STATE-OF-THE-ART REVIEW
Mic o-RNA Analysis in
Pulmona y A e ial Hype ension
Cu en Knowledge and Challenges
Cá ia A. San os-Fe ei a, MD,
a,d
Mónica T. Ab eu, PHD,
b,c,d
Ca la I. Ma ques, PHD,
b,c,d
Lino M. Gonçal es, MD, PHD,
a,b,c,d
Rui Bap is a, MD, PHD,
a,b,c,d,e
Hen ique M. Gi ão, PHD
b,c,d
HIGHLIGHTS
The ole o miRNAs in PAH is as expanding, and i is inc easingly di ficul o iden i y which molecules ha e he highes
ansla ional po en ial.
This e iew discusses he challenges in miRNA analysis and in e p e a ion in PAH and highligh s 4 p omising miRNAs in
his field.
Addi ional p e-clinical s udies and clinical ials a e u gen ly needed o b ing miRNAs om he bench o he bedside soon.
SUMMARY
Pulmona y a e ial hype ension (PAH) is a a e, ch onic disease o he pulmona y ascula u e ha is associa ed wi h poo
ou comes. I s pa hogenesis is mul i ac o ial and includes mic o-RNA (miRNA) de egula ion. The unde s anding o he ole
o miRNAs in PAH is expanding quickly, and i is inc easingly di ficul o iden i y which miRNAs ha e he highes ans-
la ional po en ial. This e iew summa izes he cu en knowledge o miRNA exp ession in PAH, discusses he
challenges in miRNA analysis and in e p e a ion, and highligh s 4 p omising miRNAs in his field (miR-29, miR-124,
miR-140, and miR-204). (J Am Coll Ca diol Basic T ans Science 2020;5:1149–62) © 2020 The Au ho s. Published by
Else ie on behal o he Ame ican College o Ca diology Founda ion. This is an open access a icle unde he CC BY-NC-
ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Pulmona y a e ial hype ension (PAH) is a a e
mul i ac o ial condi ion, hemodynamically
definedasaninc easein hemeanpulmona y
a e ial p essu e (>20 mm Hg, acco ding o he
ecen ly e ised 6 h Wo ld Symposium on Pulmona y
Hype ension defini ion), wi h no mal le en icula
filling p essu es and ele a ed pulmona y ascula
esis ance (1,2). I is cha ac e ized by s uc u al and
unc ional changes in he pulmona y a e ial ascula-
u e ha e ol e om isola ed medial hype ophy o
end-s age plexi o m fib osis, o igina ing an inc ease
in pulmona y ascula esis ance (3). The p og essi e
inc ease in igh en icula (RV) a e load leads o
RV hype ophy and, ul ima ely, RV ailu e and
dea h (4).
Despi e conside able p og ess in he unde s and-
ing o he epidemiology, pa hophysiology, and man-
agemen o PAH, he p ognosis emains poo ,
especially in pa ien s wi h se e e disease (i.e., Wo ld
Heal h O ganiza ion [WHO] unc ional class IV) (1,5).
ISSN 2452-302X h ps://doi.o g/10.1016/j.jacb s.2020.07.008
F om he
a
Ca diology Depa men , Cen o Hospi ala e Uni e si á io de Coimb a, Coimb a, Po ugal;
b
Uni e si y o Coimb a,
Coimb a Ins i u e o Clinical and Biomedical Resea ch, Facul y o Medicine, Coimb a, Po ugal;
c
Uni e si y o Coimb a, Cen e o
Inno a i e Biomedicine and Bio echnology, Coimb a, Po ugal;
d
Clinical Academic Cen e o Coimb a, Coimb a, Po ugal; and he
e
Ca diology Depa men , Cen o Hospi ala En e Dou o e Vouga, San a Ma ia de Fei a, Po ugal.
The au ho s a es hey a e in compliance wi h human s udies commi ees and animal wel a e egula ions o he au ho s’
ins i u ions and Food and D ug Adminis a ion guidelines, including pa ien consen whe e app op ia e. Fo mo e in o ma ion,
isi he JACC: Basic o T ansla ional Science au ho ins uc ions page.
Manusc ip ecei ed June 3, 2020; e ised manusc ip ecei ed July 23, 2020, accep ed July 24, 2020.
JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020
ª2020 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN
COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER
THE CC BY-NC-ND LICENSE (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Pa ien s in WHO class IV ha e highe igh
a ial p essu e and pulmona y ascula
esis ance, lowe exe cise capaci y and peak
oxygen consump ion, and a 3-yea su i al
a e o 38%, compa ed wi h >60% obse ed
o pa ien s in WHO classes I o III (5–7).
Cu en PAH he apies p ima ily a ge 3
asomo o pa hways: he ni ic oxide, he
endo helin-1, and he p os acyclin pa hways
(1). Howe e , hese in e en ions migh no
accu a ely a ge he in ica e molecula p i-
mum mo ens o PAH, because gene ic sus-
cep ibili y, hypoxia, inflamma ion, DNA
damage, i al in ec ion, and shea s ess a e
all in ol ed in a ious amoun s wi hin he
pa hogenic e en s ha con ibu e o he
pa ien -specific pheno ype o disease mani-
es a ion and p og ession (8). PAH in ol es a
single o gan, he lungs, bu mul iple pulmo-
na y a e ial cell ypes—pulmona y a e y
endo helial cells (PAECs), pulmona y a e y
smoo h muscle cells (PASMCs), and pulmo-
na y a e ial ad en i ial fib oblas s (PAAFs)
(9). The inabili y o ully unde s and his
complex in e play o cells and soluble medi-
a o s has hampe ed he de elopmen o new
d ugs. In he las 15 yea s, no new d ug aimed
a no el pa hological pa hways has been
app o ed.
