Exuberant pulmonary vascular remodeling is a hallmark of. Hypertension

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1 Hypertension Proteomi nlysis Implites Trnsltionlly ontrolle Tumor Protein s Novel Meitor of Olusive Vsulr Remoeling in Pulmonry rteril Hypertension Jessie R. Lvoie, PhD; Mrk L. Ormiston, PhD; rol Perez-Irtxet, PhD; Dvi W. ourtmn, PhD; ohu Jing, MD, PhD; Eliset Ferrer, PhD; Pol ruso, PhD; Mrk Southwoo, PhD; Willim S. Foster, PHE; Nihols W. Morrell, MD ; Dunn J. Stewrt, MD, FRP kgroun Pulmonry rteril hypertension (PH) is lethl isese hrterize y exessive prolifertion of pulmonry vsulr enothelil ells (Es). Hereitry PH (HPH) is often use y muttions in the one morphogeneti protein reeptor type 2 gene (MPR2). However, the mehnisms y whih these muttions use PH remin unler. Therefore, we sreene for ysregulte proteins in loo-outgrowth Es of HPH ptients with MPR2 muttions ompre with helthy ontrol sujets. Methos n Results totl of 416 proteins were etete with 2-imensionl PGE in omintion with liqui hromtogrphy/tnem mss spetrometry nlysis, of whih 22 exhiite signifintly ltere unne in loooutgrowth Es from ptients with HPH. One of these proteins, trnsltionlly ontrolle tumor protein (TTP), ws selete for further stuy euse of its well-estlishe role in promoting tumor ell growth n survivl. Immunostining showe mrke upregultion of TTP in lungs from ptients with HPH n iiopthi PH, ssoite with remoele vessels of omplex lesions. Inrese TTP expression ws lso evient in the SU5416 rt moel of severe n irreversile PH, ssoite with intiml lesions, ololizing with proliferting Es n the ventiti of remoele vessels ut not in the vsulr mei. Furthermore, silening of TTP expression inrese poptosis n rogte the hyperprolifertive phenotype of loo-outgrowth Es from ptients with HPH, rising the possiility tht TTP my e link in the emergene of poptosis-resistnt, hyperprolifertive vsulr ells fter E poptosis. onlusion Proteomi sreening ientifie TTP s novel meitor of enothelil prosurvivl n growth signling in PH, possily ontriuting to olusive pulmonry vsulr remoeling triggere y E poptosis. (irultion. 214;129: ) Key Wors: poptosis loo vessels hypertension, pulmonry proteomis Exuernt pulmonry vsulr remoeling is hllmrk of pulmonry rteril hypertension (PH). 1 PH is ssoite with ysregulte growth n poptosis resistne of lung vsulr ells, inluing enothelil ells (Es). These hyperprolifertive ells ontriute to olusive intiml n omplex plexiform lesions. 2 4 In reent yers, there hs een intense interest in the moleulr mehnisms tht unerlie the emergene of these norml ells in PH. The ientifition of heterozygous germline muttions in the gene enoing the one morphogeneti protein type II reeptor (MPR-II), memer of the trnsforming growth ftor reeptor superfmily, in heritle PH (HPH) represente mjor vne. 5 8 lthough the geneti sis of PH is known in some ses, the unerlying mehnisms tht link loss-of-funtion muttions in MPR2 with the evelopment of PH n its hrteristi lung vsulr lesions re still unler. linil Perspetive on p 2135 E injury n poptosis re key triggers for the evelopment of PH. Tretment of rts with vsulr enothelil growth ftor reeptor ntgonist, SU5416, together with perio of hroni hypoxi, 9,1 results in moel of severe, Reeive ugust 22, 213; epte Mrh 14, 214. From the Ottw Hospitl Reserh Institute, Sprott entre for Stem ell Reserh n Regenertive Meiine Progrm, Ottw, ON, n (J.R.L.,.P.-I., D.W...,.J., W.S.F., D.J.S.); University of Ottw, Fulty of Meiine, Deprtment of ellulr n Moleulr Meiine, Ottw, ON, n (J.R.L., W.S.F., D.J.S.); University of mrige, Deprtment of Meiine, enrooke s Hospitl, mrige, UK (M.L.O., E.F., P.., N.W.M.); n Ppworth Hospitl, Deprtment of Pthology, Ppworth, UK (M.S.). Drs Lvoie n Ormiston ontriute eqully. Drs Morrell n Stewrt re joint senior uthors. The online-only Dt Supplement is ville with this rtile t /-/D1. orresponene to Dunn J. Stewrt, MD, Ottw Hospitl Reserh Institute, 51 Smyth R, Ottw, ON, K1H 8L6, n. E-mil jstewrt@ohri. 214 merin Hert ssoition, In. irultion is ville t DOI: /IRULTIONH

2 2126 irultion My 27, 214 irreversile PH tht exhiits omplex, prolifertive vsulr lesions tht re remrkly similr to those of the humn isese. 9 Importntly, this severe PH phenotype oul e rogte y inhiition of poptosis, 1 suggesting tht this is n inititing event triggering the lter emergene of poptosis-resistnt, hyperprolifertive vsulr ells The ritil role of E poptosis s trigger in experimentl moels of PH is lso supporte y the effiy of E growth n survivl ftors (eg, vsulr enothelil growth ftor n ngiopoietin-1) in monorotlineinue PH. 13,14 The possile relevne of this prigm for humn PH is supporte y the oservtion tht silening of MPR2 15 or overexpression of mutnt MPR2 in humn Es 16 inreses poptosis. The lk of vilility of lung tissue from erly-stge isese represents signifint limittion for unrveling the inititing mehnisms of humn PH. The seletion pressures exerte on resient lung Es mke it is iffiult to seprte the iret effets of MPR2 muttions on E iology from retive hnges in response to the norml growth n survivl influenes of the lung PH milieu. Reently, it hs een reognize tht ells with n enothelil phenotype nerly ientil to tht of tissue-erive Es n e otine y ifferentil ulture of irulting mononuler ells. 17 These re vriously lle lte-outgrowth enothelil progenitor ells, enothelil olony-forming ells, or loo-outgrowth Es (OEs). 17,18 These ells, whih hve een hrterize extensively y our group 19,2 Tle 1. Ptient hrteristis Sex ssy ge, y MPR2 Muttion mpp, PVR, mm Hg I, L min 1 m 2 Woos Unit 6MWD, m Tretment HPH Femle Proteomis, I, 35 W9X N/ 459 ER, PDEI fnl ssys Femle Proteomis, 51 G828R ER, PGI I, fnl ssys Mle Proteomis, 21 W9X PGI, PDEI I, fnl ssys, IH Mle Proteomis, I, fnl /5 G-T PGI, PDEI ssys Mle I, fnl ssys 45 R32X ER, PDEI Femle I, fnl ssys 36 R213X ER, PGI Femle I, fnl ssys 79 R584X PDEI Femle IH 26 N/ N/ N/ Femle IH 25 Prtil eletion 95 N/ N/ N/ N/ Femle IH 38 N93S N/ PGI Femle IH 3 R899X N/ PGI, PDEI Mle IH 48 L41S N/ ER, PDEI Men SEM IPH Mle I PGI, PDEI Femle I N/ 337 ER, PDEI Femle I PGI Femle IH 48 N/ N/ N/ N/ N/ Femle IH N/ N/ N/ PGI Femle IH N/ PGI Femle IH N/ N/ N/ N/ Femle IH N/ PGI, ER Mle IH 25 N/ N/ N/ N/ N/ Men SEM inites lium hnnel loker; I, ri inex; ER, enothelin reeptor ntgonists; fnl ssys, funtionl ssys: ell prolifertion n poptosis; I, immunolotting; IH, immunohistohemistry; mpp, men pulmonry rtery pressure; N/, not ville; PDEI, phosphoiesterse inhiitor; PGI, prostylin nlogs; PVR, pulmonry vsulr resistne; n 6MWD, 6-minute wlking istne. P<.5 vs hereitry pulmonry rteril hypertension. Ptient reeive trnsplnttion in 1997.

