Journal of Cell Science 39-63 USA 53792-4673, USA 4673, USA 53792-4673, WI Madison, CSC, neligmlclradclua ehnssfrendoglin the for However, endoglin mechanisms understood. with fully 2010). cellular not humans are and function al., in molecular seen et underlying defects (Mahmoud vascular haploinsufficiency the While 1999). of al., et some (Eng+/ Li 1999; heterozygote al., endoglin et mature (Bourdeau of network remodeling vascular inappropriate to due defects cardiovascular decreased Its angiogenesis angiogenesis. altered to during leads expression increases the in level expression Endoglin vasculature 1999). al., et Miller 1990; transforming Letarte, for receptor factor- auxiliary endothelial transmembrane an growth as vascular functions homodimeric and on (EC) kDa cells expressed 180 predominantly a glycoprotein is Endoglin/CD105 Introduction words: Key Eng+/ (EC). The cells humans. endothelial in telangiectasia-1 of hemorrhagic pathways hereditary TGF signaling cause these endoglin pro-differentiation to in and endoglin factor- Mutations of pro-proliferative growth contribution the transforming the balancing for However, receptor through auxiliary angiogenesis an regulates is (Eng) Endoglin Summary 10.1242/jcs.117275 doi: 1392–1405 126, ß Science Cell of Journal 2013 January 4 Accepted ( correspondence for *Author 3 2 1 Park SunYoung and TGF- canonical non-canonical in of participating quiescence by and endothelium activation the regulates Endoglin 1392 nolndfcet(Eng deficient humans. in Endoglin malformations 1999). vascular al., hemorrhagic decreased and hereditary et in (HHT-1) with associated telangiectasia-1 results are Li gene and protein 2000; endoglin the of of al., level allele et single a Bourdeau in 1999; Mutations al., et Bourdeau function and development vascular from EC gain retinal Eng+/ from prepared To aortas we obscure. in EC, attenuated remain of similarly properties was mechanisms angiogenesis angiogenic underlying sprouting affect endoglin Aortic the that Eng+/ of morphogenesis. endoglin mechanisms demonstrated, of haploinsufficiency regulatory and been importance that autonomous the has show cell Eng+/+ Although the development we retinopathy. into study vascular ischemic insight present and detailed oxygen-induced the during angiogenesis In neovascularization in haploinsufficiency. retinal expression endoglin of with attenuation in humans results in seen defects vascular ossetwt utie ciaino ioe-ciae rti iae(AK ahas n bratSa-eedn signaling Smad-dependent aberrant and were pathways, changes these (MAPK) Mechanistically, kinase production. protein NO and mitogen-activated synthase Eng+/ of NO in activation pathways endothelial of sustained expression with reduced consistent but VEGF of levels increased inln ahasmdltn ohteatvto n uecneo h nohlu uigangiogenesis. during endothelium the of quiescence and activation the both modulating pathways signaling eateto hraooy nvriyo icni colo eiieadPbi elh 0 ihadAeu,K/5 S,Mdsn WI Madison, CSC, K6/456 Avenue, Highland 600 Health, 53792- Public WI and Madison, Medicine CSC, of H4/444 School Avenue, Wisconsin Highland 600 of K6/456 Health, University Avenue, Public Pharmacology, Highland and of 600 Medicine Department Health, of Public School and Wisconsin Medicine of of University Pediatrics, School of Wisconsin Department of University Sciences, Visual and Ophthalmology of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. nignss nohla el,Etaellrmti,MPkns ahas Thrombospondins pathways, kinase MAP matrix, Extracellular cells, Endothelial Angiogenesis, b (TGF 2 1 2 er .DiMaio A. Terri , C ae oehr u eut nesoeteiprac fedgi nbt aoia n o-aoia TGF non-canonical and canonical both in endoglin of importance the underscore results our together, Taken EC. [email protected] b mot ie h Eng+/ The mice. Immorto Brae ta. 07 ogsand Gougos 2007; al., et (Bernabeu ) 2 / 2 2 iedea 1.