One o he playe s o his complex ne wo k o
s imulia emic o-RNAs(miRNAs).Mos o ou
genome is no di ec ly implica ed in p o ein syn hesis
bu , a he , in he p oduc ion o noncoding RNAs
(ncRNAs) wi h egula o y unc ions (10). Among he
se e al RNA species, 2 a ie ies o ncRNAs, loosely
defined as long and small RNA, ha e been desc ibed
ecen ly. Among he small RNAs, miRNAs a e sho
leng h, noncoding endogenous RNA molecules ha
consis o app oxima ely 22-nucleo ide-long se-
quences p o usely exp essed in all human cells and
a e belie ed o con ol app oxima ely 50% o all
p o ein-coding messenge RNA (mRNA) (11).
Thebiogenesiso miRNAsisclassified in o 2
pa hways: he canonical ( he dominan pa hway by
which miRNAs a e p ocessed), and non-canonical
pa hways. In he canonical pa hway, ansc ip ion o
miRNAs genes p oduces p ima y miRNAs ha a e
hai pin-like and can be >1,000 nucleo ides long; his
ansc ip ion is dependen on polyme ase II and is
egula ed by ansc ip ion ac o s (12). P ima y miR-
NAs a e clea ed in he nucleus o o m p ecu so
miRNAs, which a e a sho e hai pin app oxima ely
70 nucleo ides long, by he Mic op ocesso complex,
which consis s o an RNA-binding p o ein DiGeo ge
Synd ome C i ical Region 8 and a ibonuclease III
enzyme, D osha. P ecu so miRNAs a e hen expo ed
om he nucleus o he cy oplasm by expo in-5. In
hecy oplasm,p ecu so miRNAsa ep ocessedin o
sho , double-s anded, imma u e miRNA by ano he
ibonuclease, Dice . In he ollowing s ep, he 2
s ands a e sepa a ed, and 2 o hese s ands (ma u e
miRNA) a e ec ui ed by A gonau e p o eins o
inco po a e a mul ip o ein complex known as he
RNA-induced silencing complex (13). In addi ion o
he canonical miRNA biogenesis pa hway, mul iple
non-canonical pa hways ha e been e ealed by using
di e en combina ions o he p o eins in ol ed in he
canonical pa hway. In gene al, hey can be classified
as D osha- and Dice -independen pa hways (13).
MiRNAs a e able o modi y p o ein exp ession by
binding o complemen a y sequences on mRNA,
mainly in he 30-un ansla ed egion o he a ge
mRNA ansc ip s, he eby p omo ing ansla ional
inhibi ion and deg ada ion by al e ing he s abili y o
a ge mRNAs (14).Thema u emiRNAisin eg a ed
in o he RNA-induced silencing complex and hen
binds o i s mRNA a ge . Once bound o an mRNA,
he miRNARNA-induced silencing complex p o-
mo es he down egula ion o he p o ein ha he
mRNA encodes, mainly h ough di ec mRNA deg a-
da ion (15). A specificmiRNAcan a ge mul iple
mRNAs ( he so-called di e gen pa hway), and a
mRNAcanha emul iplebindingsi es o se e al
miRNAs. In addi ion, a ious ela ed miRNAs can
a ec a pa hway a di e en le els, e med a
con e gen pa hway (14). The in e ac ome o miRNAs
wi h mRNA is a mul idimensional ne wo k and plays
a c ucial ole in egula ing cellula pa hways and
biological unc ions in heal h and disease (16). Du ing
pa hological p ocesses, such as pulmona y ascula
p ocesses in ol ed in PAH, he dys egula ion o spe-
cific and dis inc miRNAs has p o ound consequences
in cell unc ion due o changes in p o ein syn hesis
(14). The e o e, miRNAs ha e been ecognized as
p omising bioma ke s and he apeu ic op ions o
many ca dio ascula diseases (14,17). Howe e , one
o he issues ega ding he s udy o miRNA in PAH
esides in he la ge he e ogenei y o models and cells
analyzed (18,19). miRNA egula ion is no conse ed
be ween he a ious pulmona y hype ension (PH)
animal models. These di e ences may ha e impo -
an implica ions in how p eclinical miRNA a ia ions
should be in e p e ed as po en ial bioma ke s and
he apeu ic a ge s and consequen ly ansla ed o
pa ien s wi h PAH (19).