3 Lvoie et l TTP in Pulmonry rteril Hypertension 2127 Tle 2. ontrol Sujet hrteristis ontrol Sujets Sex ssy ge, y Femle Proteomis, I, fnl ssys 37 Femle Proteomis, I, fnl ssys 45 Mle Proteomis, I, fnl ssys 28 Mle Proteomis, I, fnl ssys 3 Mle I, fnl ssys 2 Mle I, fnl ssys 4 Mle I, fnl ssys 41 Mle I, fnl ssys 39 Men 35 SEM 3 I inites immunolotting; n Fnl ssys, funtionl ssys: ell prolifertion n poptosis. Humn Lung Tissue Immunostining Explnte lung tissues were ollete from sujets with PH fter trnsplnttion. ontrol tissue ws selete from helthy region of the lung tht ws eing resete for the tretment of lung ner. Tissues were proesse s esrie in the online-only Dt Supplement. niml Stuies n SU5416 Injetions Stnr veterinry re ws use, following institutionl guielines, n the proeure ws pprove y the Institutionl niml re n Use ommittee (University of Ottw, Ottw, ON, n). single suutneous injetion of SU5416 [3-(3,5-imethyl-1H-pyrrol- 2-ylmethylene)-1,3-ihyroinol-2-one; 2 mg/kg, Sigm-lrih n Toris] or vehile (roxymethylellulose soium) ws elivere to 6-week-ol Sprgue-Dwley rts (hrles River, n), n ssessment of PH n RV remoeling ws performe 8 weeks therefter, s esrie in the online-only Dt Supplement. 9,1 n other lortories, exhiit hrteristis typil of mture Es. Therefore, we took vntge of the ility to erive OEs e novo from peripherl loo smples of HPH ptients n sex-mthe helthy ontrol sujets to etet ifferenes in protein expression levels etween OEs from ptients with HPH n ells from helthy ontrol sujets. Using proteomis, we hve ientifie trnsltionlly ontrolle tumor protein (TTP) s 1 of the 22 signifintly ysregulte proteins in HPH ells. TTP ws selete for further nlyses euse of its well-hrterize effets on mlignnt trnsformtion, 24,25 ner ell prolifertion n survivl, 26,27 n hroni inflmmtion. 28,29 Methos Isoltion n ulture of OEs OEs were isolte from sujets with HPH, sujets with iiopthi PH (IPH), n helthy ontrol sujets (Tles 1 n 2), s etile in the online-only Dt Supplement. 19,2 The humn stuy ws pprove y the mrigeshire 3 Reserh Ethis ommittee (referene No. 7/H36/134), n ll loo onors provie informe onsent in orne with the stuy protool. Two-Dimensionl PGE Whole protein lystes otine from OEs erive from 4 helthy ontrol sujets n 4 HPH ptients were sujete to 2-imensionl (2D) gel PGE. The 2D PGE seprtion of the OE protein lystes ws performe on 1% SDS-PGE gel in the DLT 6 eletrophoresis system (GE Helthre, Mississug, ON, n) t 1 m per gel n 25 for 18 hours until the romophenol lue rehe the ottom of the gel. Two tehnil replites were run seprtely for eh iologil smple for totl of sixteen 2D gels. Vriility etween the replites is shown in Figure I in the online-only Dt Supplement. The 16 gels were stine with Sypro Ruy protein gel stin following the mnufturer s instrutions (Sigm-lrih, Okville, ON, n) for totl protein stining. riefly, the gels were stine with Sypro Ruy gel stin overnight, wshe in 1% methnol n 7% eti i for 3 minutes, n imge on Vers Do 4 imger (io-r, Mississug, ON, n). Mss Spetrometry n Protein Ientifition Protein spots with ifferentil expression were exise with the EXQuest spot utter (io-r), s esrie in the online-only Dt Supplement. Sttistil nlysis Results re presente s men±sem. Sttistil nlysis ws performe with the GrphP Prism softwre, version 5.1. The mens of 2 groups were ompre with either Stuent t test or the unpire nonprmetri Mnn-Whitney test, s pproprite. The ifferenes etween multiple mens were etermine y 1-wy NOV, n when overll ifferenes were etete, the Tukey or Dunnett post ho nlysis ws use to etermine ifferenes etween iniviul mens. vlue of P<.5 ws onsiere sttistilly signifint. MPR-II/β-tin M r (k) HPH IPH HPH IPH HPH D Spheriity inex IPH MPR-II β tin HPH IPH Figure 1. Reue levels of one morphogeneti protein type II reeptor (MPR-II) in loo-outgrowth enothelil ells (OEs) from ptients with pulmonry rteril hypertension (PH)., Representtive imges of OEs from helthy sujets n hereitry PH (HPH) or iiopthi PH (IPH) ptients. Sle r, 1 μm., Representtive immunolot n () summry t (men±sem) of MPR-II levels in OEs from HPH (n=6) n IPH (n=3) ptients ompre with helthy ontrol sujets (n=5). These t re representtive of 3 seprte experiments. D, Spheriity inex of OEs from ptients with HPH (n=3) n IPH (n=3) ompre with helthy ontrol sujets (n=5). Sttistil ifferenes were ssesse y 1-wy NOV followe y post ho Dunnett test () or Mnn-Whitney test (D). P<.1, P<.5 vs helthy.

