween
elome e leng h and ail y, e en i some aspec s ha a e ela ed o ail y, such as oxida i e
s ess o in lamma ion con ibu e o elome e sho ening, hey may no ep esen he
p edominan ac o s in luencing he complex and mul icomponen synd ome o ail y [180].
The egula ion o gene exp ession by epigene ics was al eady commen ed o be implica ed in
he biology o aging. Some au ho s a e also wo king on he ques ion o whe he epigene ic
modi ica ions, such as DNA me hyla ion in luences he incidence o ail y. The in es iga ions
pe o med on he DNA me hyla ion pa e ns indica e ha ail y could be ela ed o accele a ed
epigene ic aging [181,182], and e en speci ic di e ences could be obse ed be ween wins
wi h a dis inc ail y index [183]. Howe e , he cos and complexi y o DNA me hyla ion
pa e n s udies in compa ison o di ec ed gene exp ession analyses should be aken in o
accoun o e alua e he applicabili y o hese me hods in he clinics. Ano he s udy app oach
ha ocuses on he ac o s ha in luence gene exp ession and unc ion is he iden i ica ion o
SNPs associa ed wi h ail y. In his sense, polymo phisms in genes in ol ed in in lamma ion,
muscle biogenesis o apop osis egula ion among o he s ha e been ela ed o ail y [7,184].
No ably, he associa ion be ween gene exp ession and ail y is p obably one o he mos
widely in es iga ed ea u es, as i englobes all he cellula pa hways as well as he s udies ha
measu e he exp ession o housands o ansc ip s o only a single one. In consequence, he e
a e hund eds o publica ions ha in es iga ed he exp ession o ce ain genes o pa hways,
and now also ‘omics’-based app oaches a e being de eloped [185,186]. Recen ly, a
comp ehensi e e iew o he bioma ke s o ail y was published, in which genes bu also
p o eins and sec e ed ac o s ela ed o aging we e included [187]. They di e en ia ed he
bioma ke s in se en ca ego ies: in lamma ion, mi ochond ia and apop osis, calcium
homeos asis, ib osis, neu omuscula junc ion and neu ons, cy oskele on and ho mones, and
o he p inciples. The au ho s also classi ied he bioma ke s depending on hei p io i y, wi h
highes sco es being a ibu ed o he ac o s associa ed wi h ail y and wi h mo e han one
hallma k o aging, and wi h a conside able amoun o e idence ha he ma ke is no equally
exp essed in ail e sus non- ail indi iduals. Finally, hey p oposed a panel o ail y
bioma ke s composed o 19 high p io i y, plus 22 medium p io i y and 3 low p io i y ma ke s.
Mos we e p o eins o genes, bu o he eme ging bioma ke candida es such as miRNAs and
mic opa icles we e also included (Figu e 13). Impo an ly, he in es iga ion o he eme ging
bioma ke s o ail y con inues o de elop, as illus a ed by he publica ion o a wo k ocused
on miRNAs nea ly a he same ime ha he commen ed e iew [188]. Besides, as men ioned
be o e, he case o mic opa icles (o ex acellula esicles) in age- ela ed p ocesses and also
In oduc ion | 65
in ail y is o cen al in e es in ou wo k, and he e o e, he ollowing sec ion is dedica ed o
hese pa icles (in oduc ion sec ion 2).
Figu e 13. The p oposed bioma ke s o ail y. The panel is composed o a co e o high p io i y ac o s
and complemen ed by medium and low p io i y ma ke s [187].
66 | In oduc ion
2. Ex acellula esicles
EVs a e memb ane-coa ed pa icles sec e ed by almos all cell ypes. Thei i s iden i ica ion
was al eady epo ed in 1946, as p ocoagulan pla ele -de i ed pa icles in no mal plasma
[189] and mo e han 20 yea s la e , in 1967, hey we e e e ed as “pla ele -dus ” [190]. Since
hen, se e al publica ions s a ed o epo no el pa icle sou ces and unc ions and by he end
o he 20 h cen u y hey we e al eady known o play a ole in ele an p ocesses, such as
an igen p esen a ion [191]. Impo an ly, a he beginning o he p esen cen u y, he esea ch
on EVs gained in e es among he scien i ic communi y, as hey we e also ound o he
implica ed in o he cen al issues, including he immune sys em media ed an i umo esponse
[192], and due o he disco e y ha EVs ans e mRNAs and miRNAs om he dono cell ha
can induce unc ional changes in ecipien cells [193].
In he las decade, housands o wo ks ha e con inued desc ibing he cha ac e is ics, unc ions
and implica ions o EVs in in e cellula communica ion. Thanks o all o hem, we can now s a e
ha EVs a e impo an playe s in mos biological p ocesses. Howe e , as i usually happens in
scien i ic esea ch, he mo e we know, he mo e complex he pic u e is, and he mo e we need
o in es iga e o unde s and he molecula p ocesses ha go e n ou sel es and he es o
li ing o ganisms.
2.1. Biological cha ac e is ics o ex acellula esicles
The e m EVs is used o e e o all he pa icles ha cells sec e e o he ex acellula media.
The e a e wo main ca ego ies o EVs: exosomes and mic o esicles. Besides, apop o ic bodies
a e also conside ed EVs. Indeed, apop o ic bodies play an essen ial ole in he p ope clea ance
o he dying cell as well as o he signalling o his p og ammed cell dea h o su ounding cells
and o he egene a ion o he issue [194]. Howe e , mos o he wo ks s udying EVs a e
ocused on exosomes and mic o esicles, due o hei mul iple unc ions and implica ions.
Exosomes a e sec e ed pa icles o igina ed by he usion o a mul i esicula body and he
plasma memb ane, while mic o esicles a e o med by he di ec budding and ission o he
plasma memb ane. Mo eo e , apa om hei dis inc biogenesis, exosomes and
mic o esicles ha e also classically been di e en ia ed based on hei size. Exosomes we e
de ined o be a ound 50-100 nm in diame e , and mic o esicles om 100 nm up o 1 m [195].
Howe e , e en i his classi ica ion was o me ly accep ed, nowadays we know ha he e a e
la ge pa icles o igina ed a mul i esicula bodies, as well as smalle esicles ha e agina e
om he plasma memb ane. Consequen ly, he In e na ional Socie y o Ex acellula Vesicles
(ISEV) discou ages he use o hese e ms i he biogenesis pa hway o he esicles is no
In oduc ion | 67
known, and ecommends he use o EVs o jus small, medium o la ge EVs i we wan o e e
o hei size [196].
Wi h ega d o he molecules ca ied by EVs, we ha e o conside bo h hei memb ane and
inne ca go. The memb ane o EVs consis s mainly o p o eins and lipids, bu each EV has
dis inc ypes o p o eins and lipids depending on hei o igin and unc ion. Fu he mo e, he
composi ion o he EV memb ane in luences he a e and in e naliza ion by ecipien cells
[197]. The componen s o he EV lumen a e e en mo e di e se and include p o eins and many
di e en nucleic acids. Apa om he abo e-men ioned mRNA and miRNAs, EVs ca y o he
ypes o small and long ncRNAs, ci cRNAs and dsDNA agmen s [198,199] (Figu e 14).
Impo an ly, he in es iga ions abou EV sec e ion and hei ca go e ealed mo e han a decade
ago, ha he so ing o componen s in o a o ming pa icle is a con olled mechanism and no
a andom packaging o he a ailable molecules in he sec e ing cell [193,200].
Simila ly, he up ake o EVs is hough o be a con olled p ocess. Many au ho s ha e s udied
he binding and in e naliza ion o EVs by ecipien cells and mul iple molecules, such as
e aspanins, in eg ins, lipids and lec ins, ha e been iden i ied o media e he up ake. Besides,
he in eg a ion o EVs can be pe o med by he usion o he EV and cellula memb anes, o by
dis inc endocy ic pa hways (Figu e 14). An ex ensi e and comple e e iew on he biogenesis,
elease and a ge ing mechanisms o EVs was ecen ly published by Niel and co-au ho s, and
i is a ecommended ead o go in o his subjec mo e in dep h [197].
Figu e 14. Simpli ied ep esen a ion o he sec e ion (mic o esicles and exosomes) and up ake o EVs.
Fo he in e naliza ion, EVs can (1) dock and (2) use wi h he plasma memb ane, o (3) ge endocy osed
and (4) e en ually use wi h a memb ane o he endocy ic compa men [195].
68 | In oduc ion
Due o he ocus o his wo k, we p esen he e he main cha ac e is ics and unc ions o EVs
om human cells, bu he in e cellula communica ion media ed by EVs is also p esen in many
o he o ganisms. Fu he mo e, i has been ound ha unicellula o ganisms like bac e ia also
sec e e EVs. In e es ingly, he bac e ia p oduce EVs o mul iple pu poses, including ho izon al
ans e be ween in a-species cells, s ess esponse, o bio ilm o ma ion. In addi ion, in
mammals, he communica ion be ween hos and bac e ial cells o he mic obio a is media ed,
a leas in pa , by EVs [201].
2.2. Isola ion and cha ac e iza ion o ex acellula esicles
The i s s ep o ake in o conside a ion o ob ain EVs is o decide he sample om which we
wan o isola e he pa icles, and o pe o m a p ope collec ion, handling and s o age. EVs can
be isola ed om biological luids, including blood, u ine o ce eb ospinal luid and om cell
cul u e media [202–205]. Depending on he selec ed sou ce, speci ic ecommenda ions ha e
been p oposed [206]. Howe e , he e a e many a iables ha can in luence EV sec e ion and
ha canno be comple ely con olled. Fo example, when aking blood samples, i has been
desc ibed ha age, sex, die , in ec ions, ea men s o e en ci cadian a ia ions can a ec he
EVs in ci cula ion [196]. Besides, as we will desc ibe below, he e a e plen y o EV isola ion
me hods and he choice would depend on he sample cha ac e is ics, s udy objec i e and
a ailable echniques. Thus, i is essen ial o collec and epo all he possible in o ma ion abou
he dono s, samples and applied s eps so ha we can ake in o conside a ion all ou a iables,
and also o enable he po en ial eplica ion by o he au ho s.
Rega ding EV isola ion, i is impo an o men ion ha in mos o he cases, i no in all o hem,
i is no possible o achie e a comple e sepa a ion o he esicles o in e es . The e o e, we ha e
o conside ha e en i he e m isola ion is commonly applied, we a e p obably en iching ou
samples o EVs. Mo eo e , his issue is no exclusi e o EVs, as o he echniques also used o
cells, such as so ing, p ecipi a ion o immunocap u e p esen good bu no pe ec yields.
The me hods o EV sepa a ion a e di e se, and besides, each echnique can ha e dis inc
se ings depending on he sub ype o EVs aimed o en ich. Fo ins ance, di e en ial
cen i uga ion is one o he mos widely applied me hods, bu he cen i uga ion sequences,
o ces and imes a y among s udies. The i s s eps a e usually simila , wi h cen i uga ions a
low cen i ugal o ces (< 10,000 g) o pelle cells and deb is. Then, some in es iga o s apply
middle o ce cen i uga ions (15,000-30,000 g) and eco e he EVs om he pelle , while he
ones ha ocus on small EVs ake he supe na an and pe o m high speed cen i uga ions, o
ul acen i uga ions (usually 100,000-200,000 g). Besides, he e a e au ho s ha complemen
di e en ial cen i uga ions wi h densi y g adien cen i uga ions [206,207].
In oduc ion | 69
Ul acen i uga ion has been one o he mos used me hods, bu some au ho s ha e epo ed
ha i can cop ecipi a e p o ein agg ega es o i uses and can e en induce EV clumping and
damage [206,208].
The e a e o he classically applied echniques o EV isola ion ha include size exclusion
ch oma og aphy, p ecipi a ion, il a ion and immunocap u e. Fo size exclusion and il a ion,
he po e size o he ma ix and o he memb ane, espec i ely, can be selec ed. In addi ion, in
he las yea s, a di e en il a ion me hod has been in oduced: angen ial low il a ion
(TFF). In con as o he common il a ions ha p essu e he sample pe pendicula ly o he
il e , TFF consis s on he applica ion o a angen ial o ce, which minimizes p essu e and
enables he eci cula ion o he sample in o he sys em. Besides, he po e size o he
memb anes applied o TFF can also be chosen depending on he desi ed EVs. The use o TFF
is pa icula ly bene icial when la ge olumes o samples a e handled, as cell cul u e media o
u ine [209,210]. Howe e , TFF can only sepa a e he EVs based on hei diame e .
On he o he hand, he immunocap u e me hods a e a ac i e when a speci ic subpopula ion
o EVs wan o be sepa a ed. This sys em is based on he use o immobilized an ibodies ha
ecognize and bind EV-speci ic molecules, usually p o eins exposed a hei memb ane. The
selec ed an ibodies can be immobilized on a pla e, a chip o a magne ic bead, and he e a e
many comme cial ki s a ailable [207]. Ne e heless, when using immunocap u e p o ocols
unwan ed soluble ligands can also be eco e ed, o pa o he desi ed EVs los i he e a e
mo e ligands han an ibodies a ailable. In addi ion, immunocap u e will always sepa a e a
subpopula ion o EVs, as no uni e sal ma ke has been ound. Fo his eason, as an example,
we canno claim o isola e all exosomes om a complex sample when applying an an i-CD63
an ibody because no all he exosomes a e posi i e o his e aspanin. To illus a e he
complexi y o EVs, a ep esen a ion o some o he mos common molecules iden i ied o be
ca ied by EVs can be seen in Figu e 15.
In e es ingly, new me hods a e being de eloped o he isola ion o EVs. The mic o luidic and
acous ic se ings a e p omising echniques, and e en he combina ion o bo h o hem ha e
been shown o be e ec i e o isola e EVs om whole blood [211]. In any case, he elec ion o
he EV sepa a ion me hod (o combina ion o me hods) is s ongly in luenced by he objec i e
o he s udy, as well as by he equi ed ime, cos s and applicabili y, i i is di ec ed o a
po en ial clinical applica ion [202].
Wi h ega d o he cha ac e iza ion o EVs, and despi e hei small size, he e a e mul iple
me hods a ailable: he ones ha could be applied o cha ac e ize cells ha ha e been adjus ed
o EVs, and he ones ha ha e been specially de eloped o EVs. On one side, we can desc ibe
76 | In oduc ion
3. Mul iple scle osis
MS is a ch onic au oimmune disease o he CNS, cha ac e ized by pa hologic demyelina ion o
axons and subsequen neu odegene a ion. I is a he e ogeneous disease and, clinically, i can
ollow elapsing- emi ing o p og essi e o ms. The elapsing- emi ing o ms a e
cha ac e ized by ou b eaks o neu ological disabili y symp oms las ing a leas 24h ( elapses)
ollowed by eco e y pe iods ( emissions) in which symp oms imp o e pa ially o
comple ely. This is he mos common disease cou se a he ime o diagnosis, wi h
app oxima ely 85% o pa ien s ini ially diagnosed wi h a elapsing- emi ing o m o MS. In he
p og essi e o ms, he disease de elops s eadily and esul s in a apid accumula ion o
disabili y. App oxima ely 50% o pa ien s wi h elapsing- emi ing o ms, con e o a
seconda y p og essi e phase wi hin 10 yea s o disease onse [266].