–15from E10.5–11.5 at die mice ) ieaeval,te exhibit they viable, are mice ) nvivo in ) 1 e Liu Wei , nvitro in Atu ta. 2000; al., et (Arthur b n aberrant and 2 1 b inln pathways signaling huinWang Shoujian , ciiis n oeseiial ouaino Cpeoye ean elusive. remains phenotype, EC of modulation specifically more and activities, Cwr oeahrn,ls irtr,adfie oudrocapillary undergo to failed and migratory, less adherent, more were EC r-rlfrtv n r-ifrnito inln ahasof pathways TGF signaling EC. Neubauer pro-differentiation 1998; and al., pro-proliferative et TGF 1999). Hirschi al., 1989; et al., TGF on et depend (Antonelli-Orlidge membrane basement differentiation, of pericyte production of and induction migration, and proliferation nignsswieAK ciaini rwhihbtr n is and inhibitory growth is activation ALK5 while TGF angiogenesis protein non-canonical mitogen-activated on elusive. the remain endoglin pathways, including (MAPK) of pathways, impact signaling the pathways, (TGF rvosycniee naern aoia TGF canonical aberrant been have in pathways considered signaling ALK5) previously Smad-dependent of of and presence importance the downstream TGF requires (ALK1 and ALK1 the ALK5 of both mediate ALKs activation addition, which In Phosphorylated events. proteins Seki 2000; Smad 2003). al., these et phosphorylate Oh from al., 1993; al., signals et et Lutty balancing 2008; (Lu, by pathways two ALK5, and kinase-1(ALK1) aiu nigncpoessicuigrglto fEC of regulation including processes angiogenic Various L1atvto yTGF by activation ALK1 b 1 hitn .Sorenson M. Christine , b (TGF I)adtodsic TGF distinct two and RII) b b ouaeE rpristruhTGF through properties EC modulate ,wt motn oe nvsua ucin TGF function. vascular in roles important with ), b euae nignsstruhblnigthe balancing through angiogenesis regulates b 2 2 I Guase l,20) lhuhthe Although 2003). al., et (Goumans RII ie nadto,Eng+/ addition, In mice. ieaeval n xii oeo the of some exhibit and viable are mice b rmtsE ciainduring activation EC promotes 2 n ae Sheibani Nader and b I cii-eetrlike activin-receptor RI, eerhArticle Research 2 Cexpressed EC b b rcpo II -receptor b signaling activity 1,3, b b b * Journal of Cell Science ncotmc lodea 1.–15adehbtfiueof failure exhibit and E10.5–11.5 at ALK1 2001). die al., also et mice deficient (Larsson knockout ALK5 development from vascular EC supporting and abrogated migration 1996), and production fibronectin al., altered show et mice Oshima 2001; (Larsson sack al., yolk the et in defect development vascular al., to et due E10.5 (Lebrin TGF stability of and Lack maturation 2005). vessel mediate to thought oei euaiggot fnwbodvses(ufe l,2003; al., et (Duff vessels blood new a of play growth may regulating and in angiogenesis role during expressed highly Eng+/ is in Endoglin neovascularization retinal of Attenuation Eng+/ the Furthermore, observed. also were results These Eng+/ Matrigel. in alterations in with consistent also morphogenesis We OIR. capillary during Eng+/ neovascularization showed retinal of attenuation investigation. further inducing needs retinal function to EC contributes and haploinsufficiency neovascularization and endoglin How retina. become to leaky, damage the further expression causing and to hemorrhage, and fragile vitreous, retina the are into sprout VEGF vessels the these causes Unfortunately, angiogenesis. upregulating vessels blood ischemic, sufficient causes the stage, of this and During days. 5 lack vessels for oxygen) (20% air are blood retinal mice room the to high returned 12, developing of day The postnatal At vessels. the growth levels. existing the of oxygen obliteration time, further high impedes this to sensitive During oxygen highly days. al., is 75% vasculature 5 et to exposed are for (Smith mice (P7) condition oxygen 7 day prematurity postnatal model, this of In 1994). retinopathy human the angiogenesis of model reproducible highly autonomous understood. cell poorly remains phenotype, specifically EC TGF more of 1997). modulation to and Pepper, endoglin activities, 1996; of contribution angiogenesis al., exact et the (Krupinski However, angiogenesis in development. effects cell TGF factor, muscle proliferation TGF smooth vascular Although in defects and exhibit vascular mutants angiogenesis and ALK1 differentiation However, EC mice. on in minimal development is mutation Effect 2000). ALK1 al., et of (Oh vessels blood dilated and network capillary Results during and EC canonical of both TGF of state non-canonical engagement quiescence through and perhaps activation angiogenesis is both endoglin of for expression essential appropriate that demonstrate results our TGF TGF to to Smad2/3 reduced respond the TGF with of not expression consistent phosphorylation did Smad1/5 and enhanced TGF Smad1/5, of phosphorylated effect level, anti-proliferation (NO) Eng+/ in oxide oxide (iNOS) NOS nitric inducible nitric endothelial and of (eNOS) in