4 2128 irultion My 27, 214 Results MPR-II Levels re Reue in OEs From Ptients With HPH The seline hrteristis of the stuy sujets re presente in Tles 1 n 2. The men ge ws similr for HPH ptients n helthy ontrol sujets unergoing proteomi nlysis (35±6 n 35±4 yers, respetively; n=4 per group), with similr femle to mle istriution (1:1). Men pulmonry rteril pressure in these HPH ptients ws 61±5 mm Hg, n pulmonry vsulr resistne ws 12±2 Woos units, whih ws in keeping with the rest of the HPH ptients. In ontrst, IPH ptients exhiite slightly higher men pulmonry rteril pressures (78±6 mm Hg; P<.5) n pulmonry vsulr resistne (17.6±3.5 Woos units; P=NS). OEs exhiite typil enothelil morphology (Figure 1) n surfe mrker expression (ie, vsulr enothelil growth ftor reeptor-2, D31, von Willern ftor, D146) s previously pulishe. 19,2 The mjority of the MPR-II muttions in the HPH group oe for premture termintion of trnsltion (ie, R32X, R584X, R213X, n W9X [2 ptients]) n were preite to ffet ifferent omins of the MPR-II protein (W9X, lign ining/extrellulr; R213X, R32X, n R584X, kinse omin; Tle 1). n itionl muttion-ering line possesse oule sustitution muttion in the 5 untrnslte region of MPR2. 3 MPR-II levels in oth the HPH n IPH groups were reue y lmost 3-fol y Western lot nlysis ompre with helthy ontrol sujets (Figure 1 n 1). Interestingly, OEs from ptients with HPH n IPH were more elongte ompre ru Inorportion (OD 45) D leve spse-3/gpdh (Fluoresene Intensity) TNF-α + HX HPH HPH SD 1 2 Dys # TNF-α + HX Totl Live ells E HPH HPH Erly poptoti ells (% nnexinv + PI - ) Dys HPH NM SD with the helthy ontrol sujets n thus h signifintly lower spheriity inex (Figure 1D), wheres there ws no ifferene in overll ell size (Figure II in the online-only Dt Supplement). Greter Prolifertion ut Reue Survivl of OEs From HPH Ptients OEs from HPH ptients showe >2-fol greter ru inorportion over 48 hours (Figure 2) n signifintly greter totl live ell ounts ompre with ells from helthy ontrol sujet (Figure 2). Interestingly, levels of leve spse-3 lso tene to e inrese in OEs from HPH ptients ompre with helthy ontrol sujets in poptosis-inuing onitions (Figure 2 n 2D). Similrly, the perentge of erly-stge poptoti ells (nnexin V positive n propiium ioie negtive) mesure y flow ytometry ws signifintly greter in HPH ells ompre with ells from helthy ontrol sujets (Figure 2E). Quntittive nlysis of HPH Ptient Derive OE Proteins Using 2D PGE Whole OE protein lystes otine from 4 helthy ontrol sujets n 4 HPH ptients were seprte y 2D PGE. totl of 416 proteins were etete, of whih 22 were ifferentilly expresse t vlue of P<.5 (unorrete) in HPH ompre with helthy ontrol ells (Figure 3 n 3): 11 ownregulte (fol ifferene of <.8) n 11 upregulte (fol ifferene of >1.2; Figure 3). These proteins were ientifie y mss spetrometry (liqui hromtogrphy/tnem mss spetrometry). Tles 3 n 4 present the sores of -SP-3 GPDH TNF-α + HX Figure 2. loo-outgrowth enothelil ells (OEs) from ptients with hereitry pulmonry rteril hypertension (HPH) show inrese prolifertion n poptosis., ru inorportion (6 hours, 1 μmol/l) n () totl live ells for OEs from helthy sujets (n=5) n HPH ptients (n=4). These t re representtive of 2 seprte experiments in whih eh smple ws one in triplite., Representtive imges n (D) summry t (men±sem) of humn leve spse-3 (sp175) infrre immunossy showing the effet of 4 hours of tumor nerosis ftor-α (TNF-α) n yloheximie (HX) fter 16 hours of inution in SD meium. These t re representtive of 1 experiment in whih eh smple ws one in triplite. E, Summry t (men±sem) of nnexin V/propiium ioie (PI) stining n flow ytometry (n=5 for ll groups). NM represents norml meium. The t re representtive of 2 seprte experiments. Signifint ifferenes were ssesse y the Stuent t test. P<.5, P<.1, #P=.6, HPH vs helthy.

5 Lvoie et l TTP in Pulmonry rteril Hypertension pi 7 HPH 4 pi 7 1 kd D -Log 1 (P-vlue) M r (k) Log 2 (HPH/) HPH the mthing proility of etete pepties with theoretil tse pepties (Msot tse). TTP ws mong the upregulte proteins (fol inrese of 1.35), n representtive 2D gels showing the spot orresponing to TTP re presente in Figure 3, with the orresponing intensity plot in Figure 3. Upregultion of TTP protein expression ws onfirme y immunolotting (Figure 3D n 3E), with 1.7-fol inrese in HPH ompre with helthy ontrol ells (.46±.7 versus.26±.4; P<.5; Figure 3E). TTP Meites the Hyperprolifertive Phenotype of HPH ells n ts s Prosurvivl Ftor in OEs OEs were trnsfete with TTP sirn n ontrol (srmle) sirn (Figure 4). Immunolotting ws performe 48 hours fter trnsfetion of OEs, n the most effetive TTP sirn sequene ws use in susequent experiments (Figure III in the online-only Dt Supplement). TTP silening erese TTP protein levels y >9% (Figure 4). TTP knokown in OEs from HPH ptients reue ru inorportion over the 4 ys ompre with the ontrol sirn (Figure 4), wheres in ells from helthy ontrol sujets, TTP silening proue TTP (log 2 unne) E TTP β-tin TTP/β-tin HPH HPH Figure 3. Trnsltionlly ontrolle tumor protein (TTP) is upregulte in loo-outgrowth enothelil ells (OEs) from ptients with hereitry pulmonry rteril hypertension (HPH)., Representtive 2-imensionl PGE from helthy ontrol n ptient with HPH. rrows inite the spot orresponing to TTP protein for the respetive OE protein smple., In volno plot, 22 proteins were signifint in ifferentil expression (lue ots, P<.5; horizontl line represents P=.5)., Quntifition of spot intensity orresponing to TTP. Eh spot inites the men intensity of tehnil uplites of eh smple (n=4 in eh group). D, Representtive immunolot n summry t (men±sem; E) showing TTP levels in helthy or HPH OEs (n=5 for oth groups). These t re representtive of 3 seprte experiments. Sttistil ifferenes were ssesse y the Stuent t test. P<.5. moest n nonsignifint erese in prolifertion (Figure IV in the online-only Dt Supplement). This is onsistent with TTP ontriuting importntly to the hyperprolifertive phenotype of mutnt OEs. In ontrst, silening of TTP inrese erly-stge poptosis in OEs similrly in ells from helthy ontrol sujets (42.5±4.78% versus 29.81±6.11%; P<.5) n HPH ptients (4.14±2.86% versus 25.76±2.76%; P<.1) ompre with their respetive ontrol sirn onitions (Figure 4D). Silening of MPR2 Inrese TTP n Reue mir-27 OEs from helthy ontrol sujets were trnsfete with sirn trgeting MPR2 or ontrol sirn (Figure 5). MPR2 silening resulte in 95% reution in MPR-II gene n protein expression tht persiste to 72 hours fter trnsfetion (Figure 5 n 5), ssoite with elevte TTP protein (Figure 5 n 5) n gene expression (Figure 5D) ompre with ontrol sirn. mir-27, whih hs een reporte to regulte TTP gene expression, 31 ws foun to e signifintly reue in response to MPR2 silening s erly s 24 hours fter trnsfetion (Figure 5E). ssessment of sl mir-27 expression in helthy ontrol ells versus HPH