Among he a ious echniques a ailable o s udy
miRNAs egula ion in cell lines, animal models, and
pa ien s, mic oa ay analysisiso g ea use,because
ABBREVIATIONS
AND ACRONYMS
BMPR2 =bone mo phogene ic
p o ein ecep o ype 2
EPC =endo helial p ogeni o
cell
HIF =hypoxia-inducible ac o
HPAH =he edi a y pulmona y
a e ial hype ension
lncRNA =long noncoding RNA
MCT =monoc o aline
miRNA =mic o-RNA
mRNA =messenge RNA
ncRNAs =noncoding RNAs
PAAF =pulmona y a e ial
ad en i ial fib oblas
PAEC =pulmona y a e y
endo helial cell
PAH =pulmona y a e ial
hype ension
PASMC =pulmona y a e y
smoo h muscle cells
PH =pulmona y hype ension
RV = igh en icle
SU/Hx/Nx =associa ion o
Sugen 5416 wi h ch onic
hypoxia ollowed by no moxia
WHO =Wo ld Heal h
O ganiza ion
San os-Fe ei a e al.JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020
Mic oRNA in Pulmona y A e ial Hype ension NOVEMBER 2020:1149–62
1150
i allows he de ec ion o genome-wide gene exp es-
sion (20). The de elopmen o hese powe ul, e ec-
i e, and high- h oughpu da a me hodologies
enables he unbiased sc eening o mechanisms
al e ed in a specific da ase and he sea ch o
di e en ially exp essed genes, acco ding o he
expe imen al design (e.g., heal hy con ol subjec s
s. pa ien s wi h disease). They ha e been b oadly
applied o in es iga e he pa hophysiology o di e se
ca dio ascula diseases (14,17).
Recen ly, he ole o long non-coding RNAs
(lncRNA) in he pa hobiology o PAH has also been
explo ed. LncRNAs a e longe han 200 nucleo ides
and a e abundan ly p esen in he genome (21). Based
on hei subcellula localiza ion, lncRNAs a e able o
egula e gene exp ession h ough di e en mecha-
nisms. In con as o miRNA, lncRNA can yield hei
egula o y e ec s, bo h a he ansc ip ional and
pos - ansc ip ional le els, h ough in e ac ions wi h
DNA, ch oma in, and o he species o RNA. In addi-
ion, hey can ac as epigene ic modula o s (22).
Eme ging e idence sugges s ha hese molecules
play an impo an ole in PAH pa hogenesis as
undamen al d i e s and ga ekeepe s in he egula-
ion o key cellula and molecula a ficking in
PASMC and PAEC dys egula ion (21).
In his e iew, we summa ize he cu en knowl-
edge on miRNA exp ession in expe imen al models o
PH and pa ien s wi h PAH, ocusing ou analysis on
hosemiRNAs ha ha ebeen ound oha ea
conse ed egula ion ac oss animal models and hu-
man PAH samples using mic oa ay analysis (Cen al
Illus a ion). Finally, we highligh u u e pe spec-
i es and po en ial challenges conce ning miRNA
applica ion in he clinical a ena, namely, in he
de elopmen o no el diagnos ic and he apeu ic
ools o PAH.
CHALLENGES IN MICRO-RNA ANALYSIS AND
INTERPRETATION IN PAH
The fi s e idence ha sugges ed ha miRNAs
con ibu e o he pa hogenesis o PAH esul ed om
he obse a ion ha bone mo phogene ic p o ein e-
cep o ype 2 (BMPR2) p o ein, bu no mRNA le els,
was educed in animal models o PH (23). Since hen,
miRNAs ha e been implica ed in a wide ange o
pulmona y ascula p ocesses in ol ed in PAH (14).
Howe e , many challenges limi he alida ion o
miRNAs disco e ed in expe imen al models.
LACK OF CONCORDANCE AMONG ANIMAL PH
MODELS. Fi s , al hough all PH animal models a e
cha ac e ized by an inc ease in RV a e load and
p og ession o RV ailu e, he igge is subs an ially
di e en be ween hem (19,24). The e o e, he e is a
lack o conco dance in he pa e n o miRNA exp es-
sion among di e en expe imen al models o PAH
(19,24). Fo example, adminis a ion o monoc o a-
line (MCT) o a s mos ly a ge s he pulmona y
ascula endo helium and elici s a s ong pulmona y
inflamma ion p ocess, especially monocy e ec ui -
men , which plays an impo an ole in human idio-
pa hic PAH (24). Con e sely, in he ch onic hypoxia
mu ine model, pe sis en hypoxia causes ascula
s uc u al emodeling in all 3 laye s o he pulmona y
a e iola wall, wi h hype ophy o PASMCs being a
majo his ological finding and ha ing less inflamma-
ion compa ed wi h he MCT model (19,25). The
model induced by he associa ion o ascula endo-
helial g ow h ac o ecep o an agonis , Sugen 5416,
wi h ch onic hypoxia ollowed by no moxia (SU/Hx/
Nx) causes p o ound and sus ained PH, along wi h
inflamma ion and angio-obli e a ion, culmina ing in
RV emodeling and ailu e, in bo h a s and mice (24).
The subjacen mechanism is belie ed o in ol e
dea h o PAECs, wi h subsequen p oli e a ion o an
apop osis- esis an cell ype. The es i u ion o no -
moxia esul s in he de elopmen o neoin imal le-
sions ex emely simila o human plexogenic
a e iopa hy (24). Al hough he SU/Hx/Nx model has
been sugges ed as a mo e ele an model o human
PAH (19,26), miRNAs ha e p edominan ly been
s udied in he MCT o ch onic hypoxia models.