modestly synthase expression were decreased decrease Src with and significant MAPKs, consistent Akt p38 A phosphorylated and Eng+/ of affected. ERK levels the JNK, the a of Mechanistically, while and activation expression apoptosis. VEGF sustained increased of exhibited to due rate part, in reduced rate, Eng+/ faster the a However, at production proteins. ECM in various changes of with concomitant interactions cell-matrix xgnidcdicei eioah OR ntemuei a is mouse the in (OIR) retinopathy ischemic Oxygen-induced eew hwdta alisfiinyo nolnrsle in resulted endoglin of haploinsufficiency that showed we Here 2 b b Caels irtr n alt undergo to fail and migratory less are EC b a engnrlyrgre sa anti- an as regarded generally been has a o fetdi Eng+/ in affected not was b b I nteecls oee,tersos of response the However, cells. these in RII I rAK nue mroi ehlt at lethality embryonic induces ALK5 or RII inln pathways. signaling b hw ohsiuaoyadinhibitory and stimulatory both shows b xrse oe eesof levels lower expressed , 2 2 nohla elcl and cell-cell endothelial Cwr eitn othe to resistant were EC nvivo in 2 2 C Collectively EC. n recapitulates and Cproliferated EC b 2 -mediated 2 C was EC, 2 mice b EC by ie ssoni i.1,, n++adEng+/ and Eng+/+ 1A,B,E Fig. Eng+/ in and shown Eng+/+ compared in As we OIR mice. during neovascularization, neovascularization and during retinal endoglin of development role physiological vascular the into insight further gain Du ifrn otaa tgso eeomn.Tertnlvsesof vessels retinal Eng+/ The of development. vasculature of stages retinal postnatal the different examined next Eng+/ in We vascularization retinal postnatal Defective aclrcl ulipeeto h irossd ftertn eertn h inner the penetrating retina the of of number side (*** mean membrane vitreous limiting the the represent on bars present in nuclei Data cell vascular section. Methods and mice. four Materials the of non- eyes in mean eights the in retina are each bar of ( each area in whole to Data relative hypoxia. area perfused to response in days areas 5 perfused for oxygen) (20% air room and days 5 ( for oxygen 75% to exposure ( Eng+/+ ( 12-day-old Eng+/ from in prepared neovascularization were retinal wholemounts of Attenuation 1. Fig. hw nFg F hs ossetwt rvoswr,endoglin angiogenesis. work, during previous with essential are consistent is data Thus, expression 1F. the Fig. of in assessment shown quantitative The Eng+/ (supplementary neovascularization. contrast, OIR In S1). during retinal Fig. mice enhanced material of and Eng+/+ upregulation vasculature in significant retinal neovascularization with in consistent expression into is endoglin retina the This from vitreous. Eng+/+ sprouting vessel air, the tufts room vascular oxygen-mediated to numerous return have after high mice that show to 1C,D Fig. sensitivity obliteration. similar exhibited acltr (magnification vasculature F C B uniaieassmn frtnlnoaclrzto efre sdescribed as performed neovascularization retinal of assessment Quantitative ) wle l,20;Jri ta. 06;tnDjee l,20) To 2008). al., et Dijke ten 2006b; al., et Jerkic 2007; al., et ¨wel xoe ohg xgn(5)fr5dy,o otaa a 7(1)following (P17) 17 day postnatal or days, 5 for (75%) oxygen high to exposed ) )and( nolnadrglto fagoeei 1393 angiogenesis of regulation and Endoglin D .Tertnswr tie ihclae Vt iulz h retinal the visualize to IV collagen with stained were retinas The ). P , 6 0.001; 5.( 25). 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Fig. increased material in (supplementary mice observed cells Eng+/+ with compared mice vascular we Eng+/+ tertiary significant proliferating and However, as secondary a of without number rate branches. and mice, diameters similar Eng+/+ vessel on as Eng+/ impact a density The vascular at S2A–D). similar Fig. retina material the (supplementary of periphery Eng+/ 1394 tts n eosre infcn hnei opooyof confluent morphology Cell in and change dishes. significant sub-confluent a culture observed at and we gelatin-coated 2003) and examined states, al., on was et Eng+/ cultured and (Su morphology Eng+/+ us were from EC by Methods. mice and described Material method in detailed novel a using 2 iesrue rmteotcnreadetne othe to extended and nerve optic the from sprouted mice ora fCl cec 2 (6) 126 Science Cell of Journal 2 mot mice Immorto 2 iehad mice 2 mice 2 Eng+/ iiihdcl-elitrcinwihbcm oeovosat obvious more morphology, became which confluence. shape