3.1. E iopa hology o mul iple scle osis
MS is a complex disease, and i s e iology is no comple ely unde s ood. I has been ound ha
a combina ion o gene ic, epigene ic and en i onmen al ac o s, inc ease he isk o de eloping
MS. Among he gene ic ac o s, he HLA-DRB1*15:01 allele in he MHC class II is he ea lies ,
and mos dominan isk ac o iden i ied, while ciga e e smoking, highe la i udes, low sun
exposu e, low i amin D le els and Eps ein-Ba i us in ec ion a e he p incipal
en i onmen al isk ac o s. Besides, in he las yea s, he implica ion o he mic obiome is being
in es iga ed, as se e al s udies ha e shown i s in luence on he immune sys em egula ion, and
some di e ences in he gu mic obiome be ween MS pa ien s and heal hy con ols ha e also
been epo ed [267].
The pa hological p ocess o MS is ini ia ed by an in lamma o y p ocess media ed by
au o eac i e T cells. The igge o he au oimmune a ack is hough o be an au oan igen, bu
i has no been iden i ied ye . The au o eac i e T cells ge ac i a ed in he pe iphe y, s a o
p oduce p oin lamma o y molecules and o exp ess adhesion molecules ha a ou hei
a achmen o endo helial cells o he BBB. In addi ion, he BBB is usually damaged in MS
pa ien s and, as a esul , he au o eac i e cells a e able o i s i mly adhe e o endo helial cells,
and hen mig a e h ough he BBB in o he CNS [268]. Once in he CNS, T cells a e eac i a ed
by as ocy es o mic oglia, in lamma ion sp eads and inally, e ec o T cells damage he myelin
shea h o axons, and mac ophages and glial cells pa icipa e in he diges ion p ocess. As a
esul , he e is an impai ed isola ion o axons and abno mally slow ac ion po en ial
ansmission [269]. An illus a ion o his p ocess is p esen ed in Figu e 17.
In oduc ion | 77
Figu e 17. P oposed mechanisms and implica ed cells and molecules in demyelina ion and
emyelina ion p ocesses [269].
In he i s s ages o MS, he neu ologic unc ion is pa ially o comple ely es o ed a e a
demyelina ing e en . This p ocess is media ed by oligodend ocy e p ecu so cells ha ge
ac i a ed, mig a e o he lesion, p oli e a e and di e en ia e o ma u e myelin-p oducing
oligodend ocy es. Howe e , he newly p oduced myelin shea h is usually hinne han he
o iginal, and besides, he egene a i e p ocess becomes less e icien wi h inc easing age.
Consequen ly, he pa hologic au oimmune a acks can esul in a axonal degene a ion and
subsequen neu odegene a ion, which a ec s he neu ologic unc ion o MS pa ien s and
inc eases disabili y [270].
78 | In oduc ion
3.2. Mul iple scle osis and p ema u e aging
Mos o MS pa ien s expe ience he i s symp oms a hei 20s o 30s, bu i should be
men ioned ha he e a e also paedia ic o ju enile [271] and la e-onse MS cases [272].
Impo an ly, in he las decades, e ec i e disease-modi ying ea men s ha slow he
p og ession o MS ha e been de eloped [267]. Thanks o he bene icial e ec s o hese
ea men s, MS pa ien s ha e a slowe a e o disabili y accumula ion and hus, a be e quali y
o li e han MS pa ien s o p e ious gene a ions [273]. In consequence, and ollowing he same
end as he gene al popula ion, he li e expec ancy o MS pa ien s is inc easing, and wi h i , he
mean age o MS pa ien s is also ge ing highe . The epo s o he MSBase egis y [274] show
ha al eady mo e han 20% o MS pa ien s a e aged ≥60 yea s (msbase.o g). The e o e, an
ele a ed numbe o pa ien s su e om he in e ac ions be ween he MS disease and he aging
p ocess.
Howe e , in mos o he cases, i is no possible o dis inguish be ween he cha ac e is ics o
MS and aging in a pa ien o ad anced age. This is due o simila i ies be ween he ypical
ea u es o he wo p ocesses, which include cogni i e and ca dio ascula p oblems, bowel and
bladde dys unc ion, o educed mobili y, among o he s. O cou se, no all he MS pa ien s o
elde s p esen hese p oblems, bu hey a e common in he wo cases. The simila i ies be ween
MS and aging a e also epo ed a he biological le el, as immune sys em exhaus ion and
ch onic in lamma ion occu in bo h p ocesses [275]. Fu he mo e, i would no he possible o
disc imina e be ween he causes o each ea u e, as he o ganism has o be unde s ood as a
whole en i y, in which he dys unc ions accumula e and can in luence he o he sys ems (like
explained o he biologic hallma ks o aging).
In a di e en app oach, he possible de elopmen o p ema u e aging in pa ien s wi h
au oimmune diseases like MS, ype 1 diabe es o heuma oid a h i is has been p oposed [276–
279]. Pa icula ly o MS pa ien s, he ch onic and in ense implica ion o he immune sys em
du ing MS pa hology, as well as he e ec s o immunomodula o y d ugs p esc ibed, ha e been
sugges ed o p omo e he p ema u e exhaus ion o he immune sys em [279]. Indeed, some o
hese wo ks also alluded o he possibili y o an in e se ela ion, wi h inc eased isk o
de eloping au oimmune diseases unde p ema u e immunosenescence.
Wi h ega d o immunosenescence, some wo ks epo ed inc eased le els o CD4+CD28- T
cells [280,281], hymic in olu ion [282], al e ed T cell homeos asis [283] and dis u bed
egula o y T cell de elopmen and unc ion [284] in adul MS pa ien s. In ela ion o
in lamma ion, ele a ed le els o TNF- and IL-6 among o he in lamma o y ma ke s ha e been
ound in he ce eb ospinal luid and se um o MS pa ien s du ing emission, indica ing ha
In oduc ion | 79
some signs o ch onic in lamma ion, simila o he ones obse ed he age-associa ed
in lammaging could be p esen [285,286].
These publica ions poin o he possible p ema u e immune decline in MS. In con as , he e
a e o he wo ks ha did no ind immunosenescen ea u es in MS pa ien s, o ha epo ed
di e ences in immune cha ac e is ics depending on he immunomodula o y d ug ecei ed by
he MS pa ien [277,287,288]. In any case, he link be ween p ema u e aging and MS should be
u he in es iga ed o elucida e whe he he e is a causa i e ela ion, and i such, which s eps
could be aken o p e en o app op ia ely ea his si ua ion.
JUSTIFICATION
Jus i ica ion | 83
One o he majo conce ns o ou socie y is he aging o he popula ion. The apid inc ease o
li e expec ancy and he consequen ising incidence o age-associa ed diseases and dependency
ha e made us awa e o he in e disciplina y challenge we ace.
Many e o s a e conduc ed in nume ous ields o manage he socioeconomic impac o aging
and we, as pa o he scien i ic communi y, a e wo king o desc ibe and unde s and his
complex p ocess om he biological, biochemical and biomedical poin o iew.
In his con ex , he p esen p ojec was ou lined in 2015, aiming o ad ance knowledge and
con ibu e o he ul ima e goal o imp o ing he quali y o li e du ing he na u al p ocess o
aging.
HYPOTHESIS AND OBJECTIVES
92 | Chap e one
Common me hods
Pa icipan s and F ail y classi ica ion
Fo he p esen s udy, samples om 3 di e en coho s o elde dono s and samples om
heal hy adul s we e used. We ob ained he samples o he elde coho s in collabo a ion wi h
he P ima y Ca e Uni o Biodonos ia Heal h Resea ch Ins i u e and he Neu ology depa men
o Donos ia Uni e si y Hospi al. Pa icipan s a e om he p o ince o Gipuzkoa (Basque
Coun y, Spain) and mee he c i e ia shown in Table 1.
Table 1. Inclusion c i e ia o he coho s.
All pa icipan s comple ed a ques ionnai e and dono s wi h acu e illness we e excluded. The
s udy was app o ed by he hospi al’s e hics commi ee and all pa icipan s p o ided w i en
in o med consen be o e blood sampling. F ail y s a us o elde pa icipan s was assessed by
p ima y ca e se ices. A ba e y o es s was conduc ed. The ansla ed e sion o ail y es s
was applied. A sho desc ip ion o ail y es s is shown in Table 2 and he main cha ac e is ics
o each coho in Table 3.
Coho Desc ip ion
Pa icipan s om E en e ia and Pasaia
Su i o s o a p e ious s udy we e con ac ed and in i ed o pa icipa e
No u he inclusion c i e ia
Samples ob ained July 2014 – May 2015
Pa icipan s o m E en e ia, I un and Honda ibia
Aged 70 o o e , communi y-dwelling and au onomous (Ba hel > 90)
Samples ob ained May 2015 – July 2016
Pa icipan s om Ge a ia, U nie a, Zumaia and Zes oa
Aged 70 o o e , communi y-dwelling, including au onmomus and
non-au onomous
Samples ob ained Augus 2016 – May 2017
Coho 1
Coho 2
Coho 3
Chap e one | 93
Table 2. Sho desc ip ion o he es s applied o measu e ail y.
F ail y assessmen es
Desc ip ion
Tilbu g F ail y Indica o (TFI) [15]
A use - iendly ques ionnai e based on a
mul idimensional app oach. I is composed o a physical, a
psychological and a social domain.
Gai speed (GS) [17]
Exp essed in me e s pe second (m/sec). Pa icipan s
we e asked o walk a hei usual pace. The es was
pe o med wice and GS was calcula ed based on he
sho e ime.
Timed up-and-go (TUG) [18]
The ime needed o s and up om a chai , walk 3 me e s,
u n a ound, walk back and si down, wi h he help o
hei usual walking aid, i any.
Sho Physical Pe o mance Ba e y
(SPPB) [19]
A unc ional capaci y es composed o gai speed, es o
balance and ime needed o s and up om a chai 5
consecu i e imes.
Ge on opole F ail y Sc eening Tool
(GFST) [16]
Based on clinical judgemen . 6 yes/no ques ions ha help
he physician o e alua e he exis ence o ail y.
Ba hel Index (Ba hel) [291]
A mul ipa ame ic es measu ing he pe o mance in
ac i i ies o daily li ing and mobili y.
Table 3. In o ma ion o s udy pa icipan s.
Adul s
Coho 1 Coho 2 Coho 3 -
Pa icipan s 53 295 91 57
Female/Male 30/23 153/142 56/32 30/27
Age (mean) 79-92 (82.51) 71-91 (79.83) 70-96 (76.98) 24-46 (33.51)
F ail y assesmen -
TFI X X
GS X X X
TUG X X X
SPPB X X
GFST X
Ba hel X X X
Elde s
94 | Chap e one
Blood sampling
Pe iphe al blood was collec ed by expe ienced nu ses by enipunc u e wi h a 21-gage needle
in 8 ml se um sepa a o ubes and 4 ml EDTA ubes (Vacu aine , BD Biosciences) and di ec ly
deposi ed in he Basque Biobank o hei p ocessing and s o age. Se um sepa a o ubes we e
allowed o clo o 30 min and cen i uged a 1258 g o 20 min o eco e se um om he
supe na an . EDTA ubes we e kep up igh and cen i uged a 1258 g o 20 min o eco e
plasma. To ob ain RNA, samples om EDTA ubes we e incuba ed wi h Bu e EL (Qiagen) o
e y h ocy e lysis and hen RNA om leukocy es was ex ac ed wi h QIAamp RNA Blood Mini
Ki (Qiagen) ollowing manu ac u e ’s ins uc ions. Fo DNA, a second EDTA ube was used and
he ex ac ion was pe o med wi h FlexiGene DNA Ki (Qiagen) ollowing he manu ac u e ’s
ins uc ions. RNA and DNA quan i y and quali y we e assessed wi h a Nanod op 1000
spec opho ome e (The mo Fishe ). The ob ained se um, plasma, RNA and DNA samples we e
aliquo ed and s o ed a -80 °C. When needed, co esponding eques o ms we e ul illed o
ob ain he samples om he Basque Biobank.
Chap e one | 95
In lamma ion
In oduc ion
Ch onic low-g ade in lamma ion is one o he bes desc ibed cha ac e is ics o aging. I has
been widely shown ha elde s p esen ele a ed le els o in lamma o y ma ke s in ci cula ing
blood, in he absence o o e in ec ion [137,138,292]. This accumula ion o p oin lamma o y
molecules, e med in lammaging, has also been p oposed as a po en ial bioma ke o ail y.
P e ious s udies ha e measu ed he concen a ion o p oin lamma o y ma ke s such as IL-6,
TNF- and CRP among o he s in dono s wi h di e en deg ees o dependency. Howe e , he
ob ained esul s a e con o e sial, and e en i some esea che s ha e ound an inc eased
concen a ion in ail and non-au onomous elde s, o he s udies did no epo signi ican
di e ences [159,169,171,174]. O he molecule ela ed o in lamma ion and p oposed as a
ail y bioma ke is albumin. The a e o albumin syn hesis is a ec ed by bo h nu i ion and
in lamma ion, and in lamma ion alone is associa ed wi h a g ea e ca abolic a e o albumin.
Dec eased albumin le els ha e been p oposed as a isk ac o o ail y, bu simila o he
abo e-men ioned in lamma o y ma ke s, he e is no consensus on i s alidi y [293,294].
The aim o ou s udy is o in es iga e he alidi y o in lamma o y media o s as bioma ke s
ha could complemen he unc ional and clinical e alua ion o elde s o he iden i ica ion o
ail y. To ha end, we i s compa ed he concen a ion o he abo e ci ed molecules be ween
adul s and elde dono s, and hen, based on he ail y classi ica ion o elde s, e alua ed
whe he hese molecules show di e en le els wi h ail y and dependency in ou coho s.
Ma e ials and me hods
CRP ELISA and TNF- Luminex
Plasma samples om elde s (coho 2, n=111) and adul s (n=39) we e hawed on ice. CRP
concen a ion was measu ed wi h Quan ikine ELISA (R&D) ollowing he manu ac u e ’s
ins uc ions. Plasma samples we e dilu ed 1:150 o i he s anda d cu e o he ki . On he
o he side, a panel o 6 in e leukins was designed o luminex measu emen : IL-6, IL-10, IL-2,
IL-1, IL-1Ra and TNF-. The Milliplex Map #HCYTOMAG-60K ki (Me ck) was used.