Despi e ep oducing impo an cha ac e is ics o hu-
man PAH, hese models gene ally ail o ep oduce
he se e e pulmona y a e iopa hy usually p esen in
pa ien s wi h PAH (19). O he models ha e been
de eloped o be e ep oduce he na u al his o y o
human PAH. Recen ly, a a model wi h a monoallelic
dele ion o 71 bp in exon 1 in he Bmp 2 gene was
de eloped. This model showed no only some o he
pi o al cellula and molecula dys unc ions desc ibed
in human PAH, bu also he g adual pheno ype seen
in humans. I also showed myoca dial abno mali ies,
suppo ing he hypo hesis ha he RV migh also be
a ec ed in PAH besides he inc eased a e load
imposed by pulmona y ascula disease (27).
Conside ing hese pa hophysiological di e ences,
i is no su p ising he e is di e gence in miRNA
egula ion among he di e en animal models e-
po ed in he li e a u e (18,19). Schlosse e al. (19)
explo ed hese dispa i ies in he pa e n o exp ession
o a se o miRNAs causally implica ed in PAH in he
plasma, lung, and RV o di e en animal models and
in plasma om pa ien s wi h PAH (19). They ound
ha mos o he miRNA in es iga ed had disco dan
pa e ns ac oss he di e en issues and models (19).
Mo eo e , a e analyzing he mRNA a ge s o
JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020 San os-Fe ei a e al.
NOVEMBER 2020:1149–62 Mic oRNA in Pulmona y A e ial Hype ension
1151
miRNAs, hey also ound disco dan egula ion in he
di e en models, which sugges ed ha in hese sce-
na ios miRNA migh no be he only o he mos sig-
nifican egula ion mechanism (19).
HETEROGENEITY IN REGULATION AMONG
DIFFERENT HUMAN PAH ETIOLOGIES. Second,
pheno ypic a iabili y be ween p edominan ly
hypoxic and nonhypoxic human o ms o PAH
ega ding miRNA le els migh he e o e be ecapi u-
la edbywha is oundin heanimalmodels,which
sugges s di e en pa hological mechanisms o dis-
ease.Asinanimalmodels, hedi e en o mso
human PAH a e unlikely o be equi alen , and i e-
mains unclea whe he he a ious ypes o PAH sha e
he same pa hophysiology (26). Al hough hey ha e
CENTRAL ILLUSTRATION Key Mic o-RNAs Associa ed Wi h Ca dinal Fea u es o Pulmona y A e ial Hype ension
San os-Fe ei a, C.A. e al. J Am Coll Ca diol Basic T ans Science. 2020;5(11):1149–62.
The miRNA dys egula ion plays an impo an ole in he hype p oli e a i e and apop osis- esis an pheno ype o pulmona y ascula cells in PAH, including PAECs,
PASMCs, and PAAFs. This figu e illus a es he lack o conco dance in he pa e n o exp ession o mos miRNAs be ween human PAH and di e en animal PH models
and highligh s 4 miRNAs ha migh ul ima ely ep esen a g ea e po en ial o ansla ing in o he clinical a ena. miRNA ¼mic oRNA; PAAF ¼pulmona y a e ial
ad en i ial fib oblas s; PAEC ¼pulmona y a e y endo helial cell; PAH ¼pulmona y a e ial hype ension; PASMC ¼pulmona y a e y smoo h muscle cell; PH ¼
pulmona y hype ension; SU/Hx/Nx ¼associa ion o Sugen 5416 wi h ch onic hypoxia ollowed by no moxia.
San os-Fe ei a e al.JACC: BASIC TO TRANSLATIONAL SCIENCE VOL. 5, NO. 11, 2020
Mic oRNA in Pulmona y A e ial Hype ension NOVEMBER 2020:1149–62
1152
simila his opa hological eflec ions in he pulmona y
a e y, he e a e di e en o ms o PAH wi hin he
same classifica ion; he e o e, di e en ial miRNA
p ofiles may eflec di e en mechanisms o disease
(18). We ha e p e iously epo ed ha in pa ien s in
whom he hypoxic pheno ype is p edominan (e.g.,
Eisenmenge synd ome, lung-disease ela ed PH, o
ad anced igh hea ailu e, in which low ca diac
ou pu leads o hypoxia a a pe iphe al issue le el),
he up egula ion o miR-424(322) p omo es hypoxia-
inducible ac o (HIF)-1
a
accumula ion,which,in
u n, con ibu es o inc eased miR-424(322) le els
(28). Al hough in his hypoxic pheno ype—exposed o
high HIF-1
a
le els—miR-424(322) likely p omo es
some amoun o pulmona y ascula p oli e a ion, i
is also necessa y o main ain he li e-sa ing
compensa o y mechanisms ha mi iga e he pa ho-
physiological mani es a ions o ch onic hypoxia (e.g.,
e y h ocy osis). This migh explain why lowe le els
o miR-424(322) we e associa ed wi h a poo e p og-
nosis in pa ien s wi h Eisenmenge synd ome in ou
coho , because hey migh be a cause (o a conse-
quence) o a de imen al lowe global ac i a ion o
HIF-1
a
compensa o y pa hways (28). Fo example, i
is well ecognized ha ela i e anemia due o i on
deficiency is a ma ke o poo p ognosis in Eisen-
menge synd ome (29,30).