6 213 irultion My 27, 214 Tle 3. Liqui hromtogrphy/tnem Mss Spetrometry Dt of Proteins Foun to e Sttistilly Upregulte in OEs From HPH Ptients No. Protein Ientity SWISS-PROT No. MS Sore Nominl Mss, Mr lulte pi Signifint Peptie Mthes, n ov, % F (HPH/H) 1 Uroporphyrinogen eroxylse P S protese regultory suunit 7 P L-ltte ehyrogense hin P opine-1 Q Trnsltionlly ontrolle tumor protein P Glutthione synthetse P DN replition liensing ftor MM7 P α-tinin-1 P Perilipin-3 O Eukryoti peptie hin relese ftor P GTP-ining suunit ERF3 11 Protesome tivtor omplex suunit 1 Q OE inites loo-outgrowth enothelil ell; n HPH, hereitry pulmonry rteril hypertension. The protein ientities for eh spot re liste. The other olumns epit the following: SWISS-PROT No., SWISS-PROT ession numer; MS Sore, mss spetrometry sore initing the signifine of protein ientifition from peptie mss fingerprint oring to MSOT softwre (sore vlue >5 for P<.5); Nominl Mss, Mr, theoretil moleulr weight of the mthing protein; lulte pi, theoretil isoeletri point of the mthing protein; Signifint Peptie Mthes, numer of sttistilly signifint peptie mthes mthing to the protein; ov, %, perent of ientifie sequene to the omplete sequene of the known protein; F (HPH/H): fol hnge of the unne rtio of the spot etween HPH n helthy ontrol smples. P vlue ws lulte y the Stuent t test (2 tile); flse isovery rte=.8. P OEs lso emonstrte signifintly reue mir-27 levels in MPR2 muttion ering HPH ells (Figure 5F). Inrese TTP Protein Expression in Humn PH Lungs We nlyze TTP expression in explnte lungs of ptients with HPH n IPH unergoing trnsplnttion ompre with ontrol onor lungs. Setions from norml onor lungs showe miniml TTP immunoretivity tht ws osionlly lolize to ells tht pper to e within the ir spes (Figure 6 n 6, jent hemtoxylin n eosin stining in Figure 6). In ontrst, immunohistohemil stining of HPH n IPH lung setions revele n inrese in TTP immunoretivity in the ells lining the luminl surfe of remoele vessels (Figure 6 n 6e n Figure 6g n 6h, respetively, n jent hemtoxylin Tle 4. Liqui hromtogrphy/tnem Mss Spetrometry Dt of Proteins Foun to e Sttistilly Downregulte in OEs From HPH Ptients No. Protein Ientity SWISS-PROT No. MS Sore Nominl Mss, Mr lulte pi Signifint Peptie Mthes, n ov. (%) F (HPH/H) 1 Gunine nuleotie-ining protein G(I)/G(S)/G(T) P suunit β-2 2 MP-epenent protein kinse type I-α regultory P suunit 3 Protein isulfie-isomerse 3 P α-tinin-4 O WD repet-ontining protein 61 Q9GZS S protese regultory suunit 6 P Lmin-1 P V-type proton TPse suunit, rin isoform P Vinulin P Lmin-2 Q Proollgen-lysine,2-oxoglutrte 5-ioxygense 3 O OE inites loo-outgrowth enothelil ell; n HPH, hereitry pulmonry rteril hypertension. The protein ientities for eh spot re liste. The other olumns epit the following: SWISS-PROT No., SWISS-PROT ession numer; MS Sore, mss spetrometry sore initing the signifine of protein ientifition from peptie mss fingerprint oring to MSOT softwre (sore vlue >5 for P<.5); Nominl Mss, Mr, theoretil moleulr weight of the mthing protein; lulte pi, theoretil isoeletri point of the mthing protein; Signifint Peptie Mthes, numer of sttistilly signifint peptie mthes mthing to the protein; ov, %, perent of ientifie sequene to the omplete sequene of the known protein; n F (HPH/H), fol hnge unne rtio of the spot etween HPH n helthy ontrol smples. P vlue lulte y the Stuent t test (2 tile); flse isovery rte=.8. P

7 Lvoie et l TTP in Pulmonry rteril Hypertension 2131 sittp sitrl ru Inorportion (OD 45) sitrl sittp HPH ### Dys n eosin stining in Figure 6f n 6i, respetively). Immunofluoresent stining revele tht TTP-positive intiml ells (Figure 6g) oexpresse D31 (Figure 6i), n E mrker, wheres TTP stining ws ompletely sent from the intiml surfe of vessels from ontrol lungs (Figure 6). Interestingly, TTP stining ws notly sent in the vsulr mei (Figure 6g), whih stine strongly for α-smooth musle tin in oth remoele rteries from HPH setions n norml vessels from ontrol lungs (Figure 6e n 6, respetively), In the PH tissue, TTP ws lso strongly expresse in ventitil regions surrouning remoele vessels, onsistent with the known expression pttern of TTP in immune ells (Figure 6g), 29 whih re usully unnt in humn setions from ptients with en-stge isese. Inrese TTP Expression in the SU5416 Rt Moel of Severe PH single injetion of SU5416 (2 mg/kg) in Sprgue-Dwley rts from hrles River proue severe PH (Figure 7) n mrke right ventriulr remoeling (Figure 6), s well s omplex pulmonry rteril olitertive lesions (Figure 7). In NM OP DH M r (k) D TTP/ β -tin (% sitrl) Erly poptoti ells (% nnexinv + PI - ) sitrl sitrl sittp sirn sittp HPH TTP β tin Figure 4. Trnsltionlly ontrolle tumor protein (TTP) silening reues loo-outgrowth enothelil ells (OEs) prolifertion n survivl., Representtive imges of OEs fter trnsfetion with ontrol sirn (sitrl; n ) or TTP sirn (sittp; n ) t 1 nmol/l for 48 hours. Sle r, 1 μm., Representtive immunolot (top) showing TTP silening fter trnsfetion with sitrl n summry t (men±sem; ottom). These t re representtive of 5 ifferent OE smples. DH inites Dhrmfet; NM, norml meium; n OP, Optimem., ru inorportion in OEs from ptients with HPH (n=4) trnsfete with sitrl or sittp. These t re representtive of 2 seprte experiments performe in triplite. D, nnexin-v n propiium ioie (PI) stining y flow ytometry in helthy n HPH OEs trnsfete either with sitrl or sittp. Results re expresse s men±sem (helthy ontrol sujets, n=3; HPH, n=4). These t re representtive of 2 seprte experiments. Signifint ifferenes were ssesse y 1-wy NOV followe y the Tukey multiple-omprison test for (P<.5, P<.1 vs respetive ontrol sirn; ###P<.1 sitrl t y 4 vs 1) or pire Stuent t test for n D (P<.5, P<.1 vs respetive ontrol sirn). the ontrol lungs, there ws snt TTP immunofluoresene stining in the enothelium of smll rterioles n pillries (Figure 7). In ontrst, SU5416-trete rts showe mrke inrese in TTP ssoite with remoele rterioles n plexiform-like lesions (Figure 7e). TTP immunoretivity ws ololize to proliferting ell nuler ntigen positive, proliferting ells within vsulr lesions (Figure 8), n these intiml ells expresse the enothelil mrker von Willern ftor (Figure 8). TTP stining ws lso seen in the ventiti of remoele rteries, often ololizing with the mrophge mrker D68, onsistent with its expression y inflmmtory ells (Figure 8). Interestingly, inrese TTP expression ws pprent s erly s 1 week fter SU5416 ministrtion (Figure V in the online-only Dt Supplement), efore the evelopment of omplex vsulr lesions in this moel. Disussion PH is progressive vsulr isorer hrterize y ysregulte E growth n survivl, leing to remoeling n olitertion of pulmonry rterioles. 32 E injury n poptosis hve inresingly een reognize s triggers 11,33,34 resulting in the emergene of prolifertive n poptosis-resistnt resient lung vsulr ells 1,12 y mehnisms tht re lrgely unknown. To te, few stuies hve exmine the iret impt of MPR2 muttions on Es, whih ply key role in oth the initition n progression rteriolr remoeling in PH. OEs re highly prolifertive n show typil E olestone morphology n moleulr phenotype. 35 Unlike resient lung Es, OEs hve not een iretly expose to the isese environment of the PH lung; however, MPR-II expression ws reue not only in OEs from ptients with heterozygous MPR2 muttions ut lso in ells erive from ptients with IPH without ientifile muttions in MPR2. This is lso onsistent with reports showing erese MPR-II expression in lung tissue in IPH 36 n niml moels of this isese. 36 In ition, ells erive from HPH or IPH ptients exhiite phenotypi ifferenes in ell shpe, eing signifintly more elongte thn ells from helthy ontrol sujets. OEs from HPH ptients lso exhiite greter suseptiility to poptosis while showing inrese prolifertion ompre with helthy ontrol ells, in keeping with previous report 36 n onsistent with role for enothelil MPR2 muttions in oth the initition n progression of omplex lung vsulr lesions tht re hllmrk fetures of PH In this stuy, we ientifie 22 proteins tht were ifferentilly regulte etween OEs from HPH ptients n helthy ontrol sujets. Downregulte proteins inlue gunine nuleotie ining protein suunit β2 n MP-epenent protein kinse type-iα regultory suunit, oth of whih meite homeostti signling in Es. 37 Downregultion of gunine nuleotie ining protein suunit ws lso foun y 1-imensionl proteomi nlysis of PH lung tissue onute y ul-slm et l 38 ; however, in this se, it ws the α1 rther thn the β2 suunit. hlorie intrellulr hnnel proteins 1 n 4 were lso inrese in PH lung tissue in the previous stuy. lthough hlorie intrellulr hnnel protein expression ws not foun to e ltere in OEs from HPH in our stuy, hlorie intrellulr hnnel protein 1 phosphoryltion levels were inrese in seprte nlysis

8 2132 irultion My 27, 214 Figure 5. Silening of one morphogeneti protein type II reeptor (MPR-II) in loo-outgrowth enothelil ells (OEs) les to inrese trnsltionlly ontrolle tumor protein (TTP) n erese mir-27 expressions., Gene expression of MPR2 in helthy ontrol OEs (n=3) t 24, 48, n 72 hours fter trnsfetion with sirn towr MPR2 (simpr2) or sirn ontrol pool (sip). These t re representtive of 1 experiment performe in triplite., Representtive immunolot for MPR-II, TTP, n β-tin protein levels in helthy ontrol OEs (n=3) t 72 hours fter sirn trnsfetion., Quntifition of TTP protein levels reltive to β-tin in helthy ontrol OEs (n=3) t 72 hours fter sirn trnsfetion. D, TTP gene expression in helthy ontrol OEs (n=3) t 72 hours fter sirn trnsfetion. These t re representtive of 1 experiment performe in triplite. E, Gene expression of mir-27 in helthy ontrol OEs (n=3) t 24, 48, n 72 hours fter sirn trnsfetion. These t re representtive of 1 experiment performe in triplite. F, seline mir-27 expression in OEs from helthy onors (n=8) n HPH ptients (n=7). These t re representtive of 1 experiment performe in triplite. Sttistil ifferenes were ssesse y the Stuent t test. P<.5. of the phosphoproteome using the sme smples (unpulishe oservtions). opine ws inrese in oth stuies, ut gin ifferent isoforms were ientifie y ul-slm et l n our group, opine-3 n opine-1, respetively. In ition, we ientifie numer of proteins involve in ell prolifertion suh s DN replition liensing ftor MM7, omponent of the prereplitive omplex, 39 n the eukryoti peptie hin relese ftor GTP-ining suunit ERF3, whih were foun to e inrese in HPH-erive ells. One of the upregulte proteins in HPH ells ws TTP (lso lle fortilin). 26 TTP is highly networke protein tht promotes ell growth n survivl n hs een implite s meitor of mlignnt trnsformtion. 4 TTP interts with multitue of signling pthwys to inhiit poptosis (p53 n l-xl) 41,42 n to promote ell survivl n prolifertion (EF1, EF18β, n Rhe). 43,44 TTP is lso ritil regultor of the murine oule minute 2 p53 xis 45 y preventing uto-uiquitintion of murine oule minute 2, therey promoting murine oule minute 2 meite uiquitintion n trnsriptionl inhiition of p Interestingly, inhiition of the murine oule minute 2 p53 intertion with the ntiner rug Nutlin3 hs reently een shown to e n effetive tretment for experimentl moels of PH y ttenuting pulmonry rtery smooth musle ell prolifertion. 