© 2010 Nature America, Inc. All rights reserved. authors authors contributed equally to this work. should Correspondence be addressed to F.L. or C.L.M. ([email protected]). ([email protected]) 6 de la Santé et de la Recherche Médicale U711, Paris, France. Recherche Scientifique UMR144, Paris, France. 1 over the mucosal entire disseminated severe anemia. Multiple lesions and intense in result that hemorrhages nasal gastrointestinal and chronic cause can lesions Such wall. vascular the of thinning also but lumen vascular the of dilation only by not characterized are lesions Telangiectasic treated. and recognized not if complications severe cause can these ; intervening without and and liver. Large AVMs consist of direct connections between septum, oral mucosa nasal and gastrointestinal the tract to in large AVMs dilatation) in the , of (regions telangiectases include arteriovenous malformations (AVMs), which range from small cate to the nucleus after activation to the same downstream Smad signal proteins, and second messengers that cells translo endothelial by primarily expressed are receptors growth factor- the transforming ( (ALK-1) the either in mutations by caused is It individuals. 10,000 in 1 about of prevalence a with disease vascular dominant autosomal an is HHT malformations. vascular of treatment the for strategy therapeutic a as useful be may which maturation, vessel induce to thalidomide of ability the demonstrate findings Our humans. in treatment thalidomide of effects the explain may mechanisms similar that showed thalidomide with not or treated HHT with individuals from epithelium nasal of Biopsies pericytes. to cells endothelial from signaling PDGF with interference genetic or pharmacological by reversed partially were treatment thalidomide of effects The activation. cell mural stimulated and cells endothelial in expression (PDGF-B) factor-B growth platelet-derived increased treatment Thalidomide defects. wall vessel rescued thus and coverage cell mural stimulated treatment HHT, of thalidomide model experimental an endoglin), (encoding the in mutation null a for heterozygous mice In stabilization. vessel blood enhanced and hemorrhage reduced of result a as rose individuals treated the of levels hemoglobin blood The tested. HHT with subjects of group small a of majority the in (epistaxis) nosebleeds of frequency and severity the reduced thalidomide with treatment that here treat. to We report difficult clinically are and life of quality their affect severely can which nosebleeds, recurrent develop affected individuals Many malformations. vascular by characterized disorder inherited an is (HHT) telangiectasia hemorrhagic Hereditary L Christine Mummery J Cornelis J Westermann Bréant Christiane Franck Lebrin telangiectasia hemorrhagic reduces epistaxis in hereditary with individuals Thalidomide stimulates vessel and maturation nature medicine nature Received 18 September 2009; accepted 8 March 2010; published online 4 April 2010; St. St. Antonius Hospital, Nieuwegein, The Netherlands. Institut National de la Santé et de la Recherche Médicale U833, Collège de France, Paris, France. ENG gene or the gene encoding activin receptor-like kinase-1 kinase-1 receptor-like activin encoding gene the or gene ACVRL1 1 , Samly Srun

). These genes encode receptors for members of of members for receptors encode genes These ). advance online publication online advance 1 , Thomas Mathivet 7 , 8 6 , Frans Disch β (TGF- 3,4 1 , Karine Raymond . Clinical manifestations .HHT of manifestations Clinical β 3 ) ) family of ligands Hubrecht Hubrecht Institute, Biology Developmental and Stem Cell Research, Utrecht, The Netherlands. 7 1 Department Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands. 6 , Bruno Larrivée , Johannes J Mager 5

Institute Institute of Human Genetics, Centre International for Life, Newcastle University, Newcastle, UK. 2 , Martin Sabrina 1,2 . These 1 - , Jean-Léon Thomas