Con e sely, in o he pa ien s wi h PAH, such as
hose wi h idiopa hic PAH, he edi a y PAH,
d ug-induced PAH, o connec i e issue
diseaseassocia ed PAH, he hypoxic componen is
likely no as ele an o he mechanism o disease.
Fo ins ance, in pa ien s wi h he edi a y PAH, he
BMPR2 pa hway can be down egula ed by mu a ions
in se e al genes (BMPR2, ACVRL1, ENG, SMAD1,
SMAD4, SMAD9)(31). In addi ion, e en i he e a e no
iden ified mu a ions, he pa hway can be down-
egula ed in pa ien s wi h idiopa hic PAH (32). PAECs
subjec ed o hypoxia condi ions show down-
egula ion o miR-424(322) le els, whe eas, in
PASMCs, no di e ences ha e been ound compa ed
wi h con ol cells (28). Pa adoxically, we (28)and
o he s (19) ound ha ci cula ing miR-424(322)/503
le els we e ele a ed in pa ien s wi h PAH and pa-
ien s wi h ch onic h omboembolic PH. Howe e ,
hose pa ien s we e hemsel es in a ious phases o
hediseasea he imeo analysis.Onceagain, he
imepoin o miR-424(322) assessmen and o gan-
specific di e ences (lung, hea ) migh ha e an
impo an ole in hese appa en ly con adic o y e-
sul s (19,28). Toge he , hese findings sugges ha he
balance o hypoxic and inflamma o y and/o gene ic
s imuli in each pa ien will de e mine he specific
pa ien -le el de egula ion o miRNAs, again adding
complexi y o ansla ional app oaches.
DIFFERENT miRNA EXPRESSION PROFILE BETWEEN
RV HYPERTROPHY AND RV FAILURE. Finally, he e
a edi e encesin hemiRNAexp essionp ofile be-
ween RV hype ophy and RV ailu e, which sugges s
specific changes in he signa u e o miRNAs du ing
PAH p og ession (33,34). The compa ison o he
a ailable da a on miRNA exp ession in he RV e eals
ew o e laps among he s udies (35–37). The di e en
app oaches o induce RV hype ophy and ailu e
(pulmona y a e y banding s.ch onichypoxia),
di e en su gical in e en ions leading o di e en
banding g adien s, and he use o di e en species
(mouse, a ,o ine)a elikely easons(34). The
mechanisms unde lying he ansi ion om
TABLE 1 Human Mic oa ay S udies in PAH
Pa ien Popula ion Tissue Mic oa ay Pla o m Analysis Me hod Obse a ions Da ase
6 pa ien s wi h PAH-SSc, 20 SSc
pa ien s wi hou PAH and 9
heal hy con ol subjec s
EPCs A yme ix Human Exon 1.0
ST A ay
A linea model was fi ed o he
log- ans o med exp ession
alues using he limma
package
2,175 genes we e ound
di e en ially exp essed
be ween con ol and SSc
PAH
GSE73674
15 pa ien s wi h PAH (6 IPAH, 4 PAH
CTD-PAH, 4 PAH seconda y o
CHD and 1 CTEPH) and 11 no mal
subjec s
Lung A yme ix Human Gene 1.0
ST A ay
A linea model was fi ed o he
log- ans o med exp ession
alues using he limma
package
12,834 genes we e ound
di e en ially exp essed in
he PAH coho compa ed
wi h con ol subjec s
GSE113439
6 pa ien s wi h PAH and 3 heal hy
con ol subjec s
PASMCs A yme ix Mul ispecies
miRNA-4 A ay
A linea model was fi ed o he
log- ans o med exp ession
alues using he limma
package
—GSE108707
14 pa ien s wi h PAH and 14 sex-/
age- ma ching con ol subjec s
PBMCs A yme ix GeneChip
Human Gene 1.0 ST A ay
A linea model was fi ed o he
log- ans o med exp ession
alues using he limma
package
2,624 genes we e ound
di e en ially exp essed
be ween con ol subjec s
and pa ien s wi h PAH
GSE131793
CHD ¼congeni al hea disease; CTD-PAH ¼connec i e issue disease-associa ed pulmona y a e ial hype ension; CTEPH¼ch onic h omboembolic pulmona y hype ension; EPC ¼endo helial cells de i ed
om ci cula ing endo helial p ogeni o ; IPAH ¼idiopa hic pulmona y a e ial hype ension; miRNA ¼mic o-RNA; PASMC ¼pulmona y a e y smoo h muscle cell; PAH ¼pulmona y a e ial hype ension;
PBMC ¼pe iphe al blood mononuclea cell; SSc ¼sys emic scle osis.
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compensa ed RV hype ophy o RV ailu e emain
unclea , and u he in es iga ions a e needed o gain
a be e unde s anding o miRNA exp ession pa e ns
in RV pa hological emodeling (34,36).
MICROARRAY TECHNOLOGY AND
DATA ANALYSIS
Genome-wide s udies, as mic oa ays and RNA
sequencing, deli e sc eening echnologies ha can
apidly iden i y genes, clus e s o co- egula ed genes,
o pa hways ha a e engaged in pa hological p o-
cesses. Mic oa ay echnology has been ex ensi ely
applied in he s udy o complex diso de s, including
cance , diabe es, and ca dio ascula diseases (38).