46 Thus, TTP is promising nite ftor in the pseuomlignnt hnges in vsulr ell phenotype tht hve reently een esrie in PH. TTP (histmine-relesing ftor) 29 is lso potent meitor of hroni inflmmtion n hs een implite in the lk of resolution of irwy inflmmtion in sthm. 47 Moreover, reent report emonstrte tht TTP ws enrihe in poptoti nnovesiles erive from humn umilil vein Es, meiting ell-ell survivl signling in systemi rteril smooth musle ells. 48 This rises the possiility tht TTP my represent link etween pulmonry rteriolr E poptosis s triggering event n the susequent retive vsulr ell prolifertion n inflmmtion tht hrterizes estlishe PH. role for TTP in PH ws supporte y inrese expression y immunostining in lung setions of ptients with HPH n IPH, ssoite with omplex rteriolr intiml n plexiform lesions. Expression of TTP ws lso oserve in rt moel of severe ngioprolifertive PH. TTP-positive ells within the plexiform-like lesions oexpresse proliferting ell nuler ntigen, mrker of ell prolifertion. Inrese vsulr TTP expression ws evient s erly s 1 week fter SU5416 injetion (Figure V in the online-only Dt Supplement), preeing the evelopment of omplex lesions or hemoynmi normlities in this moel. t 8 weeks, there ws unnt TTP stining, whih ws lolize to severely remoele rterioles, minly in the intim n ventiti (Figure V in the onlineonly Dt Supplement). Interestingly, no inrese in TTP stining ws seen in the monorotline rt moel of PH, even t vne stges of isese (Figure VI in the onlineonly Dt Supplement). euse the monorotline moel oes not exhiit intiml vsulr lesions, the lk of upregultion of TTP my provie further support speifi role for this protein in the olitertive intiml hnges n omplex plexiform-like lesions tht re foun in oth the SU5416 moel n humn PH. However, it is importnt to reognize tht these t re only orreltive n tht the iret role of TTP in the progression of vsulr remoeling n hemoynmi normlities nees to e onfirme in stuies using phrmologil or moleulr mnipultions to trget this pthwy. TTP silening mrkely reue prolifertion preferentilly in HPH OEs, onsistent with role for this protein in

9 Lvoie et l TTP in Pulmonry rteril Hypertension 2133 TTP ontrol e HPH g h IPH RVSP (mmhg) RV/(LV+S) Rtio ontrol SU5416. ontrol SU5416 f i TTP α-sm TTP α-sm DPI TTP α-sm DPI DI H&E ontrol α-sm D31 TTP e f g h ontrol SU5416 PH e f g h Figure 6. Trnsltionlly ontrolle tumor protein (TTP) expression is elevte in lung vsulr lesions of ptients with pulmonry rteril hypertension (PH)., Lung setions from ontrol sujets (n=6) showe little to no TTP immunoretivity (rown) t the luminl surfe of loo vessels (highlighte with rrows; n ) with hemtoxylin n eosin stining of n jent setion (). Lung setions from ptients with hereitry PH (HPH; n=6; n e) n iiopthi PH (IPH; n=6; g n h) showing efinite immunoretivity for TTP in the ells lining the luminl surfe of the remoele vessels (highlighte with rrows) with hemtoxylin n eosin stining on n jent setion (f n i, respetively). Originl mgnifition 1 (,, n g) or 2 (,, e, f, h, i). Sle r, 1 μm., Immunofluoresene imges (α-smooth musle tin [α-sm], re; D31, green; TTP, purple; DPI, lue) from ontrol sujet ( ) n n IPH ptient (e i). ololiztion of TTP-positive ells with D31 enothelil ell mrker from the PH ptient is shown in i. Sle r, 1 μm. meiting the hyperprolifertive phenotype of HPH ells. In ontrst, knokown of TTP inrese poptosis similrly in OEs from ptients with HPH n helthy ontrol sujets. These finings re in greement with erlier gene-silening stuies tht supporte n importnt role for TTP in ell prolifertion n poptosis in vrious ner ells, inluing from prostte ner, metstti rinom ell lines (MF7), n skin squmous ell rinom. 24,49,5 Interestingly, OEs from HPH ptients exhiite inrese poptosis ompre with helthy ells espite hving higher levels of TTP expression. This my suggest tht moest inreses in sl TTP expression nnot fully ompenste for the loss of MPR-II survivl signling in these ells. We lso foun tht sirn silening of MPR2 in OEs inrese TTP expression. This ws i Figure 7. Trnsltionlly ontrolle tumor protein (TTP) immunostining in vsulr plexiform lesions of rt moel of severe pulmonry rteril hypertension (PH)., Right ventriulr systoli pressure (RVSP) n () rtio of right ventrile weight to left ventrile plus septum weight [RV/(LV+S)] in vehile (roxymethylellulose)-trete ontrol (n=3) or SU5416-trete (2 mg/kg; n=3) Sprgue-Dwley rts. Signifint ifferenes were ssesse y the Stuent t test. P<.1., TTP (green) n α-smooth musle tin (α-sm; re) immunofluoresene stining in smll pulmonry rteries from ontrol ( ) n SU5416-trete (e h) rts. Nulei re ounterstine with DPI (lue). Differentil interferene ontrst (DI) imge merge with TTP, α-sm, n DPI in n h. rrow highlights TTP-positive ells within the lumen of smll rteriole. Results re representtive setions from 3 nimls per group performe in triplite. Sle r, 5 μm. ssoite with signifint reution in mir-27, whih hs previously een shown to regulte TTP expression in the ontext of orl ner. 31 Further exmintion of seline mir-27 expression in HPH OEs emonstrte signifint reution ompre with helthy ontrol ells. These finings lign well with previous report y Drke n ollegues, 51 whih emonstrte reue mir-27 in pulmonry rteril Es from HPH ptients, n provie potentil link etween reue MPR-II signling in PH n elevtion of enothelil TTP. Using nonise proteomi pproh, we now report tht, mong numer of ysregulte proteins, MPR2 muttions re ssoite with inrese TTP expression in ptient-erive, enothelium-like ells. This potent prosurvivl protein ws lso mrkely inrese in omplex rteriolr lesions in oth humn n experimentl PH. This is the first time tht TTP hs een implite in PH, n these t support potentil role for this tumor protein s link etween E poptosis n the trnsformtion of vsulr Es to hyperprolifertive n pseuomlignnt phenotype. knowlegments We thnk Dr Lwrene Puente for performing the mss spetrometry nlysis n Dr Jmes Gomes for the use of his PDQuest softwre n 2D gel imging n utting systems.