6 ing, a major feature of HHT that increases in frequency with age with frequency in increases that HHT of feature major a ing, bleed to prone become and weaken to vessels cause abnormalities These cells. mural surrounding the from the of ment disengage in resulting degradation, and production matrix cellular extra to and to migration and lead proliferation to cell endothelial thought abnormal is factors angiogenic key these of Deregulation production (VEGF) factor growth endothelial vascular increased TGF- decreased to attributed been has HHT with effects side have all and intervals hemorrhage-free ited lim best, offer, at options However, these drugs. anti-inflammatory of application topical or skin, with epithelium surgical nasal of pathways, replacement fibrolytic and coagulation the of manipulation difficult. To limit blood loss, the options used few include therapeutic treatment local making individuals, affected in common are surface viduals indi affected in anomalies vascular these from resulting treating symptoms in beneficial be therefore could of Inhibitors , Repke J Snijder The development of vascular malformations in individuals individuals in malformations vascular of development The doi:10.1038/nm.213 1 1 , Stieneke van den Brink 0 . However, antiangiogenic drugs may also affect normal normal affect also may drugs antiangiogenic However, . 2 Institut Institut Curie, Centre de Recherche, Centre National de la 6 4 , Anne Eichmann 1 , Helen M Arthur 3 , FreitasCatarina

1 5 , 8 , &

Eng s e l c i t r a 4 β Institut Institut National activation and and activation gene gene 5 . 1 , 8 These These

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4,9  - - - - - . . © 2010 Nature America, Inc. All rights reserved. administration administration of 100 mg of thalidomide daily significantly ( either in mutations had and years-old 75- and 48- between were HHT with pregnancy in nausea treat to 1960s the in recurrent prescribed that to and comparable was severe given dose The with thalidomide. with HHT epistaxis with subjects seven treated We Thalidomide lowers nosebleed frequency in subjects with HHT RESULTS malformations. vascular treating for utility therapeutic broader a have may thalidomide that possibility the depth explore more to in remodeling vessel and angiogenesis affects thalidomide which by mechanisms the investigated also We have (epistaxis). nosebleed ing of severity the on effects monitoring specifically HHT, vessels immature of cells pathway are as such the VEGF that angiogenesis, or inhibiting pathways in involved stimulating signaling of components of expression regulate cular vas from bleeding inhibits thalidomide which by mechanisms The thalid received who diseases gastrointestinal individuals in certain bleeding with inhibits treat reportedly to it used now pathologies: is various it criteria, inclusion guidelines controlled strict carefully Under and years. recent in renaissance a enjoyed has defects limb cause to thalido enough of was mide tablet 100-mg one Just gestation. of 36 day and 20 day between window time-sensitive short, a within place took exposure development times of specific antiangiogenic at tissues of and organs fetal to result supply blood a the reduced that newborns, activity in evident became defects severe when market the from removed was 1960s, early action. of mechanism described a well- and preferably implementation clinical cautious fore requires reduce may HHT there and physiology, normal with affect also may but symptoms disease individuals in agent this of use drug label VEGF, to cause both reported been has targets that antibody an bevacizumab, and processes, physiological Subject Table 1 A  its and subjects seven the of six in epistaxis of frequency the lowered # We treatedsubjects1–7withthalidomide;8–10wereuntreatedcontrols. 10 indicatesthesubjectsin 2 1 6 5 4 3 9 8 7

# # # Here we have examined the effects of thalidomide on subjects with with on Here subjects we thalidomide of have the effects examined the in pregnancy in nausea treat to used originally Thalidomide, # s e l c i t r

malformations are unknown, although it has been reported to reported been has it although unknown, are malformations

2 Clinical characteristics Clinical of characteristics subjects with HHT after treatment with thalidomide. ENG 0 Severity of . More recently, it has been shown to target endothelial tip endothelial . to target More shown recently, been it has bleeding *** *** *** *** ** ** ** ** ** ** or or 1 monthbeforethalidomide ACVRL1 Figure 4 o mide as an antiangiogenic cancer therapy cancer antiangiogenic an as mide Number per 1 c week 4 ( . *,mild;**,moderate;***,severe.NA,notavailable.§ NA 32 32 26 18 35 21 35 59 28 . Table 13,14 1 . when drug occurred These defects and and Duration 15–20 15–20 25–30 10–90 20–25 20–60 (min) Nosebleeds 10,15–17 NA NA NA 30 1 1 Supplementary Fig. 1 Fig. Supplementary 13,14 and reduce epistaxis reduce and . However, thalidomide thalidomide However, . and in HHT patients patients HHT in and Severity of bleeding After 3monthsofthalidomide ** ** ** * * * * 1 3 . The subjects subjects .The Number per