The in ica e pa hogenesis o PAHisasui able a ge
o mic oa ay echnology, which p o ides an addi-
ional ool o u he explo e he exp ession o key
genes and egula o y ne wo ks (18). In addi ion, high-
h oughpu sc eening is use ul in he de ec ion o
no el molecula a ge s o diagnosis, p ognosis, and
ea men . The e o e, in addi ion o he iden ifica ion
o coding RNAs, he exp ession o miRNAs can also be
analyzed by mic oa ay echnologies. These echnol-
ogies ely on nucleic acid hyb idiza ion a ge miR-
NAs and hei co esponding complemen a y p obes
(39). Fi s , complemen a y DNA is syn hesized om
isola ed miRNAs by e e se ansc ip ion, labeled
wi h a fluo escen dye, and hen hyb idized o he
mic oa ay. A e elimina ing any unbound comple-
men a y DNAs wi h a se ies o washing s eps, he
mic oa ay is scanned o measu e he fluo escence
in ensi y on each p obe spo , which is ansla ed in o
he ela i e amoun o each a ge miRNA om he
o iginal sample (39).
MiRNA a ays ha e expe ienced subs an ial ech-
nological p og ess in ecen yea s, wi h inno a ions
in p obe design, immobiliza ion echniques, sample
labeling, and signal de ec ion me hods (40). Al hough
genome-wide miRNA mic oa ay pla o ms con ey an
inexpensi e way o analyze a la ge numbe o
simul aneous measu emen s, hey a e unable o
iden i y new miRNAs (41), which is only possible wi h
RNA sequencing (42). Addi ional disad an ages o
hese pla o ms a e ela ed o miRNA in insic cha -
ac e is ics, such as a small ma gin o op imize he
hyb idiza ion condi ions seconda y o hei sho
leng h. Ano he disad an age is he limi ed speci-
fici y o miRNAs ha ha e simila sequences and
maydi e byasli leasasinglenucleo ide,which
dec eases he sensi i i y and specifici y o mic o-
a ays. Ano he pi all o he mic oa ay is he
inabili y o quan i y absolu e miRNA abundance (39).
Despi e all he limi a ions, mic oa ays a e s ill he
bes a ailable ool o compa ing he ela i e abun-
dance o p e-defined miRNAs be ween 2 condi ions
(e.g., pa ien s wi h disease and heal hy con ol sub-
jec s) (39). Rega ding PAH, he in o ma ion on
exp ession, unc ion, and ole o miRNA in human
issues is s ill limi ed. O e all, he a ay s udies p o-
ide a solid cha ac e iza ion o end-s age damage in
PAH (18). Al hough he cu en goal o mic oa ay
analysis in PAH has been o explo e pa e ns o
exp ession ha iden i y no el bioma ke s o help o
unde s and he pa hophysiology behind he disease
and hee ec so d ugs, heya ealsouse ul
a confi ming he ele ance o possible
e iologies un eiled in expe imen al models in
humans (18).
Conside ing his, we pe o med an analysis o hu-
man mul i issue mic oa ay da ase s (Table 1). These
da ase s included endo helial cells de i ed om
ci cula ing endo helial p ogeni o (EPCs) (GSE73674),
lung (GSE113439), PASMCs (GSE108707), and pe iph-
e al blood mononuclea cell (GSE131793) samples o
bo h pa ien s wi h PAH and heal hy con ol subjec s.
We used GEOque y lib a y o ex ac GEO da ase s.
Di e en ially exp essed genes we e de e mined by
TABLE 2 Dys egula ed miRNAs in Pulmona y Hype ension O ganized by Thei Mechanis ic Role
Mechanism miRNA
P oli e a ion miR-210; miR-204; miR-424; miR-503; miR-130/301; miR-193; miR-17w92; miR-145; miR-21; miR-124;
miR-140; miR-500b-3p; miR-206; miR-200; miR-338-3p; miR-141-3p; miR-133a-3p: miR-29a;
miR-214; miR-223; miR-100; miR-222; miR-25; miR-138; miR-1; miR-497, miR-1268, miR-665; miR-98;
miR-191; miR-30a-5p; miR-593-5p; miR-203; miR-429; miR-371b-5p; miR-760; miR-1181; miR-143;
miR-92b-3p; miR-135a-5p; miR-23a; miR-1281; miR-361-5p; miR-195-5p; miR-150; miR-4632;
miR-221-3p; miR-125; miR-34; miR-103/107 miR-322: miR-199b-5p; le -7a-5p; miR-20a; miR-328
Vasocons ic ion miR-130/301; miR-328; miR-190; miR-29b; miR-1; miR-543; miR-27b; mR-328
DNA damage miR-223; miR-204
Es ogen signaling and sex-specific miR-96; miR-29
Angiogenesis miR-208; miR-126; miR-495 miR-206
Inflamma ion miR-181a/b-5p; miR-146b; miR-135; miR-124
Abb e ia ion as in Table 1.