10 2134 irultion My 27, 214 jent setions e Soures of Funing Dr Lvoie is supporte y Fons e l reherhe en snté u Quée sholrship. Dr Ormiston is fune y the ritish Hert Fountion n the nin Institutes of Helth Reserh. This work ws fune y wrs to Dr Stewrt from nin Institutes of Helth Reserh (No ) n the Ontrio Reserh Exellene Fun (REST No. GL2-1-42). itionl funing ws provie y ritish Hert Fountion Progrm Grnt to Dr Morrell. Infrstruture support ws provie y the mrige Ntionl Institute for Helth Reserh iomeil Reserh enter. None. TTP TTP vwf TTP f PN α-sm α-sm D68 TTP α-sm DPI Dislosures TTP D68 DPI Referenes 1. Pietr GG, Ewrs WD, Ky JM, Rih S, Kernis J, Shloo, yres SM, ergofsky EH, runge H, Detre KM, Fishmn P, Golring RM, Groves M, Levy PS, Rei LM, Vreim E, Willims GW. Histopthology of primry pulmonry hypertension: qulittive n quntittive stuy of pulmonry loo vessels from 58 ptients in the Ntionl Hert, Lung, n loo Institute, Primry Pulmonry Hypertension Registry. irultion. 1989;8: TTP PN DPI vwf α-sm DPI Figure 8. Trnsltionlly ontrolle tumor protein (TTP) immunostining ssoite with proliferting enothelil ells n with mrophges. Lung setions re from SU5416-trete rts., Strong immunoretivity for TTP () ololizing with proliferting ell nuler ntigen (PN) positive ells (). rrow inites ololiztion for intrluminl ells in through. Results re representtive setions from 3 nimls, eh experiment performe in uplite., TTP immunostining () orresponing to von Willern ftor (vwf) stining (e) in jent setions, representtive of 3 nimls, eh experiment performe in uplite., rrows inite ololiztion of TTPpositive ells () with mrophges (D68; ) in the ventiti of remoele vessel. Results re representtive setions from 3 nimls. Originl mgnifition 1 or 2 s pproprite. Sle r, 5 μm. α-sm inites α-smooth musle tin. g h 2. Morrell NW, Yng X, Upton PD, Journ K, Morgn N, Sheres KK, Tremth R. ltere growth responses of pulmonry rtery smooth musle ells from ptients with primry pulmonry hypertension to trnsforming growth ftoret(1) n one morphogeneti proteins. irultion. 21;14: Zho YD, ourtmn DW, Ng DS, Ro MJ, Deng YP, Trogis J, Hn RN, Stewrt DJ. Mirovsulr regenertion in estlishe pulmonry hypertension y ngiogeni gene trnsfer. m J Respir ell Mol iol. 26;35: Tuer RM, Mreki J, Rihter, Fijlkowsk I, Flores S. Pthology of pulmonry hypertension. lin hest.me. 27;28:23 42, vii. 5. Lne K, Mho RD, Puiulo MW, Thomson JR, Phillips J 3r, Loy JE, Nihols W, Tremth R. Heterozygous germline muttions in MPR2, enoing TGF-et reeptor, use fmilil primry pulmonry hypertension. Nt Genet. 2;26: Thomson JR, Mho RD, Puiulo MW, Morgn NV, Humert M, Elliott G, Wr K, You M, Mikhil G, Rogers P, Newmn J, Wheeler L, Higenottm T, Gis JS, Egn J, rozier, Peok, llok R, orris P, Loy JE, Tremth R, Nihols W. Spori primry pulmonry hypertension is ssoite with germline muttions of the gene enoing MPR-II, reeptor memer of the TGF-et fmily. J Me Genet. 2;37: Mho RD, Puiulo MW, Thomson JR, Lne K, Morgn NV, Wheeler L, Phillips J 3r, Newmn J, Willims D, Gliè N, Mnes, MNeil K, You M, Mikhil G, Rogers P, orris P, Humert M, Donni D, Mrtensson G, Trnejerg L, Loy JE, Tremth R, Nihols W. MPR2 hploinsuffiieny s the inherite moleulr mehnism for primry pulmonry hypertension. m J Hum Genet. 21;68: Deng Z, Morse JH, Slger SL, uervo N, Moore KJ, Venetos G, Klhikov S, ynis E, Fisher SG, rst RJ, Hoge SE, Knowles J. Fmilil primry pulmonry hypertension (gene PPH1) is use y muttions in the one morphogeneti protein reeptor-ii gene. m J Hum Genet. 2;67: e K, To M, lzoui, Ito M, Fgn K, ool D, Voelkel NF, MMurtry IF, Ok M. Formtion of plexiform lesions in experimentl severe pulmonry rteril hypertension. irultion. 21;121: Trseviiene-Stewrt L, Kshr Y, lger L, Hirth P, M Mhon G, Wltenerger J, Voelkel NF, Tuer RM. Inhiition of the VEGF reeptor 2 omine with hroni hypoxi uses ell eth-epenent pulmonry enothelil ell prolifertion n severe pulmonry hypertension. FSE J. 21;15: Jursz P, ourtmn D, ie S, Stewrt DJ. Role of poptosis in pulmonry hypertension: from experimentl moels to linil trils. Phrmol Ther. 21;126: Tuer RM, Groves, esh D, Voelkel NF. Exuernt enothelil ell growth n elements of inflmmtion re present in plexiform lesions of pulmonry hypertension. m J Pthol. 1994;144: mpell I, Zho Y, Snhu R, Stewrt DJ. ell-se gene trnsfer of vsulr enothelil growth ftor ttenutes monorotline-inue pulmonry hypertension. irultion. 21;14: Zho YD, mpell I, Ro M, Ng D, Stewrt DJ. Protetive role of ngiopoietin-1 in experimentl pulmonry hypertension. ir Res. 23;92: Teihert-Kuliszewsk K, Kutryk MJ, Kuliszewski M, Kroui G, ourtmn DW, Zuo L, Grnton J, Stewrt DJ. one morphogeneti protein reeptor-2 signling promotes pulmonry rteril enothelil ell survivl: implitions for loss-of-funtion muttions in the pthogenesis of pulmonry hypertension. ir Res. 26;98: Yng X, Long L, Reynols PN, Morrell NW. Expression of mutnt MPR-II in pulmonry enothelil ells promotes poptosis n relese of ftors tht stimulte prolifertion of pulmonry rteril smooth musle ells. Pulm ir. 211;1: Yoer M. Defining humn enothelil progenitor ells. J Throm Hemost. 29;7(suppl 1): Lvoie JR, Stewrt DJ. Genetilly moifie enothelil progenitor ells in the therpy of riovsulr isese n pulmonry hypertension. urr Vs Phrmol. 212;1: Ormiston ML, Deng Y, Stewrt DJ, ourtmn DW. Innte immunity in the therpeuti tions of enothelil progenitor ells in pulmonry hypertension. m J Respir ell Mol iol. 21;43: Geti I, Ormiston ML, Rouhni F, Toshner M, Movssgh M, Nihols J, Mnsfiel W, Southwoo M, rley, Rn, Vllier L, Morrell NW. prtil n effiient ellulr sustrte for the genertion of inue pluripotent stem ells from ults: loo-erive enothelil progenitor ells. Stem ells Trnsl Me. 212;1: Hur J, Yoon H, Kim HS, hoi JH, Kng HJ, Hwng KK, Oh H, Lee MM, Prk Y. hrteriztion of two types of enothelil progenitor