interaction cobblestone cell-cell diminished packed closely Eng+/ showed EC nldn CM1 CM2 CM1 EF1 and of markers, expression VEGFR1, in EC decrease a VCAM-1, other observed also of We ICAM-2, We 2B). (Fig. levels EC. VEGFR2 ICAM-1, expression Eng+/+ in including with the downregulated compared was determined and expression 2008), integrity B4-lectin al., EC the et sustained of Eng+/ Eng+/ one for (DiMaio PECAM-1, proteins in angiogenesis Moreover membrane decreased EC. essential Eng+/+ were with levels compared expected, As expression analysis. FACS PECAM-1 endoglin by including B4-lectin markers and EC endoglin, various (CD31), of level expression the odtrietepoete fEng+/ of properties the determine To 2 2 2 Ccmae ihEg/ C(i.2) hl Eng+/+ While 2A). (Fig. EC Eng+/+ with compared EC C(i.2) hs el eeas espstv for positive less also were cells These 2B). 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EC similar retinal of isolations different the Note Eng+/ (** from section. mice aortas methods Eng+/+ of and sprouting Materials in the decrease in significant described as performed was ( Eng+/ format. and digital Eng+/+ in from photographed were networks tube-like Eng+/ ragoei rpriso C iia eraein Eng+/ from decrease prepared ICAM-2 similar lysates S5A,B). and Fig. of retinal material PECAM-1 A (supplementary in modulation including observed markers EC. in was EC expression of of expression endoglin properties in for roles proangiogenic important role Eng+/ with significant proteins in two angiogenesis, VEGFR1, and ICAM-2 seseto eut rmAadB aaaetema ubro branch Eng+/ of by (*** number formation mean tube the (magnification in are fields decrease Data high-power B. 5 and from A points from results of (magnification assessment format digital in photographed and Eng+/ EC from retinal aortas of of morphogenesis sprouting capillary Abrogated 3. 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Cmnlyrstill monolayer EC EC ossetwith consistent , . 2 2 2 mice, EC EC 2 in Journal of Cell Science ugse htatrto ncl-elitrcin a looccur. also may interactions cell-cell in alteration that suggested Eng+/ in morphology Eng+/ altered in The junction cell-cell of Abrogation analysis FACS by We integrins properties. various adhesive including of EC expression and examined proteins also ECM of impact expression major a on has Eng+/ haploinsufficiency endoglin in Thus, reduced EC. was Eng+/+ TSP-2 and TSP-1, of IV an collagen shows and Eng+/ osteopontin 5F in tenascin-C, Fig. of 1991). production Tucker, in 1989; increase Gordon, and Sabet al., et 2009; (Lund inflammation and important repair, play wound migration, and cell EC in roles by secreted components TSP-2 ECM their major and the (TSP-1), are of thrombospondin-1 ECM Eng+/ IV, expression various and collagen of osteopontin, and Eng+/+ production proteins in the determined proteins ECM next of We cell production receptors. specific influenced the significantly through is proteins by migration Cell ECM integrins. receptor to surface adhere Eng+/ cell in proteins Endothelial ECM of production Altered each acid in intracellular cells of Eng+/ levels adherent in the The were of left measuring phosphatase. were number by cells quantified cells The was no Non-adherent BSA. well until with adhere. plate coated the to wells washing plates gently allowed 96-well by on were removed coated cells were Varying 2010). proteins and al., ECM et of (Park described concentrations previously as IV, collagen 1396 eut eecnitn ihrdcdmgaino Eng+/ of migration reduced with consistent were Moreover, results 5E). (Fig. EC Eng+/+ Eng+/ with compared IV collagen and xld h osblt fcagsi fiiyadaiiyo these of avidity and affinity not in do integrins. changes results of possibility these the However, exclude S4). Fig. material (supplementary Eng+/ and Eng+/+ 2 2 dee oclae hl n++E i o.These not. did EC Eng+/+ while I collagen to adhered ora fCl cec 2 (6) 126 Science Cell of Journal a Ccmae ihEg/ C ncnrcs expression contracts, In EC. Eng+/+ with compared EC 1, a 2, 2 a 3, Cepesdsmlrlvl fteeintegrins these of levels similar expressed EC a 5, 2 a Cwr oeahrn nF,VN, FN, on adherent more were EC v, a 2 5 2 b 1, Ccmae ihEg/ EC Eng+/+ with compared EC C eacnC fibronectin, Tenascin-C, EC. a v b 3, 2 b 2 1, Ccmae with compared EC EC b 2 3and EC 2 b .The 8. 