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using he limma package (43). A design ma ix was
c ea ed o he 2 g oups o samples (con ol subjec s
and pa ien s wi h PAH), and a linea model was fi ed
o he log- ans o med exp ession alues. The di -
e en ial exp ession was achie ed a e an empi ical
Bayes adjus men .
MiRNA WITH SIMILAR PATTERNS OF EXPRESSION IN
ANIMAL PH MODELS AND HUMAN PAH. O e he
yea s, moun ing e idence has explo ed he ole o
dys egula ed miRNAs in he hype p oli e a i e and
apop osis- esis an pheno ype o pulmona y ascula
cells, including PAECs, PASMCs, and PAAFs, in he
TABLE 3 A Lis o Well-Es ablished Dys egula ed miRNAs in PAH
miRNA Changes in PH Sample ype Ta ge s Func ion (Re . #)
miR-29 YHypoxic PH a PAAFs
a
-SMA, ex acellula ma ix collagen Fib osis (44)
MCT PH a PASMCs Collagen I (45)
(in adap a i e RV
hype ophy)
[
Fe ally-implan ed ao opulmona y shun
o ine
Collagen A1, Collagen 3A1 (37)
miR-124 YHuman PAH PASMCs NFAT PASMC p oli e a ion (46)
Human PAH PAAFs and human PAH PAECs PTPB1, MCP-1 P oli e a ion (47-49)
miR-140 Y
[
MCT PH a PASMCs TNF-
a
PASMC p oli e a ion (50)
SU/Hx/Nx and MCT PH a PASMCs SMURF1 PASMC p oli e a ion (51)
Human hypoxia PASMCs SOD2 PASMC p oli e a ion (52)
SU/Hx/Nx PH a RV MFN1 Apop osis o ca diac myocy es (53)
miR-204 YHuman PAH PASMCs HIF-1
a
PASMC p oli e a ion (54)
Human PAH PASMCs RUNX2 PASMC p oli e a ion (55)
Human PAH and MCT PH a s PASMCs SHP2, S c kinase, NFAT PASMC p oli e a ion (56)
Human PAH and hypoxia a PAECs ATG7 Au ophagy (57)
miR-210 [Hypoxic PH a PASMCs ISCU PASMC p oli e a ion (58)
E2F3 PASMC p oli e a ion (59)
MKP-1 PASMC p oli e a ion (60)
miR-1 [
Y
Plasma human PAH and Su/Hx/Nx PH a
lungs
SOD1, Cx43, CAV2, KLF4 Endo helial dys unc ion (61)
SU/Hx/Nx PH a lungs KCNA5 PASMC hype ophy (62)
Human PAH and hypoxic PH a PASMCs SphK1 PASMC p oli e a ion (63)
MCT PH a RV TGF
b
RV hype ophy (64)
miR-130/301 [SU/Hx/Nx mouse model EDN1 Vasocons ic ion (65)
MCT PH mouse PAECs BMPR2 Lung ascula emodeling (66)
Pulmona y fib osis mouse lung PPAR
g
-APOE-LRP8 Fib osis (67)
miR-138 [Hypoxic PH a PASMCs Ms 1 PASMC p oli e a ion (68)
Human PAH PASMCs MCUC PASMC p oli e a ion (69)
Human PAH PASMCs TASK-1 PASMC p oli e a ion (70)
miR-17w92 [Human PAH PAECs BMPR2 PAEC su i al (71)
Hypoxic PH mouse and MCT PH a lungs p21 PASMC p oli e a ion (72)
Human PAH PASMCs PDLIM5 PASMC dedi e en ia ion (73)
Hypoxic PH mouse PASMCs PHD2 PASMC p oli e a ion (74)
miR-21 [Human PAH PAECs BMPR2, RhoB Dec ease angiogenesis and
asodila ion
(75)
Human PAH PASMCs PTEN PASMC p oli e a ion (76,77)
Human PAH lung issue DDAH1 PASMC p oli e a ion (78)
Hypoxia PH mouse lung BMPR2 PASMC p oli e a ion (79)
miR-214 [Human PAH PASMCs PTEN, CCNL2 PASMC p oli e a ion (80,81)
Hypoxia PASMCs ARHGEF12 PASMC p oli e a ion (82)
Human PAH PASMCs MEF2C, MYOCD, SMC PASMC p oli e a ion (83)
miR-223 YHuman PAH PASMCs PARP-1 PASMC p oli e a ion (84)
Hypoxia PH mouse and a lung and hypoxia
PASMCs
RhoB, MYPT1, MLC2 PASMC p oli e a ion (85)
MCT PH a PASMCs ITGB3 PASMC p oli e a ion (86)
miR-424 [Human plasma and MCT PH a PAECs SMURF1, BMPR2 RV hype ophy (28)
YSU/Hx/Nx, MCT PH a and human PAH
PAECs
FGF2, FGFR1 PASMC p oli e a ion (87)
MCT ¼monoc o aline; PAAF ¼pulmona y a e ial ad en i ial fib oblas s; PAEC ¼pulmona y a e y endo helial cell; PASMC ¼pulmona y a e y smoo h muscle cell; PH ¼pulmona y hype ension; RV ¼ igh
en icle; SU/Hx/Nx ¼associa ion o Sugen 5416 wi h ch onic hypoxia ollowed by no moxia; o he abb e ia ion as in Table 1.