Manu ac u e ’s ins uc ions we e ollowed and plasma samples we e assayed undilu ed, bu
only he measu emen s o TNF- we e abo e he lowes poin o he s anda d cu e. We
pe o med a second ial wi h he same ki and ob ained simila esul s. In o de o sol e his
issue, we epea ed he assays using he high sensi i i y ki #HSTCMAG-28SK (Me ck) p o ided
96 | Chap e one
by he manu ac u e , bu mos o he samples we e s ill non-de ec able. Las ly, we also ied a
high sensi i i y luminex ki om ano he b and, #FCSTM09-04 (R&D) o IL-6, IL-10, IL-2 and
IL-1. Wi h his ki he measu emen o analy es was also non-de ec able in many samples
(65/160). A e he ob ained nega i e esul s, we decided no o measu e mo e plasma samples
wi h he luminex echnique and we analysed only he esul s om TNF- he only analy e ha
ob ained de ec able and eliable esul s (elde s n=37 and adul s n= 39).
CRP, TNF-, IL-6 and albumin ELISA
Wi h he objec i e o es se um samples and o s eng hen ou esul s, samples om elde s
(coho 3, n=91) and adul s (n=18) we e used. Samples we e hawed on ice, CRP, TNF- and
IL-6 we e measu ed wi h Quan ikine ELISAs (R&D) and albumin wi h an ELISA ki (In i ogen,
The mo) ollowing he manu ac u e ’s ins uc ions. Se um samples we e dilu ed o i he
s anda d cu es o each ki : dilu ed 1:100 o CRP, undilu ed o TNF-, undilu ed o IL-6 and
dilu ed 1:500000 o albumin.
S a is ical analysis
S a is ically signi ican di e ences be ween he s udy g oups and co ela ions be ween
a iables we e es ed wi h G aphPad P ism e sion 6.01 o Windows (G aphPad So wa e,
www.g aphpad.com). D’Agos ino-Pea son no mali y es was applied and non-Gaussian
dis ibu ion was con i med o all samples. Consequen ly, Mann-Whi ney es s we e applied o
e alua e di e ences be ween wo s udy g oups. Fo co ela ion analysis, Spea man coe icien
was calcula ed. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.
Resul s
In lamma o y ma ke s in plasma
To e alua e he in lamma o y s a us di e ences be ween he s udy g oups, CRP and TNF-
we e measu ed in plasma samples om coho 2. Fi s , esul s om elde s we e compa ed o
heal hy adul s, and we con i med an inc eased concen a ion o bo h CRP and TNF- in aged
indi iduals (Figu e 18A and Figu e 19A). Then, he co ela ion be ween age and
in lamma o y ma ke s was e alua ed, bu no signi ican co ela ions we e ound (Figu e 18B
and Figu e 19B). Simila ly, no di e ences we e ound based on gende (Figu e 18C and
Figu e 19C). Conside ing o he possible con ounde ac o s and aking ad an age o he
a ailable da a abou he d ug numbe p esc ibed o he dono s in his coho , we also e alua ed
he co ela ion be ween he numbe o d ugs each pa icipan akes and he concen a ion o
CRP and TNF-. We ound a signi ican posi i e co ela ion be ween CRP and he d ug numbe
– which was co ec ed by linea eg ession be o e u he analysis –, while no co ela ion was
ound o TNF- (Figu e 18D and Figu e 19D).
Chap e one | 97
Finally, he concen a ion o bo h in lamma o y ma ke s was compa ed be ween obus and
ail indi iduals. The eco ded ail y scales o coho 2 we e: TFI, GS, TUG, SPPB and GFST.
The classi ica ion o each ail y scale was conside ed, and we did no ind any signi ican
di e ences (Figu e 18E-I and Figu e 19E-I). To pe o m a mo e obus compa ison, he
pa icipan s ha a e classi ied as obus o ail o all he es ed scales (n=40) we e compa ed,
bu no di e ences we e epo ed (Figu e 18J and Figu e 19J). In he las app oach, he elde s
wi h he same classi ica ion o he 3 scales ha e alua e he unc ional s a us (GS, TUG, SPPB,
n=63) we e b ough in o compa ison, and as o he p e ious analyses, no di e ences we e
ound (Figu e 18K and Figu e 19K).
Figu e 18. Concen a ion o CRP in plasma. (A) The e is ele a ed CRP in elde s compa ed o adul s. (B)
Among elde s, CRP concen a ion has no co ela ion o age and (C) he e is no signi ican di e ence
be ween emales and males. (D) A posi i e co ela ion be ween CRP concen a ion and d ug numbe
was ound and co ec ed. (E-I) No di e ences in CRP le els be ween obus and ail indi iduals we e
ound o he 5 analysed ail y scales. (J) We also compa ed he indi iduals classi ied as obus o ail
wi h all he a ailable es s o (K) wi h he 3 unc ional scales (GS, TUG, SPPB), bu no di e ences we e
ound.
98 | Chap e one
Figu e 19. Concen a ion o TNF- in plasma. (A) The e is ele a ed TNF- in elde s compa ed o adul s.
(B) Among elde s, CRP concen a ion has no co ela ion o age, (C) he e is no signi ican di e ence
be ween emales and males and (D) no co ela ion be ween TNF- concen a ion and d ug numbe was
ound. (E-I) No di e ences in TNF- le els be ween obus and ail indi iduals we e ound o he 5
analysed ail y scales. (J) We also compa ed he indi iduals classi ied as obus o ail wi h all he
a ailable es s o (K) wi h he 3 unc ional scales (GS, TUG, SPPB), bu no di e ences we e epo ed.
In lamma o y ma ke s in se um
Fo he cha ac e iza ion o in lamma o y ma ke s in se um, samples om coho 3 we e used.
We s udied he p e iously measu ed CRP and TNF-, as well as IL-6 and albumin. We ob ained
he same esul s as in plasma, con i ming ha he e is an ele a ed ch onic in lamma ion in
elde s when compa ed o adul s: inc eased CRP, TNF- and IL-6, while educed albumin
(Figu e 20A, Figu e 21A, Figu e 22A and Figu e 23A). Mo eo e , CRP, TNF- and IL-6
showed a posi i e co ela ion o age among elde s (Figu e 20B, Figu e 21B and Figu e 22B),
which was co ec ed by linea eg ession o each analy e. On he o he hand, no co ela ion
wi h age was ound o albumin (Figu e 23B). Rega ding gende , no di e ences we e ound
Chap e one | 99
o any o he analy es (Figu e 20C, Figu e 21C, Figu e 22C and Figu e 23C). A las , we made
use o he a ailable da a o Ba hel and TUG scales, pe o ming he compa ison be ween he
di e en dependency s a uses o pa icipan s in coho 3: obus , ail and non-au onomous.
We ound no signi ican di e ences o any o he molecules be ween he analysed g oups
(Figu e 20D, Figu e 21D, Figu e 22D and Figu e 23D), ollowing he same end as he
analyses in plasma samples.
Figu e 20. Concen a ion o CRP in se um. (A) The e is ele a ed CRP in elde s compa ed o adul s. (B)
Among elde s, se um CRP concen a ion has a posi i e co ela ion o age and (C) he e is no signi ican
di e ence be ween emales and males. (D) When compa ed based on Ba hel and TUG scales, no
di e ences in CRP le els be ween obus , ail and non-au onomous indi iduals we e ound.
Figu e 21. Concen a ion o TNF- in se um. (A) The e is ele a ed TNF- in elde s compa ed o adul s.
(B) Among elde s, se um TNF- concen a ion has a posi i e co ela ion o age and (C) he e is no
signi ican di e ence be ween emales and males. (D) When compa ed based on Ba hel and TUG scales,
no di e ences in TNF- le els be ween obus , ail and non-au onomous indi iduals we e ound.
100 | Chap e one
Figu e 22. Concen a ion o IL-6 in se um. (A) The e is ele a ed IL-6 in elde s compa ed o adul s. (B)
Among elde s, se um IL-6 concen a ion has a posi i e co ela ion o age and (C) he e is no signi ican
di e ence be ween emales and males. (D) When compa ed based on Ba hel and TUG scales, no
di e ences in IL-6 le els be ween obus , ail and non-au onomous indi iduals we e ound.
Figu e 23. Concen a ion o albumin in se um. (A) The e a e educed albumin le els in elde s compa ed
o adul s. (B) Among elde s, se um albumin concen a ion has no co ela ion o age and (C) he e is no
signi ican di e ence be ween emales and males. (D) When compa ed based on Ba hel and TUG scales,
no di e ences in albumin le els be ween obus , ail and non-au onomous indi iduals we e ound.
Discussion
In lammaging is one o he main biological cha ac e is ics o human aging. This e m was
p oposed in 2000 by F anceschi e al. [136], al hough a wo k showing he accumula ion o
in lamma ion wi h age and i s ela ion o mo ali y was al eady published in 1991 by
Moo adian e al. [295]. Since his e m was in oduced, many wo ks ha e in es iga ed he
ela ionship be ween in lamma o y ma ke s and aging, dependency and mo ali y. Howe e ,
he ob ained esul s a e di e se and many imes disco dan , so no consensus has been eached
[138,144].
Chap e one | 101
The s udies in es iga ing he po en ial ole o molecules linked o in lamma ion as ail y
bioma ke s encoun e he same p oblem. Many wo ks ha e been ca ied ou , bu no clea
associa ion has been ound, as some ound inc eased concen a ions o p oin lamma o y
ma ke s in ail subjec s, whe eas o he s did no epo any signi ican di e ences
[170,173,296]. I should also be conside ed ha he s udy designs, echniques and he
cha ac e is ics o included pa icipan s a e dis inc in each in es iga ion. Mo eo e , he
published s udies ha e been ca ied ou in di e en coun ies, hence dis inc i e gene ic and
en i onmen al aspec s should be conside ed. In addi ion, he es s employed o ail y
assessmen e alua e he s a us o pa icipan s based on di e en aspec s, and he e o e, he
same pe son can be conside ed ail based on one scale and obus based on ano he one. Fo
example, he e a e es s ha ocus on clinical aspec s [16], while o he s gi e mo e impo ance
o he psychologic domain [15] o he unc ional pe o mance [19]. This di e si y o es s
shows he he e ogenei y o he “ ail y” e m, which makes i e en mo e di icul o iden i y a
biological ma ke o he synd ome.
Rega ding he echniques a ailable o he quan i ica ion o he molecules o in e es , we
decided o apply ELISA and Luminex. We ob ained good esul s wi h he ELISA ki s o plasma
and se um samples, while we encoun e ed de ec ion p oblems wi h Luminex ki s. We chose
Luminex because i is a echnique ha enables he de ec ion o a panel o analy es in he same
expe imen , sa ing ime and educing he amoun o sample needed. Mo eo e , we ha e
applied his p o ocol in p e ious expe imen s o o he p ojec s, and we ob ained good esul s
wi h cell cul u e supe na an s. Howe e , when plasma samples we e es ed, all he selec ed
analy es, excep TNF-, did no each de ec able le els in mos o he samples. We hough ha
he concen a ion o ou analy es could be lowe han he de ec ion limi o he ki s, bu we
dismissed his possibili y a leas o IL-6, as we ha e p e iously measu ed i wi h ELISA in
samples o he same cha ac e is ics and he ob ained concen a ions a e no ably highe han
he de ec ion limi o he Luminex ki s used. Due o his, we decided o con inue analysing ou
plasma and se um samples wi h ELISA.
We eliably measu ed CRP, TNF-, IL-6 and albumin in ou coho s, ha include aged subjec s
om he egion o Gipuzkoa (Basque Coun y, Spain) who ha e been e alua ed wi h se e al
ail y scales. In hese coho s, ou esul s con i med he p esence o in lammaging by he
inc eased low-g ade in lamma ion in elde s when compa ed o adul s. Bu ega ding ail y,
which was he main objec i e o his wo k, none o he analysed molecules showed signi ican
di e ences based on he dependency s a us.
Mo eo e , o y o o e come he abo e-men ioned issue o he he e ogenei y be ween ail y
es s, and aking ad an age o he a ailable da a in coho 2, we also compa ed he
108 | Chap e one
Table 4. The 35 genes iden i ied o be di e en ially exp essed be ween obus and ail elde s.
Gene symbol En ez Gene ID F ail A g (log2) Robus A g (log2) Fold Change p- alue
EGR1 1958 8.08 6.66 2.66 0.0014
DDX11L1 100287102 7.73 9.32 -3.01 0.0039
MIR454 768216 3.13 3.94 -1.63 0.0004
CISH 1154 6.82 7.99 -2.25 0.0033
DDX11L10 100287029 6.25 8.28 -4.11 0.0013
LOC101929775 101929775 5.82 7.22 -2.62 0.0004
LOC644172 644172 5.82 7.22 -2.62 0.0004
NSF 4905 5.58 7.43 -3.59 0.0002
TRAJ17 28738 7.18 8.57 -2.62 0.0017
TRAJ19 28736 6.52 7.6 -2.11 0.0037
TRAV8-3 28683 7.42 8.86 -2.71 0.0004
CD40LG 959 7.14 8.15 -2.01 0.0098
CLDN12 9069 3.13 4.51 -2.6 0.0151
CNTNAP3 79937 6.8 8.18 -2.6 0.0479
CNTNAP3B 728577 6.95 8.21 -2.39 0.0310
CSRNP1 64651 8.01 6.94 2.1 0.0307
CTSLP8 1518 2.14 3.25 -2.16 0.0260
CXCL8 3576 10.17 9.06 2.15 0.0190
G0S2 50486 8.89 7.46 2.69 0.0318
GCNT4 51301 5.8 6.82 -2.02 0.0217
GJB6 10804 5.18 4.09 2.12 0.0057
IGHV2-26 28455 3.63 4.88 -2.38 0.0291
LOC100505530 100505530 5.35 6.64 -2.45 0.0224
LOC105378916 105378916 6.36 7.72 -2.56 0.0333
MIR3941 100500866 3.16 4.43 -2.4 0.0059
MIR487A 619555 2.42 3.62 -2.29 0.0275
MIR626 693211 2.96 4.04 -2.11 0.0244
MTRNR2L2 100462981 5.58 6.84 -2.39 0.0334
RLN1 6013 4.65 5.71 -2.08 0.0092
TIA1 7072 7.46 8.49 -2.05 0.0462
TRAJ14 28741 8.14 9.17 -2.04 0.0128
TRAJ16 28739 7.77 8.78 -2.01 0.0300
TRAJ48 28707 7.83 8.9 -2.09 0.0185
TRAV16 28667 5 6.58 -2.98 0.0172
TRBV3-1 28619 7.21 8.39 -2.26 0.0058
Chap e one | 109
Nex , we e alua ed whe he he exp ession o he selec ed candida es is al e ed in a la ge
coho . Fo his pu pose, we selec ed 120 RNA samples om coho 1 and coho 2. To main ain
he p e iously se c i e ia, only he pa icipan s ha ob ained he same classi ica ion in he TFI,
GS and TUG es s we e selec ed ( obus s n=103, and ails n=17). The RT-qPCR analysis o
hese 120 samples con i med he inc eased exp ession o EGR1 in ail elde s, while no
di e ences we e ound o DDX11L1 and MIR454 (Figu e 27).
