Micro-RNA Analysis in Pulmonary Arterial Hypertension: Current Knowledge and Challenges

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. 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 1153 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. 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 1154 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. 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 1155 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. 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 1156 (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. 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 1157