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Fig. nvitro in A 5 nrclua ircoie(O level (NO) oxide nitric Intracellular ) ;** 3; P , b 2 0.01; ctnnwssmlri both in similar was -catenin 2 C P n noln( endoglin and ) EC elcluesse and/or system culture cell , 2 ie hsi ncnrs to contrast in is This mice. 0.01, n iewsaaye by analyzed was mice 5 ) ( 3). b 2 n ctnnadZO-1 and -catenin E 5 B h xrsinof expression The ) Cwr stained were EC 3). 2 h xrsinof expression The ) C n may and EC, D expression ) 2 P , 2 Cwas EC 0.05, EC. b 2 2 - Journal of Cell Science nolnadrglto fagoeei 1397 angiogenesis of regulation and Endoglin w ifrn sltoso Cwt iia results. similar with EC of isolations with different repeated two were J experiments Image These using software. determined of were intensities bands relative protein The the TSP-2. and TSP-1 IV, collagen osteopontin, fibronectin, tenascin-C, for n ellsts rprdfo n++and Eng+/+ from Eng+/ prepared lysates, medium cell conditioned and in proteins, ECM various n eemnda ecie nteMtrasand (*** Materials section the Methods in was described IV as collagen determined and I collagen vitronectin, Eng+/ and Eng+/+ adhesions) focal (magnification (red; vinculin and actin) (green; Eng+/+ in Eng+/ fibers and stress actin and adhesions focal onigtenme fclsmgae through (** migrated filter cells the of number the by counting determined transwell, a through migration cell frslsi (*** A in results of oioe ypoorpywti 8h 48 (magnification within photography was by closure monitored wound on and monolayers plates EC gelatin-coated of wounding scratch by in Eng+/ proteins (ECM) matrix of extracellular expression altered and enhanced adhesion migration, Attenuation 5. 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Letarte, and A. Gougos, 8 meiso 3 m sn loecn irpaerae Vca 1420 (Victa2 triplicate reader numbers. in cell microplate times to three fluorescent normalized conducted were and a Assays PerkinElmer). using Counter, nm) Multilabel 535 nm/emission 485 # ebr .P,MMrry . oasi . a,M,Ky,B . ii,V and V. Dixit, A., B. Keyt, M., Yan, J., Kowalski, A., McMurtrey, P., H. Gerber, S. Kumar, and M. J. Garland, C., Li, E., S. Duff, and B. D. Rifkin, C., H. Dietz, D., Loch, L., Zilberberg, S., Smaldone, L., Carta, M. Letarte, and E. M. Faughnan, A., M. Bourdeau, Letarte, and J. D. Dumont, A., P. Bourdeau, C. Vary, and A. B. Conley, C., Bernabeu, rn rmteNtoa ntttso elh[rn ubrP30 number [grant Health Support of Center Institutes Cancer National Carbone a the P. P30-EY016665]; from Paul and Grant [grant Wisconsin N.S.) Health of (to of Institutes University EY021357 National EY016995, the by numbers supported the the was work to edited This contributed and Funding and mice study, S.W. editing. the and the and writing manuscript generated conceived W.L. interpretation, N.S. C.M.S. T.A.D., manuscript; staining; manuscript; the retinal and collection the wrote data performed experiments, and the of analysis, majority the performed S.P. contributions Eng+/ Author the proving for Li Dean Dr thank We Acknowledgements Means appropriate. with when evaluated comparisons were multiple samples for treated and unpaired control student’s between differences Statistical analysis Statistical Du N. Sheibani, and M. C. Sorenson, A., E. Scheef, Q., Huang, S., E. Wang, A., T. Y. DiMaio, Zhang, and R. M. Derynck, B. Weinstein, and E. Tournier-Lasserve, E., Dejana, E., Torsney, I., D. Wilson, G., Renforth, J., A. Smith, J., Ure, M., H. Arthur, A. P. D’Amore, and R. S. Smith, B., K. Saunders, A., Antonelli-Orlidge, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.117275/-/DC1 online available material Supplementary in months. Deposited 12 was AstraZeneca. after S.P. from release Award for 0950057G]. PMC Predoctoral number a [grant by from grant supported Association a by Heart supported from is Award American C.M.S. Research and Foundation. 1-10-BS-160] Research a number Retina of the [grant recipient Association a Diabetes American is Research the N.S. from Blindness. award Prevent departmental to unrestricted an and CA014520]; wl . ln,N,Jri,M,Aeao . Bolan M., Arevalo, M., Jerkic, N., Eleno, A., ¨wel, .5wr osdrdsignificant. considered were 0.05 lcpoeno ua nohla cells. endothelial human of glycoprotein eurmn o l-/D activation. Flk-1/KDR for 3 Requirement phosphatidylinositol the through survival N. Ferrara, mice. (Fbn1)-null fibrillin-1 of aortas the in signaling F. 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