Figu e 27. Exp ession o 3 o he candida es in a alida ion coho . (A) The exp ession o DDX11L1 is
no di e en in obus and ail pa icipan s. (B) EGR1 is signi ican ly up egula ed in ail y. (C) MIR454
is down egula ed in ail y, bu s a is ical signi icance was no eached.
Physical in e en ion and EGR1 exp ession
Some o he dono s o coho 2 we e in i ed o pa icipa e in a physical in e en ion s udy o
3 mon hs. Blood samples we e collec ed be o e and a e he in e en ion, and he exp ession
o DDX11L1, EGR1 and MIR454 we e de e mined by RT-qPCR. No clea ends we e obse ed
o DDX11L1 and MIR454 (Figu e 28A and 28C). In con as , EGR1 le els we e educed in 9
ou o he 12 dono s wi h a p- alue o 0.06, ein o cing he po en ial o his gene as a bioma ke
o ail y (Figu e 28B). Fu he mo e, when he changes in EGR1 exp ession and TUG
pe o mance we e compa ed, we obse ed ha 8 ou o he 12 pa icipan s ob ained
conco dan esul s: a educ ion o EGR1 accompanied by a be e TUG sco e, o inc eased EGR1
accompanied by a wo se TUG sco e (Figu e 28D).
110 | Chap e one
Figu e 28. Physical in e en ion, gene exp ession and physical pe o mance. (A) No di e ences we e
epo ed o DDX11L1 exp ession. (B) EGR1 exp ession is educed in 9 ou o he 12 pa icipan s and a
p- alue o 0.06 was ob ained wi h he pai ed-samples s a is ical analysis. (C) No di e ences we e
epo ed o MIR454 exp ession. (D) When he exp ession o EGR1 and TUG sco es we e compa ed, 8
ou o he 12 pa icipan s ob ained conco dan esul s: educ ion in EGR1 exp ession and TUG
pe o mance ime, o inc eased EGR1 exp ession and TUG pe o mance ime.
Discussion
In he p esen s udy, we ha e pe o med a ansc ip omic analysis o communi y-dwelling
indi iduals om he Basque Coun y. A se o 35 di e en ially exp essed ansc ip s was ound
be ween obus and ail elde s. Among hem, he e we e genes linked o in lamma ion and
hypoxia- ela ed pa hways, immune esponse, apop osis and se e al membe s o he T cell
ecep o alpha locus. These p ocesses ha e also been ela ed o ail y in se e al p e ious
s udies, bu howe e , ou se o genes was di e en om a ecen ly p oposed panel o po en ial
ail y bioma ke s [187].
Wi hin he 35 ansc ip s, we selec ed 3 o he i s alida ion app oach: DDX11L1, EGR1 and
MIR454. The echnical alida ion con i med he mic oa ay esul s, while when we measu ed
hem in a la ge coho , only he inc eased exp ession o EGR1 in ail subjec s was con i med.
Mo eo e , we de e mined he le els o EGR1 in 12 dono s be o e and a e a 3-mon h physical
Chap e one | 111
in e en ion s udy, and 9 o he pa icipan s educed he exp ession o EGR1. Again, no
di e ences we e epo ed o DDX11L1 and MIR454. Ou esul s indica e ha EGR1 is a
p omising bioma ke o ail y ha should be u he in es iga ed.
Indeed, EGR1 is a ansc ip ion ac o ac i a ed in esponse o a b oad ange o s imuli ha
a ec s di ec ly o indi ec ly he exp ession o mul iple signalling pa hways and umou
supp esso s, and i modula es and pa icipa es in mul iple cellula p ocesses such as mi ogen
esponse, g ow h, p oli e a ion, apop osis o di e en ia ion o se e al cell ypes [300–302].
Besides, some s udies also ound EGR1 changes o be associa ed wi h aging and age- ela ed
phenomena, like senescence o immune esponse egula ion [303–306]. Thus, he ole o EGR1
in ail y is p obably complex and depending on he issue and con ex i s a ge genes and he
elici ed unc ions may di e .
Mo eo e , as discussed be o e, he iden i ica ion o bioma ke s o ail y is challenging due o
he lack o consensus i s de ini ion and he mul iple ail y sc eening ools a ailable. Thanks o
he wo k pe o med a p ima y ca e se ices, we e had he da a o 3 di e en es s (TFI, GS
and TUG) o each o he pa icipan s in ou s udy, and a e he mic oa ay analysis and he
esul s o he PCA, we decided o ocus on he subjec s ha ob ained he same classi ica ion in
all o he es s, educing he e ogenei y. In addi ion, i should be men ioned ha despi e his,
he exp ession le els epo ed by RT-qPCR we e highly a iable. In his sense, one o he main
ad an ages o longi udinal s udies is ha he exp ession o single pa icipan s can be measu ed
o e ime and he e olu ion o each o hem de e mined. Indeed, ou esul s om he physical
in e en ion pilo s udy show ha e en i he exp ession o EGR1 was di e en , mos o he
subjec s educed he le els o EGR1 a e only 3 mon hs. This is an in e es ing poin also om
he pe spec i e o he e e sibili y o ail y. I is gene ally accep ed ha ail y is a e e sible
s a e [7,307], and he exp ession o EGR1 could be used as a bioma ke o his p ocess.
Finally, we ha e o poin ou ha he p esen s udy was he only i s s ep and we ocused on
he alida ion o 3 o he ansc ip s iden i ied. We a e now analyzing mo e candida es om
he p esen ed lis o 35 di e en ially exp essed genes be ween ou obus and ail indi iduals.
Ou aim is o con inue e alua ing he exp ession di e ences and besides, o y o unde s and
he unc ions o e ec s ha hese changes could be inducing in ail elde s.
112 | Chap e one
Chap e one | 113
Ex acellula esicles
In oduc ion
EVs a e memb ane-coa ed pa icles o endosomal o plasma memb ane o igin ha a e
sec e ed o he ex acellula en i onmen . They play an essen ial ole in indi ec in e cellula
communica ion as hei memb ane and cy osolic p o eins, lipids and gene ic ma e ial can be
ans e ed be ween cells [195]. Mo eo e , almos all cell ypes elease EVs and hey can be
isola ed om plasma and o he body luids.
EVs a e eleased bo h in physiological and pa hological condi ions and hey a e implica ed in
many cellula p ocesses. In pa icula , EVs play a ole in a ious s ages o he immune esponse
and hey ha e been ela ed o in lamma o y, au oimmune and in ec ious disease pa hology.
EVs can ca y and display an igenic ma e ial and a e able o igge an igen p esen a ion and
modula e immune esponses [192]. I has also been epo ed ha he e a e inc eased
concen a ions o EVs in plasma du ing in lamma o y p ocesses, such as in cance o
au oimmune diseases [234,242].
One o he hallma ks o human aging is he ch onic low-g ade in lamma ion, he so called
in lammaging [136], a phenomenon ha modula es in e cellula communica ion. The age-
associa ed immune dys unc ion and accumula ion o senescen cells p omo e in lamma o y
signals, such as ele a ed sec e ion o p oin lamma o y cy okines and ac i a ion o NF-κB
ansc ip ion ac o . Among in lammaging, he mos widely s udied ea u e is he ci cula ing
concen a ion o IL-6. The concen a ion o his in e leukin is no mally low (o non-de ec able)
in heal hy adul s, while ele a ed le els o IL-6 ha e been epo ed in he elde ly, wi h
inc easing concen a ions in he e y old [138,140]. Mo eo e , ele a ed IL-6 has also been
associa ed wi h mo ali y in he elde ly [142].
Despi e all his knowledge, he e a e many aspec s o in lammaging ha ha e no been
elucida ed, as he implica ion o EVs in he p ocess. In he p esen s udy, and based on he
p e iously men ioned inc ease o ci cula ing EVs du ing in lamma o y episodes, we p oposed
ha his could also be obse ed in aged indi iduals as a esul o in lammaging. Fu he mo e,
we also designed an app oach o e alua e i he concen a ion o EVs in plasma could be ela ed
o he ail y s a us o old people— ail y s a us as de ined by he Ba hel Index [308] and he
Tilbu g F ail y Index [15]. These es s a e applied o e alua e and measu e he ail y and
dependence s a us o he elde ly, which could also be ela ed o an inc eased ch onic
p oin lamma o y condi ion.
114 | Chap e one
Ma e ials and me hods
S udy pa icipan s
Samples om 19 aged indi iduals ( om Coho 1: 8 males and 11 emales, mean age 83.73
yea s) and 18 adul s – classi ied in h ee age anges: 21–30, 31–40, and 41–50 yea s (3 males
and 3 emales in each g oup) we e used. Plasma and se um samples we e ob ained as
desc ibed abo e.
EV Isola ion
EVs we e isola ed as desc ibed be o e by ou g oup [202]. B ie ly, plasma was cen i uged a
13,000 g o 2 min and supe na an cen i uged again a 20,000 g o 20 min o pelle EVs. The
pelle was esuspended wi h 100 µL o il e ed DPBS (GIBCO, The mo Fishe Scien i ic), il e ed
wice h ough a 0.22 µm-po e il e . Resuspended EVs and se um samples we e s o ed a -80
°C.
Se um IL-6 ELISA Assay
IL-6 concen a ion was analysed by ELISA (BD Biosciences) ollowing he manu ac u e ’s
ins uc ions. Samples we e measu ed in duplica e and esul s ob ained wi h a mic opla e
eade (The mo Scien i ic Appliskan, The mo Fishe Scien i ic). IL-6 concen a ions we e
calcula ed and alues abo e he i s s anda d (>4.7 pg/mL) we e conside ed de ec able.
Nanopa icle T acking Analysis (NTA)
The size dis ibu ion and concen a ion o EVs we e measu ed using a NanoSigh LM10 de ice
(Mal e n) as desc ibed elsewhe e [212]. Samples we e dilu ed o app op ia ed le els o ge
accu a e acquisi ions (200–900 eco ded acks) [212] and came a se ings we e ixed and
main ained o all samples. Fil e ed DPBS was es ed and no backg ound signal was de ec ed.
Fo each sample, wo ideos o 1 min we e eco ded and analysed wi h NanoSigh NTA
so wa e 2.2 (Mal e n). Da a a e shown as he a e age coun o he wo duplica es.
S a is ical Analysis
S a is ical analysis was pe o med wi h R e sion 3.2.2 (R Co e Team (2015) [309] in RS udio
0.99.486 (RS udio Team (2015) [309]). A Shapi o-Wilk es was applied o assess no mali y.
As samples did no ollow a no mal dis ibu ion, Wilcoxon signed- ank es and non-
pa ame ic K uskal–Wallis one-way analysis o a iance we e conduc ed o e alua e IL-6 and
EV concen a ion di e ences be ween g oups.
Chap e one | 115
Resul s
F ail y S a us Classi ica ion o Aged Indi iduals
Fo he p esen s udy, adul s o di e en age anges and elde people we e en olled.
Pa icipan s we e classi ied based on hei age. Addi ionally, aged indi iduals (79–92 yea s)
we e asked o comple e he Ba hel and Tilbu g F ail y Index ques ionnai es and we e u he
classi ied as Robus , F ail o Non-au onomous, as shown in Table 5.
Table 5. Classi ica ion o en olled indi iduals based on hei age and ail y s a us. Samples o a o al numbe
o 18 adul s and 19 elde s we e analysed.
IL-6 Concen a ion Is Inc eased in he Elde ly
The le el o IL-6 in se um was measu ed and, ob ained esul s demons a ed a e y low, nea ly
non-de ec able concen a ion in adul s o di e en ages, while an ele a ed concen a ion in he
elde ly (p<0.001) (Figu e 29A). This esul con i ms he low-g ade in lamma o y condi ion o
aged indi iduals. Addi ionally, when IL-6 le els o he elde ly we e compa ed depending on
hei ail y s a us, an inc easing endency wi h dependence was ound (Figu e 29B).
Figu e 29. An ele a ed concen a ion o IL-6 is obse ed in aged subjec s. IL-6 le els we e measu ed by
ELISA and concen a ion alues abo e 4.7 pg/mL we e conside ed de ec able. (A) Elde ly indi iduals
ha e a highe concen a ion o IL-6 han adul s (***p<0.001); and (B) he e is a high a iabili y among
Robus , F ail and Non-au onomous elde ly, bu an inc easing concen a ion wi h dependency can be
obse ed.
116 | Chap e one
The Concen a ion o EVs Is No A ec ed by Age and F ail y S a us
To assess he size p o ile and concen a ion o ci cula ing EVs, NTA was conduc ed o all
samples. Resul s showed ha , ega dless o pa icle concen a ion, all samples ollowed a
simila EV size dis ibu ion, wi h mos esicles anging be ween 50 and 300 nm in all ins ances
(Figu e 30A). This esul demons a ed ha ou EV isola ion p o ocol e icien ly isola es small
EVs, emo ing la ge pa icles and pla ele s ha can be ound in plasma samples. When
compa ing he EV numbe , no signi ican di e ences we e ound be ween g oups (p = 0.505),
indica ing ha EV concen a ion is no inc eased wi h age (Figu e 30B). Mo eo e , he
concen a ion o EVs is also no a ec ed by he ail y s a us o elde dono s (p = 0.424), as
shown in Figu e 30C.
Figu e 30. Pa icle size and EV concen a ion we e measu ed by NTA. (A) Size dis ibu ion o EVs. Each
line ep esen s one sample. Despi e he pa icle concen a ion di e ence, all samples ha e a simila size
dis ibu ion— hey a e en iched in small EVs (50–300 nm); (B) EV concen a ion o di e en age anges
we e compa ed and samples om elde people (79–92 yea s) do no show an inc eased EV numbe ; and
(C) among elde indi iduals, he ail y s a us does also no al e EV concen a ion.
Chap e one | 117
Discussion
Du ing human aging, a ch onic low-g ade in lamma o y s a e called in lammaging has been
epo ed [138,140,142], and o ou knowledge, his is he i s epo in es iga ing, speci ically,
EV concen a ion in his p ocess. The esul s p esen ed in his wo k demons a e ha he e
a e ele a ed IL-6 le els in he elde ly, con i ming he basal in lammaging. In con as o wha
we hypo hesized, and despi e in lammaging, EV concen a ion in ci cula ion is no a ec ed by
human aging. Mo eo e , ail y o dependence did also no al e he EV numbe . Many au ho s
ha e p e iously s udied he implica ion o EVs in di e se in lamma o y p ocesses, including
cellula senescence, neu odegene a i e diseases and cance , indica ing ha bo h he o al
numbe o EVs in ci cula ion and also EVs om speci ic cell o igins can be inc eased
[192,230,310]. Ou esul s p esen a ch onic in lamma o y p ocess—in lammaging—in which
ci cula ing EV le els a e no a ec ed. In his wo k, and when s udying EVs, he e a e se e al
ac o s ha should be aken in o conside a ion.