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Duration 15–20 5–15 (min) without additional dietary iron supplementation in five out of six six of out five in supplementation iron dietary additional without ( dose first the of The in blood average increased concentration peripheral hemoglobin administration of month 1 within all were data whom for subjects four the of three in duration inhibited vessel formation; however, at lower however, at formation; vessel inhibited cytotoxic, being on verged and threshold solubility the (100 doses high formed. Atvery sprouts vessel of number the quantified then we marker, and cell endothelial an as (PECAM-1) molecule-1 adhesion cell platelet–endothelial for them bodies to undergo angiogenesis for a period of 3 d, we fixed and stained centrations of thalidomide in this model. After allowing the embryoid sprouting angiogenesis of VEGF and basic growth factor, embryoid presence the in I collagen onto Whenplated bodies). (embryoid cells stem embryonic mouse differentiating of els. Wespheroids used first the treated subjects, we examined its effects in several angiogenesisTo mod investigate how thalidomide might cause antihemorrhagic effects in maturation vessel promotes Thalidomide failed. have treatments other whom for epistaxis severe and HHT with subjects for option therapeutic a be Fig. Supplementary the both physi than normal concentrations hemoglobin lower had consistently analysis. statistical allow to available were values hemoglobin-concentration few too but substantial, was ( levels ( significantly dropped subjects the of two for concentrations Hemoglobin cessation. treatment after returned to epistaxis effects, side unrelated for reasons treatment who stopped others two in and individual this In shown). not (data neuropathy stopped treatment subject 19 months after the remaining first dose the because of peripheral lethargy; and drowsiness libido, of loss tion, constipa mild effects: side only minor showed treated subjects seven with treated thalidomide in being a 6-month to 5-year after follow-up ( transfusions blood additional require not did subjects four These anemia. to prevent yearly transfusions blood six and one between required week per nosebleeds 18–32 from fered 1 Fig. Supplementary ( recorded was supplementation iron whom in subjects NA NA NA 10 <5 Control subjects with HHT but not treated with thalidomide thalidomide with treated not but HHT with subjects Control Table Number ofbloodtransfusions Year before NA NA NA 1 o 6 1 0 0 8 3 0 and and logical range and the treated subjects ( subjects treated the and range logical advance online publication online advance Supplementary Fig. 1 Fig. Supplementary

1 ). Before treatment, four individuals who suf who individuals four treatment, Before ). ). These data indicate that thalidomide might might thalidomide that indicate data These ). 2 Year after 1 . We contested various adding the of effects NA NA 0 0 0 0 0 (mmol ml 12 months before Median hemoglobinconcentration 6.70 5.75 6.70 5.45 7.20 4.75 6.6 4.3 5.1 NA −1 P ); for the third, the drop drop the third, the for ); )

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© 2010 Nature America, Inc. All rights reserved. develop age-dependent vascular lesions similar to those seen in in seen those to HHT similar with individuals lesions vascular age-dependent develop maturation in individuals. these malformations vascular the of rupture by caused bleeding prevent and vessels blood as well as of increased mural cell coverage; such coverage might stabilize of of direct inhibition of endothelial cell proliferation and migration properties of thalidomide in individuals with HHT might be the result ( cov by ered sprouts endothelial of proportion the increased Thalidomide ( by surrounded was sprouts cell endothelial the of proportion small a only conditions, control Under branches. vessel (25 tion ( network a form to vessels adjacent to fused occasionally that cells tip by guided cells stalk of composed branches with treatment, lidomide tha by unchanged was sprouts vessel the of architecture general The ( cultures control)-treated (vehicle DMSO µ t ** All error bars represent s.e.m. * ( front is indicated by white arrowheads. is indicated by the white arrow, and the vascular and analyzed at P7. VSMC coverage of 150 mg per kg body weight thalidomide ( mice treated at P4 with DMSO (carrier control) or mount retinas of staining of vascular cells in whole- of endothelial cells and area. ( of the number of to reveal the vascular plexus. Right, quantification body weight ( weight ( control) ( injected intraperitoneally at P4 with DMSO (carrier from NG2DsRedBAC-transgenic mice at P7 capillaries. ( of NG2-positive pericytes to isolectin B4–positive thalidomide ( injection of DMSO or 75 mg per kg body weight Eng marking pericytes) from wild-type ( cells) and antibody specific for NG2 (green, specific for isolectin B4 (red, marking endothelial of flat-mounted retinas labeled with antibody branch points per field. ( positive surface area density and number of vessel row. Bottom right, quantification of isolectin B4– Eng higher magnifications of the vascular front in ( weight ( DMSO (carrier control) or 75 mg per kg body ( vessels in isolectin B4–stained endothelial cells in retinal and enhances pericyte/VSMC coverage. ( vessel branching in the retina of Figure 1 nature medicine nature test. NS, not significant; a, ; v, veins. e n n Supplementary Fig. 3 Supplementary Fig. 2a Fig. Supplementary g ml g ) Quantification of = 6) thalidomide at P4. At bottom left are = 13) at P7 after intraperitoneal injection with P To explore the possibility that thalidomide promotes vessel vessel promotes thalidomide that possibility the explore To < 0.01 and *** +/− +/+ d mice ( and −1 α ) Left, isolectin B4–specific (red) staining n n