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di e en PH oden models and human PAH issues.
Because o he exhaus i e lis o such molecules
(Table 2), we ini ially ocused on key miRNAs wi h a
well-es ablished ole in PAH pa hogenesis (Table 3,
Supplemen al Figu e 1). We hen cons uc ed a hea -
map ha showed he log- old change exp ession o
hese miRNAs be ween pa ien s wi h PAH and
heal hy con ol subjec s in di e en issues (Figu e 1).
As depic ed in he hea map, miRNAs could be
dis inc ly exp essed be ween he di e en issues.
Al hough miR-29, miR-17, and miR-223 we e highly
exp essed in human PAH lung samples, miR-1,
miR-124, miR-130, miR-138, miR-140, and miR-210
we e down egula ed. Rega ding PASMCs, miR-204
and miR-29 we e down egula ed; con e sely,
miR-424 was up egula ed. In EPCs, miR-1, miR-140,
and miR-21 we e up egula ed, and miR-301 showed
lowe le els o exp ession compa ed wi h hose o
heal hy con ol subjec s. Finally, miR-130, he only
miRNA wi h di e en ial exp ession in pa ien s wi h
PAH e sus heal hy con ol subjec s in pe iphe al
blood mononuclea cells, was down egula ed. In
addi ion, in line wi h p e ious epo s in he li e a u e
(19), we obse ed a lack o conco dance in he pa e n
o exp ession o mos miRNAs be ween human PAH
(Figu e 1) and di e en animal PH models (Table 3).
This sugges ed ha di e en miRNA-dependen
mechanisms migh con ibu e o expe imen al PH
and human PAH, impai ing po en ial diagnos ic and
he apeu ic applica ions. Among all his he e ogene-
i y, we highligh ed 4 miRNAs, ha due o hei simila
exp ession pa e n among he di e en models, migh
ul ima ely ep esen a g ea e po en ial o ansla ing
in o he clinical a ena, namely, miR-29, miR-124, miR-
140, and miR-204 (Figu e 2).
MiR-29. The hypoxamiR miR-29 (88)isap o-
apop o ic miRNA amily ha a ge s myeloid cell
leukemia 1 (MCL1), which is a B-cell lymphoma 2
(BCL2) amily apop osis egula o ha can be sup-
p essed by pe oxisome p oli e a o -ac i a ed ecep-
o gamma (Ppa g) agonis s in a hea s (89).
Likewise, he miR-29 amily di ec ly a ge s >16
ex acellula ma ix genes, p o iding solid e idence
o an ifib o ic e ec s in di e en o gans, including
he lungs and hea (90). In human PAH, miR-29 is
significan ly up egula ed in he lung, bu down-
egula ed in PASMCs and has shown a end o lowe
le els in EPCs and pe iphe al blood mononuclea cells
FIGURE 1 Hea map Showing logFC Exp ession o he miRNAs Be ween Pa ien s Wi h PAH and Heal hy Con ol Subjec s in
Di e en Tissues
The highligh ed boxes ep esen p alues <0.05. EPC ¼endo helial cells de i ed om ci cula ing endo helial p ogeni o ; logFC ¼log- old
change; miR ¼mic o-RNA; PAH ¼pulmona y a e ial hype ension; PASMC ¼pulmona y a e y smoo h muscle cell; PBMC ¼pe iphe al blood
mononuclea cell.
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(Figu e 1). Chen e al. (91) ound an ele a ion o miR-
29 in lung issue among pa ien s wi h he edi a y PAH,
wi h a sex-specific in e ac ion o miR-29 wi h es o-
gen me abolism. Es ogen exposu e led o significan
educ ions in PPARgand CD36 ia he up egula ion o
miR-29 in Bmp 2 ansgenic mice lungs (91), which
sugges ed a co ela ion be ween ene gy me abolism
and emale ho mone signaling, wi h a p edisposi ion
o PAH. Rega ding PASMCs, TGFb1/SMAD3signaling
nega i ely egula ed he exp ession o miR-29b and
p omo ed collagen syn hesis in an MCT a model
(45). MiR-29b ea men supp essed collagen syn-
hesis by di ec ly a ge ing collagen I and blocking
PI3K/AKT signaling (45). Ac i a ion o PAAFs also
played a ole in he pulmona y ascula emodeling o
a ch onic hypoxia a model h ough a d as ic dec ease
o miR-29a, and i induced he exp ession o
a
-smoo h
muscle ac in (
a
-SMA) and ex acellula ma ix
collagen. In con as , a miR-29a mimic ep essed he
p oli e a ion, mig a ion, and sec e ion o PAAFs
induced by hypoxia and amelio a ed pulmona y
ascula emodeling (44). miR-29 seemed o be
FIGURE 2 Ne wo ks Rep esen ing Known In e ac ions
Ne wo ks ep esen ing known in e ac ions in ol ing (A) miR-29, (B) miR-124, (C) miR-140, and (D) miR-204 ela ed o PAH. The g een a ows depic ac i a ion,
whe eas he ed lines show inhibi ion. PAAF ¼pulmona y a e ial ad en i ial fib oblas s; PAEC ¼pulmona y a e y endo helial cell; o he abb e ia ions as in Figu e 1.
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