In ou coho , a high in e -indi idual EV concen a ion a iabili y has been ound wi hin he
same g oup. Simila ly, p e ious expe imen s ha e demons a ed ha he p o ein
concen a ion and con en o EVs di e depending on he dono [311]. On he o he hand,
speci ic medica ions may also a ec EVs, as he e a e compounds ha can modi y EV
p oduc ion and elease. Fo ins ance, immunomodula o y ea men s can a ec EV p oduc ion
by immune cells and modula e EV concen a ion in ci cula ion [241,253,312]. This kind o
e ec s should be conside ed when measu ing EV le els specially in aged people, because
nea ly all o hem ha e ch onic medica ions. In his s udy, a ep esen a i e sample o
communi y-dwelling aged people was analysed and, as expec ed, all we e unde ch onic
ea men . I was e hically no possible o ask he pa icipan s in he s udy o in e up hei
medica ions. Fu he mo e, he aim o ou s udy was o e alua e whe he he low-g ade
p oin lamma o y s a us was su icien o al e EV concen a ion in he elde ly, despi e hei
medica ion. Mo eo e , e en i he o al numbe o EVs is no al e ed, EVs sec e ed om speci ic
cell ypes could be a ec ed, bo h in hei concen a ion and ca go, modula ing hei unc ion
and e ec in a ge cells, as desc ibed o o he biological p ocesses [230]. Finally, he limi ed
numbe o samples in he s udy mus be aken in o accoun and esul s should be alida ed in
a la ge coho . In b ie , hese esul s ep esen a i s epo and demons a e ha he e is no
co ela ion be ween in lammaging and EV concen a ion in ci cula ion. Mo e ex ensi e
expe imen s a e equi ed o s udy he speci ic changes ha occu o EVs in ega d o human
aging, and o u he elucida e hei ole in he p ocess.
124 | Chap e wo
Ma e ials and me hods
Ob en ion and isola ion o plasma EVs
Pe iphe al blood was collec ed by expe ienced nu ses by enipunc u e wi h a 21-gage needle
in 4 ml EDTA ubes (Vacu aine , BD Biosciences). Samples om 5 adul s (mean age 37.8 yea s,
2 emales and 3 males) and 5 elde s (mean age 85.4 yea s, 3 emales and 2 males) we e
ob ained. Tubes we e kep up igh and cen i uged a 1258 g o 20 min o eco e plasma. To
isola e EVs, plasma was cen i uged a 13,000 g o 2 min and ob ained supe na an (1 ml
pla ele ee plasma) was cen i uged again a 20,000 g o 20 min o pelle EVs. 900 l o
supe na an we e ans e ed o ano he ube and he bo om 100 l wi h he EV pelle we e
esuspended wi h 100 µl o il e ed DPBS (GIBCO, The mo Fishe Scien i ic, il e ed wice
h ough a 0.22 µm-po e il e ). The 200 l o esuspended EVs we e s o ed a -80 °C and
hawed on ice when needed.
Ob en ion and cul u e o ASCs
Abdominal liposuc ion was pe o med in a emale dono aged 49 yea s. Wi h he pa ien ’s
in o med consen , subcu aneous adipose issue was ob ained by ou pa ien umescence
liposuc ion unde local anes hesia by an expe ienced physician. ASCs we e isola ed acco ding
o Wolbank e al. [318] and cul u ed in con ol medium consis ing o DMEM-low
glucose/HAM´s F-12 (GE-Heal hca e) supplemen ed wi h 4mM L-glu amine (Sigma-Ald ich)
and 10% e al cal se um (Sigma-Ald ich) a 37°C, 5% CO2 and 95% ai humidi y. Cul u e
medium was changed h ee imes a week and cells we e passaged once a week a a spli a io
o 1:2 o 1:6 acco ding o he g ow h cha ac e is ics.
Cocul u e o ASCs wi h plasma EVs and induc ion o os eogenic di e en ia ion
ASCs we e seeded in 24-well cul u e dish wells. 3 days a e seeding, os eogenic di e en ia ion
was induced by swi ching he medium o os eogenic di e en ia ion medium consis ing o
DMEM-low glucose (GE-Heal hca e), 10% e al cal se um (Sigma-Ald ich), 4mM L-glu amine
(Sigma-Ald ich), 10nM dexame hasone (Sigma-Ald ich), 150μM asco ba e-2-phospha e
(Sigma-Ald ich), 10mM β-glyce olphospha e (Sigma-Ald ich) and 10nM 1.25
Dihyd oxy i amine D3 (Sigma-Ald ich) in a inal olume o 1 ml/well.
Be o e pe o ming cocul u e expe imen s o in es iga e he e ec o EVs, cul u e condi ions
we e op imized. Th ee di e en cell concen a ions we e assayed (4,000/9,000/14,000) and
14,000 ASC cells/well was chosen as he bes o os eogenic di e en ia ion measu emen .
Rega ding os eogenic di e en ia ion du a ion, 10 and 17 days we e es ed, and 10 days we e
chosen o subsequen expe imen s. When in es iga ing he e ec o EVs, 4 IU hepa in/ml we e
Chap e wo | 125
added o p e en cul u e medium jelli ica ion. Taken oge he , he inal expe imen al se up o
es he e ec o plasma EVs on os eogenic di e en ia ion is p esen ed below and schema ically
ep esen ed in Figu e 31. Besides, all he expe imen s pe o med and ob ained esul s o he
op imiza ion o cul u e condi ions a e p esen ed in he esul s sec ion.
▪ 14,000 ASCs/well we e seeded in 24-well cul u e dishes wi h 950 l con ol medium
supplemen ed wi h 4 IU hepa in.
▪ 3 hou s a e seeding he cells, 50 l o EVs (o 50 l o DPBS) we e added o
co esponding wells, eaching a inal olume o 1 ml/well, and ca e ully mixed o
ensu e a homogeneous EV dis ibu ion.
▪ 3 days a e seeding he cells, he cul u e medium was changed. Os eogenic
di e en ia ion (OD) medium o con ol medium was added o co esponding wells.
▪ Cells we e main ained in cul u e o 10 days mo e wi h media changes e e y 3 days.
▪ Aliza in Red o Alkaline phospha ase s aining was pe o med o measu e os eogenesis.
Figu e 31. Cell cul u e condi ions o es he e ec o EVs om plasma on os eogenic di e en ia ion o
ASCs. (A) Schema ic ep esen a ion o he cul u e p o ocol. ASCs we e seeded in 24-well cul u e dishes
in 950 l con ol medium and 3 hou s la e , when cells we e a ached 50 l o EVs o DPBS we e added.
69 hou s la e (3 days a e seeding) cul u e media we e changed, swi ched o OD medium o o esh
con ol medium in co esponding wells. Cells we e main ained in cul u e o 10 days mo e (13 days a e
seeding) wi h media changes e e y h ee days. Finally, os eogenic di e en ia ion was e alua ed by
Aliza in Red o Alkaline phospha ase (ALP) s aining. (B) Schema ic ep esen a ion o he 6 di e en
s udy condi ions. EV samples om 5 adul s and 5 elde s we e es ed, and all condi ions we e assayed in
duplica e.
126 | Chap e wo
Aliza in Red s aining
Fo quan i ica ion o calci ied s uc u es, cells we e washed 3 imes wi h PBS and hen, ixed
o 2 h in 70% e hanol (500 l/well) a -20°C. Then, cells we e washed 3 imes wi h dH2O and
s ained o 10 minu es wi h 40mM Aliza in Red S solu ion (pH 4.2, 500 l/well, Sigma-Ald ich)
in an o bi al shake a oom empe a u e. Subsequen ly, he emaining dye was emo ed by
insing he cells wi h PBS. Finally, he esidual dye was ex ac ed by 0.1M HCL/0.5% SDS
solu ion (200 l/well) o 30 min. The dye signal was quan i ied in a mic opla e eade by
de e mining he abso bance a 425 nm.
Alkaline phospha ase (ALP) s aining
To de e mine he ac i i y o ALP, cells we e washed 3 imes wi h PBS and hen, lysed by
incuba ing wi h lysis bu e o 1 h (0.25% T i on X-100, 100 l/well) a oom empe a u e.
Nex , samples we e ans e ed o 1.5 ml ubes, cen i uged a 13,000 pm 10 min a 4°C and
90 l o supe na an ans e ed o a new ube. Subsequen ly, a bu e con aining 20mM 4-
ni ophenyl phospha e disodium sal hexahyd a e, 0.5 M 2-amino-2-me hyl-1-p opanol and
0.2 mM MgCl2 (pH 10.3, 50 l/ ube) was added o each cell lysa e and incuba ed o 20 minu es
a oom empe a u e in he da k. The eac ion was s opped by adding 50 µl o 0.2M NaOH and
ALP ac i i y quan i ied by de e mining he abso bance a 405nm (620nm e ).
S a is ical analysis
S a is ically signi ican di e ences be ween he s udy g oups we e es ed wi h G aphPad P ism
e sion 6.01 o Windows (G aphPad So wa e, www.g aphpad.com). Mann-Whi ney es was
applied o e alua e di e ences be ween EVs om adul s and elde s. **p<0.01.
Resul s
Os eogenic di e en ia ion se ings
The i s expe imen s we e di ec ed o es ablish he bes cul u e condi ions o in es iga e he
e ec o EVs on os eogenesis. To his end, we es ed 3 cell densi ies (4,000/9,000/14,000 ASCs)
and wo di e en end poin s (10/17 days a e os eogenic di e en ia ion induc ion).
T iplica es we e pe o med o all he condi ions.
Calci ica ion was measu ed by Aliza in Red s aining and he ob ained esul s a e p esen ed in
Figu e 32. We con i med he induc ion o os eogenesis in all he es ed condi ions, as calcium
deposi ion was highe unde OD medium han unde con ol medium (basal calcium
deposi ion by ASCs). Mo eo e , a highe concen a ion o cul u ed cells esul ed in an
inc eased s aining and a highe di e ence be ween con ol and OD medium. Rega ding he wo
es ed end poin s, an ele a ed di e en ia ion was shown wi h he p olonga ion o os eogenesis
(17 days).
Chap e wo | 127
Wi h hese esul s, we decided o pe o m he ollowing expe imen s wi h 14,000 ASCs/well
and o measu e os eogenesis 10 days a e he induc ion. We based ou decision abou he cell
densi y on he p ominen di e ences obse ed be ween con ol and OD medium. On he o he
hand, o he os eogenesis du a ion, we ook in o conside a ion he possible e ec s o plasma
EVs: p e ious s udies ha e epo ed an enhanced di e en ia ion in he p esence o EVs, so o
be able o see he in luence o EVs, we should measu e os eogenesis a an in e media e poin
when an inc ease o calci ica ion o ALP ac i i y could be epo ed by he abso bance
measu emen .
Figu e 32. Os eogenic di e en ia ion se ings assessed by Aliza in Red s aining. 3 di e en cell
densi ies we e seeded and calcium deposi ion measu ed 10 days (A) o 17 days (B) a e os eogenic
di e en ia ion induc ion. Wells wi h con ol medium in which os eogenesis was no induced we e
main ained and assayed o measu e basal calcium deposi ion by ASCs. Inc easing s aining was obse ed
wi h mo e cells and wi h p olonged cul u e imes.
Cocul u e o ASCs wi h plasma EVs esul s in he o ma ion o jelly s uc u es
A e es ablishing he cell cul u e condi ions o ASCs, in he nex s ep we pe o med he i s
expe imen s in which EVs om plasma we e added o ASCs. Cells we e seeded in 24-well
dishes and 3 hou s a e seeding he cells, 50 l o hawed EVs we e added o each well. 3 days
a e seeding he cells, cul u e media we e changed and con ol o OD medium added o
co esponding wells. Unexpec edly, a he bo om o he wells whe e ASCs and EVs we e
cocul u ed a jelly laye had o med. Medium change was done by ca e ully pipe ing, bu e en
so, when aspi a ing he medium, he jelly laye was pa ially de ached in some wells. We s ill
decided o con inue wi h he os eogenesis p o ocol and y o measu e he e ec o EVs on
calcium deposi ion.
10 days a e os eogenic di e en ia ion induc ion, Aliza in Red s aining was pe o med.
Despi e he ca e ul pipe ing, we obse ed ha he jelly-like laye s we e de ached and i was
no possible o inse he wells a e Aliza in s aining (Figu e 33), so no esul was ob ained
om his assay.
128 | Chap e wo
Figu e 33. Fo ma ion o jelly s uc u es and cell de achmen . ASCs we e cocul u ed wi h EVs isola ed
om plasma and a jelly laye had o med a he bo om o he wells. (A) Rep esen a i e image o a well
whe e he jelly laye was de ached and lipped on op o o he cells. (B) A pic u e aken om 2 wells
cocul u ed wi h EVs. Aliza in Red s aining was pe o med, bu no concluding esul s could be ob ained.
A e obse ing his phenomenon, we pe o med an expe imen o in es iga e he o ma ion
o jelly s uc u es and whe he hepa in could p e en hem in ou cocul u es wi h plasma EVs.
We p epa ed a 24-well dish as shown in Figu e 34: EVs alone, ASCs + EVs, EVs in hepa in
con aining medium and ASCs + EVs in hepa in con aining medium we e es ed. In all cases,
14,000 ASCs and 4 U o hepa in/ml we e used, while di e en EV olumes we e added.
Figu e 34. Schema ic ep esen a ion o he 24-well cul u e dish plan. In ow A con ol medium and EVs
we e mixed, while in ow B 14,000 ASCs/well we e also added. In ow C con ol medium wi h 4 IU
hepa in and EVs we e mixed, while in ow D 14,000 ASCs/well we e also added.
3 days la e he o ma ion o gels was e alua ed. Cul u e medium was ca e ully pipe ed and
we obse ed no jelly laye s in ow A and C, con i ming ha he in e ac ion be ween con ol
medium and EVs do no p oduce he jelly s uc u es. When medium in ow B was aspi a ed,
jelly laye s we e ound in all wells, wi h hicke s uc u es in he wells cocul u ed wi h highe
olumes o EVs. In con as , medium pipe ing was pe o med no mally in ow D,
demons a ing ha he addi ion o 4 IU hepa in/ml p e en s he o ma ion o he gel o all he
EV olumes es ed. Consequen ly, we decided o inco po a e hepa in o he con ol medium
o cocul u e expe imen s.
Chap e wo | 129
E ec o plasma EVs on os eogenesis
Finally, we es ed he e ec o EVs isola ed om plasma o adul and elde dono s on os eogenic
di e en ia ion. EV samples om 5 adul s and 5 elde s we e assayed, each o hem in duplica e.