11 Lvoie et l TTP in Pulmonry rteril Hypertension 2135 ells n their ifferent ontriutions to neovsulogenesis. rteriosler Throm Vs iol. 24;24: Ingrm D, Me LE, Tnk H, Mee V, Fenoglio, Mortell K, Pollok K, Ferkowiz MJ, Gilley D, Yoer M. Ientifition of novel hierrhy of enothelil progenitor ells using humn peripherl n umilil or loo. loo. 24;14: Lin Y, Weisorf DJ, Solovey, Heel RP. Origins of irulting enothelil ells n enothelil outgrowth from loo. J lin Invest. 2;15: Tuyner M, Susini L, Prieur S, esse S, Fiui G, mson R, Telermn. iologil moels n genes of tumor reversion: ellulr reprogrmming through tpt1/ttp n SIH-1. Pro Ntl Si U S. 22;99: Tuyner M, Fiui G, Prieur S, Lespgnol, Gént, euourt S, Duflut D, esse S, Susini L, vrelli J, Mors D, mson R, Telermn. Trnsltionlly ontrolle tumor protein is trget of tumor reversion. Pro Ntl Si U S. 24;11: Li F, Zhng D, Fujise K. hrteriztion of fortilin, novel ntipoptoti protein. J iol hem. 21;276: mson R, Pee S, Mrine J, Di Fiore PP, Telermn. TPT1/ TTP-regulte pthwys in phenotypi reprogrmming. Trens ell iol. 213;23: Shroeer JT, Lihtenstein LM, MDonl SM. n immunogloulin E-epenent reominnt histmine-relesing ftor inues interleukin-4 seretion from humn sophils. J Exp Me. 1996;183: MDonl SM, Rfnr T, Lngon J, Lihtenstein LM. Moleulr ientifition of n IgE-epenent histmine-relesing ftor. Siene. 1995;269: lre M, Mho RD, Jmes V, Morrell NW, Tremth R. hrteriztion of the MPR2 5 -untrnslte region n novel muttion in pulmonry hypertension. m J Respir rit re Me. 27;176: Lo WY, Wng HJ, hiu W, hen SF. mir-27-regulte TTP s novel plsm iomrker for orl ner: from quntittive proteomis to post-trnsriptionl stuy. J Proteomis. 212;77: Humert M, Morrell NW, rher SL, Stenmrk KR, MLen MR, Lng IM, hristmn W, Weir EK, Eikelerg O, Voelkel NF, Rinovith M. ellulr n moleulr pthoiology of pulmonry rteril hypertension. J m oll riol. 24;43(suppl S):13S 24S. 33. Gine SP, Ruin LJ. Primry pulmonry hypertension. Lnet. 1998;352: Stenmrk KR, Mehm RP. ellulr n moleulr mehnisms of pulmonry vsulr remoeling. nnu Rev Physiol. 1997;59: Mein RJ, O Neill L, Sweeney M, Guuri-Fuhs J, Griner T, Simpson D, Stitt W. Moleulr nlysis of enothelil progenitor ell (EP) sutypes revels two istint ell popultions with ifferent ientities. M Me Genomis. 21;3: tkinson, Stewrt S, Upton PD, Mho R, Thomson JR, Tremth R, Morrell NW. Primry pulmonry hypertension is ssoite with reue pulmonry vsulr expression of type II one morphogeneti protein reeptor. irultion. 22;15: rreu S, Imizumi-Sherrer T, Weiss M, Fust DM. Intrellulr trgeting of the type-i lph regultory suunit of MP-epenent protein kinse. Trens riovs Me. 22;12: ul-slm V, Whrton J, upitt J, errymn M, Ewrs RJ, Wilkins MR. Proteomi nlysis of lung tissues from ptients with pulmonry rteril hypertension. irultion. 21;122: Stoeer K, Tlsty TD, Hpperfiel L, Thoms G, Romnov S, orow L, Willims ED, Willims GH. DN replition liensing n humn ell prolifertion. J ell Si. 21;114(pt 11): Telermn, mson R. The moleulr progrmme of tumour reversion: the steps eyon mlignnt trnsformtion. Nt Rev ner. 29;9: Yng Y, Yng F, Xiong Z, Yn Y, Wng X, Nishino M, Mirkovi D, Nguyen J, Wng H, Yng XF. n N-terminl region of trnsltionlly ontrolle tumor protein is require for its ntipoptoti tivity. Onogene. 25;24: mson R, Pee S, Lespgnol, Vys R, Mzzrol G, Tosoni D, ollu I, Vile G, Rorigues-Ferreir S, Wynenele J, hloin O, Hoeeke J, Mrine J, Di Fiore PP, Telermn. Reiprol repression etween P53 n TTP. Nt Me. 212;18: ns, Psser J, Shlk V, Nny-Porteois V, rile V, mzllg N, llni D, Tufino R, rgentini M, Mors D, Fiui G, Gou, Mirne M, mson R, Telermn. Trnsltionlly ontrolle tumor protein ts s gunine nuleotie issoition inhiitor on the trnsltion elongtion ftor eef1. Pro Ntl Si U S. 23;1: Hsu Y, hern JJ, i Y, Liu M, hoi KW. Drosophil TTP is essentil for growth n prolifertion through regultion of Rhe GTPse. Nture. 27;445: mson R, Krp JE, Telermn. Lessons from tumor reversion for ner tretment. urr Opin Onol. 213;25: Mourret N, Mros E, i S, Gry-oo G, Sker M, Houssini, Duois-Rne JL, oyer L, ozkowski J, Derumeux G, msellem V, not S. tivtion of lung p53 y Nutlin-3 prevents n reverses experimentl pulmonry hypertension. irultion. 213;127: Kshiwkur J, no T, Mtsumoto K, Kimur M, Kitur J, Mtho MH, Zjon DM, Ozeki T, R, MDonl SM, Sirgnin RP, roie DH, Kwkmi Y, Kwkmi T. Histmine-relesing ftor hs proinflmmtory role in mouse moels of sthm n llergy. J lin Invest. 212;122: Sirois I, Rymon M, rssr N, ilhier JF, Fejev M, Hmelin K, Lonono I, enyn M, Pshezhetsky V, Héert MJ. spse-3-epenent export of TTP: novel pthwy for ntipoptoti interellulr ommunition. ell Deth Differ. 211;18: Gnnsekr M, Thirugnnm S, Zheng G, hen, Rmswmy K. Gene silening of trnsltionlly ontrolle tumor protein (TTP) y sirn inhiits ell growth n inues poptosis of humn prostte ner ells. Int J Onol. 29;34: Wu D, Guo Z, Min W, Zhou, Li M, Li W, Luo D. Upregultion of TTP expression in humn skin squmous ell rinom inreses tumor ell viility through nti-poptoti tion of the protein. Exp Ther Me. 212;3: Drke KM, Zygmunt D, Mvrkis L, Hror P, Wng L, omhir S, Erzurum S, lre M. ltere MiroRN proessing in heritle pulmonry rteril hypertension: n importnt role for Sm-8. m J Respir rit re Me. 211;184: linil Perspetive Pulmonry rteril hypertension (PH) is omplex isese for whih the unerlying moleulr mehnisms re only inompletely unerstoo, n urrent therpies, whih ress minly vsomotor hnges, hve limite effiy. It is now reognize tht progressive inreses in pulmonry vsulr resistne re ssoite with omplex olusive rteril remoeling lolize primrily to the istl rteriolr tree n hrterize y ysregulte vsulr ell growth n survivl. In experimentl moels, it hs een shown tht enothelil ell (E) injury n poptosis trigger vsulr remoeling, resulting in the retive emergene of poptosis-resistnt n hyperprolifertive Es y s-yet unknown mehnisms. The ientifition of the geneti sis of hereitry PH hs provie further insights into the mehnisms of humn isese, with the emonstrtion tht loss-of-funtion muttions in the one morphogeneti protein reeptor 2 (mpr2) gene preispose to E poptosis through loss of survivl signling. In this stuy, we hve eluite the moleulr mehnisms unerpinning hnges in growth n survivl in loo-outgrowth Es from ptients with hereitry PH ompre with ells from helthy ontrol sujets using n unise proteomi pproh. We hve ientifie tht expression of inrese trnsltionlly ontrolle tumor protein in loo-outgrowth Es from ptients with hereitry PH is lrgely responsile for ysregulte ell growth, n we report mrkely inrese trnsltionlly ontrolle tumor protein expression in the lungs of ptients with PH, s well s in n experimentl moel of severe PH ssoite with olitertive intiml lesions. These finings suggest tht trnsltionlly ontrolle tumor protein my e key link etween E poptosis n retive rteril remoeling n is potentilly novel therpeuti trget for PH.