Ou esul s show ha , in all cases, he cocul u e o ASCs wi h plasma EVs enhance os eogenesis,
and his e ec is s onge wi h EVs om adul s (Figu e 35).
Figu e 35. Os eogenesis enhancemen by plasma EVs. ASCs we e cocul u ed wi h EVs o 3 days and
hen, os eogenic di e en ia ion was induced. 10 days a e induc ion ALP ac i i y was measu ed. Resul s
a e p esen ed in old change e sus he con ol condi ion in which no EVs we e cocul u ed. (A) G aph
showing he esul s ob ained o each EV dono . In all cases, ALP ac i i y was highe han he con ol.
(B) Box plo ep esen a ion o esul s ob ained o EVs om adul and elde dono s. EVs om adul s
enhance ALP ac i i y signi ican ly mo e han EVs om elde s. A = adul and E = elde .
Besides, we also es ed whe he EVs alone we e able o induce os eogenesis. To his end, we
cul u ed ASCs in con ol medium. The cocul u e wi h EVs was pe o med as be o e, bu ins ead
o inducing os eogenesis wi h OD medium, con ol medium was main ained. The ob ained
esul s demons a e ha he p esence o EVs alone does no induce os eogenesis (Figu e 36).
Figu e 36. E ec o EVs unde con ol medium. ASCs we e cocul u ed wi h EVs o 3 days and hen,
cul u ed o 10 days mo e wi h con ol medium. Two wells wi hou EVs we e cul u ed wi h OD medium
o 10 days as a posi i e con ol o os eogenesis. ALP ac i i y was measu ed and no di e ences we e
epo ed, indica ing ha EVs alone do no induce os eogenic di e en ia ion o ASCs. A=adul and
E=elde .
130 | Chap e wo
Discussion
In his p ojec , we ha e se he cell cul u e condi ions and in es iga ed he e ec o EVs om
plasma on he os eogenic di e en ia ion o ASCs. The s a ing poin o his wo k was based on
a publica ion by he g oup o G illa i [245], in which hey epo ed an inc eased os eogenesis
enhancemen wi h EVs isola ed om plasma o young dono s (less han 25 yea s) when
compa ed o olde ones (mo e han 55 yea s). Besides, hey ocused on galec in-3 p o ein and
showed he impo an ole o his molecule du ing he os eogenic p ocess. Rega ding he EV
expe imen s pe o med by he g oup, o isola e EVs om plasma hey il e ed he samples
h ough 0.22 m po e il e s and hen applied ul acen i uga ion a 100,000 g o 1 hou . Wi h
his p o ocol, hey isola ed small EVs and disca ded bigge EVs. Mo eo e , when conside ing
po en ial u u e applica ions, hei p o ocol could be di icul o implemen in he clinic, as mos
o he hospi als do no ha e ul acen i uges. Taken oge he , he main objec i es o ou wo k
we e o es he e ec o EVs isola ed wi h an easily applicable p o ocol [202] and o compa e
he e ec o samples coming om adul and elde dono s.
Fi s , we ob ained ASCs om a heal hy dono and conduc ed expe imen s o es ablish he
app op ia e cell cul u e se ings. In ou hands, seeding 14,000 cells/well and main aining
os eogenic induc ion o 10 days esul ed in adequa e di e en ia ion. When adding EVs o
cul u ed ASCs, a jelly laye was o med. The addi ion o only 5 l o plasma EVs was enough o
induce he o ma ion o his s uc u e. On he o he hand, a cul u e medium p epa ed wi h 4
IU hepa in/ml p e en ed jelli ica ion. The use o human plasma o cell cul u e has been widely
in es iga ed be o e, and medium clo ing was also epo ed in many cases [319]. He e we
applied EVs isola ed om plasma, and e en i we ha e demons a ed ha ou p o ocol
e icien ly isola es EVs, when handling a complex luid as plasma, o he small componen s a e
p obably cop ecipi a ed. In acco dance wi h p e ious epo s, he addi ion o a low
concen a ion o hepa in o ou cul u es p e en ed media clo ing. I should be men ioned ha
some au ho s epo ed impai ed cell p oli e a ion and di e en ia ion o ASCs unde high doses
o hepa in [320], bu we applied only 4 IU hepa in/ml o 3 days and hen, os eogenic
di e en ia ion was induced and no issues we e epo ed.
We pe o med cocul u e expe imen s wi h EVs om 10 di e en dono s: 5 adul s and 5 elde s.
In e es ingly, all EV samples boos ed os eogenic di e en ia ion, bu none o hem induced
di e en ia ion i OD medium was no used, demons a ing ha plasma EVs alone do no induce
he di e en ia ion o ASCs o os eoblas s. Mo eo e , he posi i e e ec o EVs was mo e
p ominen when samples om adul s we e applied. These esul s indica e ha EVs a ou
os eogenesis, bu an age- ela ed exhaus ion could be p esen . Ou esul s a e in acco dance
wi h he p e ious s udy by he g oup o G illa i, and impo an ly, we es ed mo e samples,
Chap e wo | 131
coming also om olde dono s (> 80 yea s, ins ead > 55 yea s), and wi h EVs isola ed wi h a
di e en p o ocol ( inal pelle ing a 20,000 g, ins ead 100,000 g).
This wo k ein o ces p e ious s udies and demons a e he po en ial applica ion o EVs o
os eogenesis enhancemen . Fu he mo e, o he au ho s showed ha EVs sec e ed by ASCs
[260,313], o by a speci ic subse o plasma EVs p omo e os eogenesis [245], bu hei
applica ion would need in i o cul u es o ASCs o p oduce EVs o complica ed p o ocols o
isola e EVs om plasma, espec i ely. In con as , ou esul s indica e ha EVs easily isola ed
om plasma, alone o in combina ion wi h cell he apies, could help os eogenesis.
Fu he mo e, o he au ho s ha applied PRP and ASCs o os eogenesis wi h posi i e esul s
we e p obably also adminis e ing EVs, as hey would be p esen in PRP samples [316,317].
Rega ding he possible clinical applica ions o p omo e os eogenesis, i should be men ioned
ha , simila o ou esul s wi h EVs, ASCs om elde s ha e a educed os eogenic po en ial
[125]. We hypo hesize ha o o e come he age-associa ed dys unc ion and o a oid allogeneic
cell ansplan a ions, egene a i e he apies in aged pa ien s could be ca ied ou wi h
au ologous ASCs and plasma EVs om a young dono . This combina ion could boos
os eogenesis while p e en ing po en ial p oblems associa ed wi h allogeneic cells.
In summa y, ASCs a e an easily accessible sou ce o MSCs and hey can be di e en ia ed in o
di e en cell ypes, including os eoblas s. Due o hei he apeu ic po en ial, many e o s a e
being made o unde s and he unde lying mechanisms o os eogenesis [321]. In pa allel, he
implica ion o EVs in os eogenic di e en ia ion is s ill s a ing o be in es iga ed. The i s
epo s, including ou s, indica e ha EVs play an impo an ole and help ASC di e en ia ion,
and we conside ha hey should be aken in o conside a ion o u u e clinical applica ions.
132 | Chap e wo
Chap e wo | 133
Myogenesis
In oduc ion
The skele al muscle is he la ges o gan in he human body. I is a highly adap able issue ha
esponds o en i onmen al condi ions and physiological challenges by changing ib e size and
composi ion. Howe e , he incidence o skele al muscle inju ies as a consequence o auma,
inhe i ed gene ic diseases, pa hology o aging is e y high and ep esen s ele an socio-
economic cos s. In he case o aging, muscle was ing, de ined by ma ked muscle mass loss and
weakening, is one o he majo p oblems leading o inc eased isk o alls and de elopmen o
physical disabili y [123,322].
I is well known ha he skele al muscle has egene a i e po en ial, which, howe e , becomes
comp omised in he case o se e e o ex ended damage as well as wi h aging [123,126]. In his
con ex , se e al me hods a e nowadays applied in he clinic o p omo e muscle epai and
egene a ion and, besides, many in es iga ions a e being conduc ed o imp o e he p esen
echniques o implemen new and mo e e ec i e me hods [126]. Among hese po en ial new
me hods, we a e specially in e es ed in he ones in es iga ing he ole o EVs in myogenesis.
Indeed, in he las yea s, many au ho s ha e in es iga ed he composi ion and unc ions o EVs
sec e ed by myoblas s and myo ubes [323]. Rega ding hei unc ions, a wo k by Fo e e e
al. epo ed ha EVs sec e ed by myo ubes educe p oli e a ion and induce myoblas
di e en ia ion [324], while a s udy by Guescini e al. did no ob ain he same esul s [325].
Howe e , he EV concen a ions and di e en ia ion endpoin we e dis inc , which could
accoun o he obse ed di e ences. Besides, i should be men ioned ha mos o he wo ks
ocusing on he e ec s o EVs on myogenesis we e ca ied ou in he C2C12 immo alized
mouse myoblas line, which is an in e es ing and use ul model o s udying many p ocesses,
bu i also p esen s some di e ences when compa ed o humans.
To ou knowledge, no wo ks ha e s udied he in luence o EVs om human plasma on
myogenesis. Ne e heless, many esea che s ha e indi ec ly applied EVs in hei
in es iga ions, as EVs a e pa o he componen s o PRP and pla ele -poo plasma (PPP). These
p epa a ions ha e been widely es ed o muscle egene a ion, as nicely e iewed by Chellini
and colleagues [326]. In any case, hey did no conside he p esence o EVs and hei e ec s as
pa o PRP and PPP a e s ill unknown.
140 | Chap e wo
We sa is ac o ily isola ed RNA om all samples and pe o med cDNA syn hesis and qPCR as
usual. Howe e , we did no ob ain cDNA ampli ica ion o some o he samples, speci ically o
he ones co esponding o con ols and o EVs om adul s unde di e en ia ion medium.
Consequen ly, no esul s could be ob ained om hese samples and, besides, he samples
cocul u ed wi h EVs om elde s wi h di e en ia ion medium we e also no analysed, as he e
was no any con ol o compa e hem wi h. The esul s om he es o he condi ions, which
included all he samples main ained in p oli e a ion medium, we e analysed and he esul s
a e shown in Figu e 42. Simila o he esul s ob ained in he i s expe imen , we saw ha
myoblas cocul u ed wi h EVs om adul s ha e ele a ed le els o myogenic di e en ia ion
ma ke s when compa ed o EVs om elde s (signi ican o MYOD1 and endency o MYOG
and DES). In con as , in his second expe imen , we did no obse e a signi ican inc ease o
he myogenic ma ke s be ween he con ol wells and he ones cocul u ed wi h EVs.
Figu e 42. Exp ession o myogenic di e en ia ion ma ke s. Myoblas s we e main ained in p oli e a ion
medium and hey we e cocul u ed wi h EVs om adul o elde dono s. The samples wi h EVs om
adul s showed a highe exp ession o MYOD1, and he same endency o MYOG and DES, while hese
wo did no each s a is ical signi icance.
Discussion
The objec i e o his sec ion was o in es iga e he e ec o EVs isola ed om plasma on
myogenesis. This is a ield ha has no been in es iga ed, bu i could ha e po en ial bene i s
o muscle egene a ion, which is o pa icula in e es o he age-associa ed sa copenia. We
decided o pe o m he expe imen s based on ou p e ious esul s on os eogenesis, as well as
on he li e a u e abou myogenic di e en ia ion. In e es ingly, a wo k by Nakamu a e al.
s udied he e ec s o EVs sec e ed by MSCs [259] and, on he o he hand, many in es iga ions
ha e e alua ed he e ec o PRP and PPP ( ha con ain EVs) on myogenesis [326]. Howe e ,
none o hem speci ically e alua ed he po en ial ole o EVs om plasma, which we conside
ha could also be playing a ole.
Chap e wo | 141
Wi h his in mind, we collabo a ed wi h a g oup om Biodonos ia ha wo ks wi h p ima y
myoblas s and pe o med wo expe imen s wi h di e en se ups. We es ed di e en cell
concen a ions, cocul u e imes and endpoin s. As in ou p e ious expe imen s, we e alua ed
he e ec o EVs no only unde di e en ia ion medium, bu also unde p oli e a ion medium,
o in es iga e whe he EVs could in luence in bo h condi ions. No ably, in he i s expe imen ,
we epo ed an ele a ed exp ession o MYOG, MYOD1 and DES myogenic di e en ia ion
ma ke s unde p oli e a ion and di e en ia ion medium. Besides, when EVs isola ed om
adul and elde dono s we e compa ed in ou wo expe imen s, a highe exp ession o he
di e en ia ion ma ke s was obse ed wi h EVs om adul s, wi h s a is ically signi ican
di e ences o MYOD1 in he second expe imen .
Ou esul s a e a i s epo indica ing ha EVs om plasma could play a ole in myogenesis
and, mo eo e , ha EVs om adul s could ha e a mo e obus e ec han he ones isola ed
om elde s. Fu he , we pe o med he expe imen s on human p ima y myoblas s, while mos
wo ks a e pe o med in mu ine immo alized myoblas s [323–325]. To ou knowledge, one
publica ion in es iga ed be o e he in luence o EVs on myogenesis wi h human p ima y
myoblas s, bu hey had a comple ely di e en objec i e, as hey s udied he e ec o EVs
p esen on he oe al bo ine se um used o cell cul u e [328].
In any case, ega ding he e ec s o EVs, we should keep in mind he complexi y o biological
p ocesses and he limi a ions o he sys ems ha we and all esea che s apply. Fo ins ance,
we y o model and s udy myogenic di e en ia ion in i o, by pla ing myoblas s, cocul u ing
hem wi h EVs om plasma and e alua ing he exp ession o ce ain genes. We conside ha
his is a good app oach o in es iga e whe he EVs ha e an e ec on he p ocess, which is a
no el ield ha is s ill in i s in ancy. Howe e , we do no eplica e he mic oen i onmen
p esen when a skele al muscle o an indi idual is egene a ing, and we would p obably ne e
be able o ep oduce i exac ly. Wi h ega d o he po en ial use EVs, he ad an age o EVs wi h
espec o cells is ha we can i s e alua e hei e ec s in in i o models, and hen, es hei
po en ial e icacy in i o wi h less sa e y conce ns.
As discussed in he p e ious sec ion o os eogenesis, we hypo hesize ha plasma EVs could
be applied in he u u e alone o in combina ion wi h au ologous s em cells o enhance
myogenesis. Howe e , and e en i some ea men s wi h PRP ha e al eady been applied o
pa ien s wi h inju ed skele al muscles wi h posi i e esul s [326], we a e s ill a om
unde s anding he implica ions o EVs. Ou app oach was jus he i s s ep and many mo e
should be aken o desc ibe he oles o plasma EVs on myogenesis and whe he aging a ec s
hem.
CHAPTER THREE
Immunosenescence and he
ole o ex acellula esicles
Chap e h ee | 145
In oduc ion
Human aging is a complex and he e ogenic p ocess, in which se e al cellula mechanisms a e
a ec ed and modula ed, leading o unc ional decline [33]. One o he mos de e mining
consequences o aging is he dys unc ion o he immune sys em, and he subsequen poo
esponse o accina ion, inc eased suscep ibili y o in ec ions and age- ela ed diseases
obse ed in he elde ly [329].
The molecula and cellula changes ha lead o immune dys unc ion ha e been ex ensi ely
in es iga ed and a e gene ally e e ed o as immunosenescence [92]. T cells a e he mos
d ama ically a ec ed immune componen s, wi h a dec ease in naï e T cells and an
accumula ion o e minally di e en ia ed T cells wi h age. Te minally di e en ia ed T cells
exhibi ea u es o eplica i e senescence and lose he exp ession o he cos imula o y
molecule CD28 om hei memb ane [98–101,330]. CD28 plays an essen ial ole in T cell
unc ion, aking pa in ac i a ion, p oli e a ion and su i al p ocesses. Hence, CD28 nega i e
T cells p esen al e ed molecula ea u es, as well as dis inc cy okine p oduc ion and e ec o
molecules [102]. The loss o CD28 a ec s ea lie and p ima ily CD8 T cells, bu i has also been
desc ibed o each CD4 T cells la e in li e [103,104]. In consequence, T lymphocy es ha e a
educed capaci y o eac agains new s imuli, con ibu ing o he a o emen ioned immune
dys unc ion. Ano he ea u e ound in immunosenescen T cells is he enhanced cy o oxici y.
Exp ession o NK cell cha ac e is ic ecep o s such as CD56 and CD57 memb ane molecules
ha e been widely epo ed in hese cells, which p omo e hei cy o oxic capaci y [105–108].
Addi ionally, many au ho s ha e ound a highe p e alence o an in e ed CD4/CD8 a io in
he elde ly, a ea u e known as immune isk pheno ype, ha p edic s sho e su i al [113–
115].
The immunosenescen p ocess and he changes ha occu in o he cell ypes du ing aging
esul in an al e ed sec e ion o molecules by cells, e med SASP [131]. The SASP componen s
ha e been classically di ided in o h ee g oups: i) soluble signalling ac o s (ILs, chemokines,
and g ow h ac o s), ii) sec e ed p o eases, and iii) sec e ed insoluble p o eins/ex acellula
ma ix componen s [132]. One o he consequences o SASP is he ch onic low-g ade
in lamma ion seen in he elde ly, he so called in lammaging [136]. The age-associa ed immune
dys unc ion and accumula ion o senescen cells p omo e in lamma o y signals, such as
ele a ed sec e ion o p oin lamma o y cy okines like IL-6 [138,140,331]. Ano he ema kable
aspec ha is a ec ed by he SASP is in e cellula communica ion. Apa om he h ee
classical SASP componen s men ioned be o e, in he las decades EVs ha e been shown o play
a cen al ole in in e cellula communica ion and immune sys em unc ion [234].
146 | Chap e h ee
EVs a e memb ane-coa ed pa icles ha a e sec e ed by almos all cell ypes and a e p esen
in mos body luids, including plasma. They can be o endosomal o plasma memb ane o igin
and hey ca y p o eins, lipids and gene ic ma e ial ha can be inco po a ed by he a ge cell.
EVs a e eleased in physiologic and pa hologic condi ions and a e implica ed in many cellula
p ocesses [195]. As s a ed be o e, EVs a e also implica ed in he immune sys em unc ion, as
hey can ca y an igenic ma e ial and modula e immune esponses [234].
Rega ding EVs in aging and senescence, he exp ession o p53 ansc ip ion ac o ha e been
ela ed o inc eased EV p oduc ion [332]. Howe e , some wo ks ha e s udied he
concen a ion o plasma EVs wi h age, wi h con adic o y esul s [331,333]. One o hese wo ks
also examined he EV p o ein ca go and in e naliza ion by immune cells and showed p o eins
di e en ially exp essed wi h age and ha EVs om olde dono s a e mo e eadily in e nalized
by B cells [333].
In spi e o ha , he e a e s ill many aspec s o EVs ha ha e no been elucida ed. Simila ly, e en
i immunosenescence a a cellula le el has been widely in es iga ed, only a ew wo ks ha e
analysed samples om nonagena ians and cen ena ians [98,334]. I is impo an o no e ha
only a small pe cen age o people each hese ad anced ages, making i e en mo e di icul o
include hei samples in s udy coho s. Wo ks ha s udied nonagena ians and cen ena ians
showed ha hei PBMCs ha e dis inc ea u es a ansc ip ional and unc ional le els when
compa ed o sep uagena ians and oc ogena ians [26,27,335].
Taking all his in o accoun , he aims o he p esen wo k we e o cha ac e ize he
immunosenescence s a us o ou coho (dono s o 20-49 and 70-104 yea s), compa ing
di e en age anges a he cellula and EV le el and o y o desc ibe he possible immune
unc ions o plasma EVs.
Ma e ials and me hods
Pa icipan s and blood sampling
Fo he p esen s udy, dono s o di e en age anges we e en olled. Heal hy adul s be ween
20-49 yea s and elde s o 70-104 yea s we e included. Elde s we e assessed a p ima y ca e
se ices and by an expe ienced neu ologis . Bo h communi y-dwelling and ins i u ionalized
pa icipan s and wi h dis inc unc ional capaci ies we e en olled, aiming o ha e a
ep esen a i e sample o age- ela ed he e ogenei y. All pa icipan s comple ed a ques ionnai e
and dono s wi h acu e illness o immunological diso de s we e excluded. Samples om 51
dono s (29 emales and 22 males), 18 heal hy adul s and 33 aged indi iduals we e collec ed a
Donos ia Uni e si y Hospi al. Pa icipan s we e classi ied based on hei age ange: 20-29
(n=6), 30-39 (n=5), 40-49 (n=7), 70-79 (n=6), 80-89 (n=10), 90-99 (n=13) and ≥100 (n=4)
Chap e h ee | 147
yea s. The s udy was app o ed by he hospi al’s e hics commi ee and all pa icipan s p o ided
w i en in o med consen be o e blood sampling.
Pe iphe al blood was collec ed by enipunc u e wi h a 21-gage needle. The i s millili e was
disca ded and hen blood collec ed in a 2.8 ml ci a e ube and 4 hepa in ubes o 4 ml
(Vacu aine , BD Biosciences).
PBMC isola ion and s o age
Wi hin 1 hou o sampling, pe iphe al blood collec ed in hepa in ubes (16 ml) was p ocessed.
PBMCs we e isola ed by densi y g adien cen i uga ion wi h Lymphop epTM (Abbo ),
ollowing he manu ac u e ’s ins uc ions. Cells we e ozen in RPMI medium 1640 wi h L-
Glu amine (Gibco, The mo Fishe ) supplemen ed wi h 10% oe al bo ine se um, 10,000 U/ml
penicillin, 10,000 μg/ml s ep omycin and 10% DMSO and s o ed in liquid ni ogen un il used.
Fo low cy ome y and cell cul u e expe imen s PBMCs we e hawed and immedia ely washed
and esuspended in he esh RPMI medium o emo e DMSO.
EV isola ion
Ci a e ubes we e immedia ely p ocessed a e blood collec ion. EVs we e isola ed as
p e iously desc ibed by ou g oup [202]. B ie ly, ubes we e cen i uged a 2,500 g o 15 min,
and he ob ained plasma was hen cen i uged a 13,000 g o 2 min and his supe na an
cen i uged again a 20,000 g o 20 min o pelle EVs. The pelle was esuspended wi h il e ed
DPBS (GIBCO, The mo Fishe ), il e ed wice h ough a 0.22 m-po e il e . Resuspended EVs
we e s o ed a -80 ºC.
C yo-elec on mic oscopy (c yoEM)
EVs we e i i ied ollowing s anda d p o ocols [336]. Glow-discha ged Quan i oil holey
ca bon ilm g ids (O hogonal A ay o 2µm Diame e Holes - 2µm Sepa a ion, moun ed on a
300M Cu g id, #657-300-CU, Ted Pella) we e i i ied in liquid e hane in Vi obo a e
deposi ion o 3 µL o he sample. C yo- ans e sample holde s o he ype GATAN Model 626
we e used o keep he sample i i ied du ing elec on mic oscopy analysis. The sample was
obse ed in a JEM-2100F UHR (80-200kV, JEOL) ield emission gun ansmission elec on
mic oscope a di e en magni ica ions. Mic og aphs we e eco ded on a s a e o he a TVIPS
F216 CMOS came a (2k x 2k).
Nanopa icle acking analysis
The size dis ibu ion and concen a ion o isola ed plasma EVs we e measu ed using a
Ze aView (Pa icle Me ix) ins umen ollowing manu ac u e ins uc ions. Samples we e
hawed on ice and dilu ed wi h il e ed DPBS o ge accu a e acquisi ions. Se ings we e ixed
and main ained o all samples. Fil e ed DPBS was es ed and no backg ound signal was
148 | Chap e h ee
de ec ed. Fo each sample, wo cycles o analysis a 11 posi ions we e pe o med and esul s
we e analysed wi h Ze aView 8.04.02 so wa e (Pa icle Me ix).
PBMC and EV cul u e
Thawed cells we e cul u ed in 96-well la -bo om pla es in RPMI medium supplemen ed wi h
10% exosome-deple ed FBS (Gibco, The mo Fishe ), 10,000 U/ml penicillin and 10,000 μg/ml
s ep omycin. 105 cells we e pla ed in each well and immedia ely a e , 100 μg o hawed EVs
(measu ed by p o ein quan i ica ion wi h Bio-Rad P o ein Assay) we e added o he
co esponding wells. Cells we e cul u ed in 200 μl medium, a a inal densi y o 106 cells pe
ml and incuba ed o 3 h a 37 °C and 5% CO2. Then, ac i a ion o cells was induced by adding
10 μg/ml phy ohemagglu inin (PHA) (Sigma-Ald ich) in co esponding wells. All cul u ed cells
we e incuba ed o 72 h a 37 °C and 5% CO2. A schema ic ep esen a ion o he cocul u e
p o ocol is p esen ed in Figu e 43. PHA was chosen o induce a polyclonal, nonspeci ic and
signi ican lymphocy e ac i a ion, simila o he one p oduced agains in ec ion agen s [337].
The 10 μg/ml concen a ion o PHA was es ablished a e i a ion. In a sample om a heal hy
adul 8 di e en concen a ions o PHA (1.25-50 μg/ml) we e es ed and 10 μg/ml was chosen
as he bes s imula ion (Figu e 44).
Figu e 43. Cell cul u e p o ocol o es he in luence o EVs on T cell ac i a ion. (A) 105 PBMCs we e
pla ed in 96-well dishes and hen, 100 μg o EVs we e added, while DPBS was added in con ol wells. 3
hou s la e , 10 μg/ml PHA we e added o induce T cell ac i a ion in hal o he wells, while he es was
main ained wi h no s imula ion. 3 days a e pla ing, T cell ac i a ion was e alua ed by low cy ome y.
(B) Schema ic ep esen a ion o he di e en s udy condi ions.
Chap e h ee | 149
Figu e 44. T cell ac i a ion unde PHA s imula ion, measu ed by low cy ome y. PHA was i a ed wi h
a heal hy adul lymphocy e sample and he 10 μg/ml concen a ion was chosen as he bes s imula ion.
Flow cy ome y
Fo he low cy ome ic analysis o PBMCs, he ollowing luo och ome-conjuga ed an i-human
monoclonal an ibodies we e used: an i-CD3 APC-Fi e750, and an i-CD56 APC om Biolegend;
An i-CD8 FITC, an i-CD28 PE, an i-CD4 PE-Cy7 and an i-CD25 PE om BD Biosciences; o cell
iabili y assessmen 7-aminoac inomycin D (7-AAD) dye (The mo Fishe ). Di e en an ibody
panels we e designed. To assess he T cell popula ion pe cen ages and he senescence s a e o
T cells, he combina ion o an i-CD3 APC-Fi e750, an i-CD56 APC, an i-CD8 FITC, an i-CD28 PE,
an i-CD4 PE-Cy7 and 7-AAD was used. T cells we e iden i ied by CD3+ s aining, NK cells by
CD3-/CD56+ s aining and B cells as double nega i e CD3-/CD56-. The same panel wi hou 7-
AAD was applied o he low cy ome y o plasma EVs. Fo cul u ed PBMC ac i a ion
measu emen an i-CD8 FITC, an i-CD4 PE-Cy7, an i-CD25 PE and 7-AAD we e combined.
Di ec ly hawed PBMCs and PBMCs om cell cul u e we e s ained ollowing he same p o ocol.
Cells we e washed and esuspended in DPBS wi h 5 % bo ine se um albumin (BSA) (Sigma-
Ald ich) o block Fc ecep o be o e s aining. Co esponding an ibodies we e added and
samples incuba ed o 20 min a oom empe a u e in he da k. Then, cells we e washed o
emo e unbound an ibodies and acqui ed in a FACS Can o II low cy ome e (BD Biosciences)
o in a Gua a EasyCy e 8HT low cy ome e (Millipo e, Me ck). Single s aining and luo escence
minus one (FMO) con ol ubes we e used o adjus compensa ions and se he ga ing s a egy.
A e ga ing o single s, lymphocy es we e ga ed based on FSC and SSC and 20,000
lymphocy es we e acqui ed o each sample. Then, lymphocy e popula ions we e
dis inguished based on luo escence and analysis o ob ained esul s was pe o med wi h FACS
Di a 8.0.1 (BD Biosciences) and InCy e 3.1 (Millipo e, Me ck) so wa e espec i ely. The ga ing
s a egy o senescen T cells and ep esen a i e do plo s a e p esen ed in Figu e 45.
156 | Chap e h ee
The cocul u e o PBMCs and EVs imp o es iabili y and in luences cy okine sec e ion
Nex , cocul u e expe imen s o PBMCs and plasma EVs we e pe o med. Cell and EV samples
o all ages we e es ed. Fou di e en condi ions we e assayed: PBMCs alone, PBMCs + EVs,
PBMCs + PHA and PBMCs + EVs + PHA. The PHA was applied o s imula e T cell ac i a ion, and
o es he e ec o EVs bo h unde non-s imula ed and s imula ed condi ions. To es whe he
he addi ion o EVs a ec s cell iabili y, we analysed cells by low cy ome y and compa ed he
7-AAD nega i e e en s be ween g oups a e h ee days in cul u e. The di e en condi ions o
each PBMC dono we e no malized o he con ol wells whe e only cells we e pla ed.
In e es ingly, we obse ed ha cell iabili y imp o es when EVs a e p esen (Figu e 50A).
Mo eo e , PHA s imula ion signi ican ly educes iabili y and his e ec is pa ially escued
when EVs a e added (Figu e 50A). In a u he analysis o hese esul s, we compa ed he e ec
o plasma EVs on cells o adul (20-49 yea s) and aged (>80 yea s) dono s. The posi i e e ec
o EVs is s onge in PBMCs om adul s o all condi ions es ed (Figu e 50B).
In o de o check whe he he cocul u e wi h EVs could also a ec cy okine p oduc ion in i o,
we pe o med a luminex assay o TNF-α, IL-6, IL-10, IL-1β and IL-2. Cell condi ioned media
om all condi ions o 2 di e en indi iduals (one adul and one aged cell dono , cocul u ed
wi h EVs om adul s and elde s) we e es ed. Impo an ly, cy okine concen a ions we e non-
de ec able in he wo condi ions whe e PHA was no added, demons a ing ha he only
addi ion o EVs does no induce cy okine p oduc ion. When compa ed o PHA s imula ion
alone, we obse ed ha EV addi ion in luences cy okine p oduc ion. The sec e ion o he
p oin lamma o y TNF-α, IL-6 and IL-1β cy okines was educed, while an i-in lamma o y IL-10
was inc eased and IL-2 no signi ican ly a ec ed (Figu e 50C).
Chap e h ee | 157
Figu e 50. (P e ious page) E ec o ex acellula esicles om plasma on PBMC iabili y and cy okine
sec e ion in i o. PBMCs om dono s o all age anges we e cul u ed o 72h in he p esence o no o
PHA o /and plasma EVs and hen analysed by low cy ome y. (A) Cell iabili y is educed a e
s imula ion wi h PHA, while he cocul u e wi h EVs imp o es iabili y. (B) The posi i e e ec o plasma
EVs on cell iabili y is s onge in cells om adul s (20-49 yea s) han aged (80-101 yea s) indi iduals.
(C) The analysis o condi ioned media by luminex showed a educed sec e ion o p oin lamma o y
cy okines TNF-α, IL-6 and IL-1β and an inc eased sec e ion o an i-in lamma o y IL-10 by s imula ed
cells cocul u ed wi h EVs compa ed o s imula ed cells wi hou EVs.
T cell ac i a ion unde PHA s imula ion is a ec ed by he cocul u e o plasma EVs and
depends on he age o he EV dono
Finally, he e ec o plasma EVs on lymphocy e ac i a ion was assessed. Polyclonal ac i a ion
o T cells was induced wi h PHA and measu ed by CD25 exp ession by low cy ome y. The
cocul u e o lymphocy es wi h plasma EVs o 72h did no induce T cell ac i a ion (Figu e 51C-
D) demons a ing ha plasma EVs alone a e no immunogenic o non-s imula ed cells. PBMC
samples o 22 indi iduals (12 adul s and 10 elde s) we e es ed, and each cell dono was
assayed wi h di e en EV dono s (up o 12 di e en EVs o one PBMC dono , each one in
di e en wells and always in duplica e). The pe cen age o T cells ac i a ed unde he same
PHA s imula ion (and wi hou EVs) was highly he e ogeneous o each PBMC dono (33-92 %
o CD25+, Figu e 51A-B). Fo no maliza ion, con ol wells wi h PBMCs + PHA wi hou EVs
we e used and old change was calcula ed.
Figu e 51. T cell ac i a ion measu ed by low cy ome y. (A-B) PBMCs we e s imula ed wi h 10 μg/ml
PHA and 72h la e CD25+ cells measu ed. The pe cen age o ac i a ed CD4 and CD8 cells is
he e ogeneous and no co ela ed o age. (C-D) PBMCs we e cocul u ed wi h plasma EVs. The cocul u e
o EVs alone do no induce T cell ac i a ion.
158 | Chap e h ee
Ou esul s show ha EVs modula e T cell ac i a ion, bu he e ec is e y he e ogeneous and
is in luenced by he age o he EV dono (Figu e 52A-D). Taking his in o conside a ion, we
pe o med a sepa a ed analysis o plasma EVs om each age ange. EVs om adul s
signi ican ly inc ease CD4 cell ac i a ion, while he ones om nonagena ians and cen ena ians
educe he ac i a ion (Figu e 53A). In he case o CD8 cells, EVs om adul s also enhance cell
ac i a ion (Figu e 53B). Impo an ly, when he endency o he whole coho was analysed,
we saw ha he ac i a ion enhancemen capaci y o EVs signi ican ly dec eases wi h age
(Figu e 53).
Figu e 52. Analysis o ac i a ed lymphocy es unde PHA s imula ion and he in luence o he EV dono
age. (A-B) The cocul u e o PBMCs wi h EVs unde PHA s imula ion a ec s cell ac i a ion in a
he e ogeneous manne . Fo each cell dono , wells wi hou EVs we e aken as e e ence o old change
calcula ion. (C-D) Bo h CD4 and CD8 cells ge mo e ac i a ed in he p esence o EVs om adul dono s
when compa ed o EVs om elde dono s. Adul s 20-49 and elde s 70-104 yea s.
Chap e h ee | 159
Figu e 53. T cell ac i a ion unde PHA s imula ion and he e ec o plasma ex acellula esicles. PBMCs
om dono s o all age anges we e cul u ed o 72h in he p esence o PHA and plasma EVs and hen
analysed by low cy ome y. Wells wi hou EVs we e aken as e e ence o old change calcula ion and
Wilcoxon es s. (A) The p esence o EVs om adul dono s esul ed in he p omo ion o CD4 cell
ac i a ion, an e ec ha dec eases g adually wi h EV dono age (in ed, Jonckhee e es ****). (B) In a
simila way, CD8 cells cocul u ed wi h EVs om adul s ge mo e ac i a ed, bu his e ec dec eases wi h
EV age (in ed, Jonckhee e es ****). Age ange in yea s.
Discussion
The p esen s udy analysed pe iphe al blood samples om adul s and elde s o di e en age
anges, 20-49 and 70-104 yea s. Fi s , he lymphocy e subse s we e compa ed and an
inc eased T cell and dec eased B cell and NK cell p opo ions we e ound wi h age. Se e al
wo ks ha e s udied he lymphocy e subse s wi h aging, bo h a he o al numbe and
pe cen age le el. Dis inc aging pa e ns ha e been ound be ween coun ies and popula ions,
poin ing ou he complexi y and he e ogenei y o he immune sys em and immunosenescence
[334,338–342]. To ou knowledge, ou wo k is he i s in es iga ing lymphocy e popula ions
wi h age in he Basque Coun y (in he no h o Spain).
On he o he hand, some aging- ela ed ea u es ha e been widely epo ed, such as he loss o
he cos imula o y molecule CD28 om T cell memb ane and a subsequen gain o NK
cha ac e is ic ma ke s [100,102,107,343]. This p ocess has been shown o a ec bo h CD8 and
CD4 T cells, bu ea lie and o a g ea e ex en o CD8 cells [104,344,345]. Ou esul s a e in
acco dance wi h p e ious epo s. Ne e heless, p e ious s udies epo ed a g adual
accumula ion o CD4 CD28- cells wi h age [98,103,346] and he e we showed a highe senescen
CD4 cell pe cen age in he 80-89 age ange, and in e es ingly a lowe pe cen age in
nonagena ians and cen ena ians, ollowing a quad a ic e ec . We hypo hesize ha a he han
CD28 exp ession eco e y, indi iduals eaching >90 yea s could be he ones ha p esen ed
lowe senescen cell p opo ions also ea lie in li e. Howe e , a longi udinal s udy wi h a la ge
160 | Chap e h ee
sample size would be needed o con i m his p og ession. As men ioned abo e, o he wo ks
p e iously desc ibed he loss o CD28 exp ession in CD4 cells in elde s, bu o ou knowledge,
ou s udy is he i s one analysing his e ec in nonagena ians and cen ena ians and
demons a es ha hey ollow a dis inc aging p og ession in some aspec s.
To u he cha ac e ize immunosenescence, we ocussed on plasma EVs. They a e known o
ca y many molecules in hei memb ane and among hem, EVs can also bea ma ke s o he
sec e ing cell [347]. To es whe he plasma EVs esemble he senescence s a us o T cells, we
measu ed T cell memb ane ma ke s on EVs. Ou esul s showed ha plasma EVs ca y T cell
speci ic molecules, while he e is no an inc eased p opo ion o “senescen -like EVs” wi h age.
E en i no signi ican di e ences we e ound be ween age anges, a highe pe cen age o CD28-
EVs was obse ed among CD8 EVs when compa ed o CD4 EVs, which could be linked o he
inc eased CD28- CD8 T cells. Impo an ly, we also iden i ied he cha ac e is ic e aspanins
CD9, CD63 and CD81 o EVs by low cy ome y. A small pe cen age o ci cula ing EVs in plasma
ca y hese molecules, bu i should be no ed ha obse ed numbe s could be unde es ima ed
by o he co-isola ed pa icles and ha EVs exp essing a single o ew copies o he su ace
an igen o in e es canno be de ec ed, as desc ibed in p e ious s udies [218,348]. Mo eo e ,
i should be men ioned ha he de ec ion o EV p o eins by low cy ome y is a di ec measu e
ha iden i ies p o eins a hei physiologic s a e – a he EV memb ane in his case –, in con as
o echniques such as wes e n blo ing o p o eomics app oaches whe e esicles a e lysed, and
he numbe s o posi i e pa icles o each p o ein canno be measu ed.
Rega ding cocul u e expe imen s o PBMCs and plasma EVs, we showed ha EVs om a
di e en dono a e no immunogenic o ecep o lymphocy es, and in con as , hey a ec cell
iabili y and cy okine sec e ion. Speci ically, plasma EVs enhance cell iabili y and pa ially
escue he dele e ious e ec o PHA, he well-known ac i a ion-induced cell dea h [349]. This
posi i e e ec is s onge on cells om adul s, when compa ed o elde s. Ou esul s indica e
o he i s ime ha he p esence o plasma EVs in cul u e can pa ially escue he ac i a ion-
induced cell dea h and mo eo e , ha EVs enhance T cell iabili y when compa ed o he
cul u e o cells alone. Fu he , plasma EVs educe he sec e ion o TNF-α, IL-6 and IL-1β
p oin lamma o y cy okines and inc ease an i-in lamma o y IL-10 in PHA s imula ed cells, bu
EVs alone do no al e cy okine sec e ion o PBMCs. I has been widely desc ibed ha PHA
s imula es cy okine p oduc ion [350], bu he e ec o EVs is s ill no unde s ood. A p e ious
s udy epo ed a simila e ec o mesenchymal cell-de i ed EVs on IL-10 p oduc ion [351],
and some au ho s ha e also s udied he e ec o EVs on lymphocy es and ILs [333,352].
Howe e , hey wo ked wi h EVs om o he issues o p oduced in cul u e, which can lead o
dis inc ou pu s.
Chap e h ee | 161
And e en i plasma EVs a e no immunogenic, hey in luence T cell ac i a ion unde PHA
s imula ion, and his e ec is di e en depending on he age o he EV dono . EVs om adul s
p omo e T cell ac i a ion and his e ec dec eases wi h age. These esul s highligh he
in luence o ci cula ing EVs on T cells and in e es ingly, also demons a e he dis inc e ec s o
plasma EV and T cell in e ac ions depending on age. The cocul u e expe imen s enable us o
mo e closely esemble he in e ac ion be ween ci cula ing cells and EVs. Much wo k is s ill
needed o elucida e he complex pool o pa icles p esen in plasma and he igge s o
obse ed e ec s, bu he p esen wo k gi es a i s desc ip ion o he ole ha EVs om plasma
ha e on T cells du ing aging.
In sho , ou wo k desc ibes he educed CD28 loss o CD4 cells in nonagena ians and
cen ena ians, he p esence bu no accumula ion o senescen ma ke s on plasma EVs and he
dis inc in e ac ions be ween T cells and plasma EVs wi h age.
CHAPTER FOUR
Mul iple scle osis and
p ema u e aging
Chap e ou | 165
In oduc ion
Aging is a uni e sal p ocess. I a ec s bo h heal hy indi iduals as well as he ones ha p esen
o he synd omes and/o diseases. Impo an ly, aging and he concomi an heal h p oblems
in e ac and in luence each o he . These in e ac ions a e obse ed in diseases ha de elop in
elde people, such as cance , bu he in luence o aging also eaches ch onic diseases as pa ien s
age, like in he case o MS [275].
MS is a ch onic au oimmune disease o he CNS cha ac e ised by pa hologic demyelina ion o
axons and subsequen neu odegene a ion. I is a he e ogeneous disease and, clinically, i can
ollow elapsing- emi ing o p og essi e o ms [353]. Mos o MS pa ien s expe ience he i s
symp oms a hei 20s o 30s, bu he e a e also paedia ic o ju enile [271] and la e-onse MS
cases [272]. In he las decades, e ec i e disease-modi ying ea men s ha slow he
p og ession o MS ha e been de eloped [267], and hanks o hem, pa ien s p esen inc easing
age a disabili y miles ones [273]. Consequen ly, he mean age o MS pa ien s is inc easing,
wi h al eady mo e han 20% o hem aged 60 yea s o o e (msbase.o g [274]). The e o e, an
ele a ed numbe o pa ien s su e om he a o emen ioned in e ac ions be ween MS and
aging p ocesses.
I should be no ed, ha he cha ac e is ic ea u es o MS a e e y simila o he ones obse ed
du ing aging, as in lamma ion, and immune al e a ions. Mo eo e , he consequences such as
mobili y and cogni i e p oblems a e also ound in bo h p ocesses, which makes i e y
complica ed o sepa a e he e ec s o MS and aging [275].
In addi ion, some au ho s ha e sugges ed ha se e al au oimmune diseases, including MS,
show p ema u e aging, specially wi h ega ds o he immune sys em. They obse ed educed
numbe s o naï e CD4 T cells in pedia ic MS [354] and inc eased le els o CD4+CD28- T cells
in adul MS pa ien s [280,281]. Mo eo e , he cha ac e is ic loss o he cos imula o y molecule
CD28 om CD4 T cells due o he epea ed s imula ion and ac i a ion is p oposed as a sign o
senescence and e minal di e en ia ion, bu i has been shown ha in MS pa ien s hese cells
emain unc ional and show inc eased cy o oxici y [355]. Fu he mo e, CD4+CD28- T cells
ha e also been ound in MS lesions in he CNS, sugges ing ha hey could be implica ed in MS
pa hogenesis [356]. Rega ding in lamma ion, ele a ed le els o TNF- and IL-6 among o he
in lamma o y ma ke s ha e been ound in he ce eb ospinal luid and se um o MS pa ien s
du ing emission, indica ing ha some signs o ch onic in lamma ion a e p esen [285,286].
The aim o his wo k was o pe o m a pilo s udy o e alua e whe he di e ences a e ound in
age- ela ed ea u es due o he p esence o MS disease. We es ed he abo e-men ioned
in lammaging (by measu ing TNF-, IL-6 and CRP concen a ions) and T cell senescence (by