-SMA)-positive cells (vascular smooth muscle cells (VSMCs)). α n ), thalidomide enhanced endothelial sprouting compared to compared sprouting endothelial enhanced thalidomide ), = 4) or thalidomide at 150 mg per kg = 15) or 150 mg per kg body weight Thalidomide normalizes excessive Eng = 4), thalidomide at 75 mg per kg body µ -SMA Eng g ml g c n n ) Left, confocal images of retinas n in vivo in +/+ = 7) and stained with isolectin B4 = 15). Right, the ratio of total area = 13) at P7 after intraperitoneal

+/−

Eng (WT) ( pericytes per isolectin-B4 surface −1 + mice from the images in the top cells compared to control (DMSO-treated) cultures cultures (DMSO-treated) control to compared cells P ) stimulated the recruitment of mural cells to the the to cells mural of recruitment the stimulated ) < 0.001, results from unpaired Acta1 +/+

, we took advantage of of advantage took , we ( n advance online publication online advance α n b = 14) and ). These results suggest that the antihemorrhagic -SMA–specific (green) = 6) and ) Left, confocal images 22,23 expression in retinas. ). Notably, thalidomide at a low concentra low a at thalidomide Notably,). . We first focused on the neonatal retina, retina, neonatal the on . focused We first P Eng < 0.05, n Eng = 14) and Eng +/−

mice +/− +/− a n

) Top, ( = 6) mice n = 13)

Supplementary Fig. 2a Supplementary

Eng

+/− mutant mice that that mice mutant d b c a α Isolectin B4 -SMA Isolectin B4 NG2DsRed Isolectin B4 NG2 Isolectin B4

-smooth muscle muscle -smooth α

Eng Eng Eng Eng a +/ +/ +/ +/ + + + + a v v 13,14 , a b ). - - - 500

DMSO DMSO DMSO DMSO DMSO 100 100 100 20 µ µ proteoglycan NG2, which marks VSMCs and pericytes ( pericytes However, and even at a low dose (75 VSMCs mg per kg body thalidomide weight), marks which NG2, proteoglycan sulfate chondroitin for by staining revealed as littermates, wild-type P7 of in retinas normal appeared coverage cell Mural bodies. on embryoid seen thalidomide of to effects lar the simi sprouting, vessel reduce to required concentrations angiogenesis, high with retinal on effect dose-dependent a had thalidomide in angiogenesis reduced markedly and ( doses lower with treated pups or termates the of weight lower the by evidenced as growth, retarded weight) body kg P7 of retinas in sprouting Eng vessel excessive and inappropriate normalize to the sufficient was P4 at once administered weight) body wild- ( to littermates compared type plexus vascular branched highly more denser, However, formed cells ( tip plexus the of front sprouting the at endothelial protrusions and filopodial retina, the in veins and arteries (P7) 7 day angiogenesis that agents modulate of effects the testing to amenable is that bed vascular a µ µ µ m m m m m Eng Eng Eng Eng +/− a +/ +/ +/ +/ Eng mice ( mice – – – – +/− Eng a Eng v pups treated at this dose compared to untreated lit untreated to compared dose this at treated pups 75 mgperkg Thalidomid Fig. 1 Fig. +/− v +/− a DMSO Fig. 1 Fig. eia soe ecsie nignss wt a with angiogenesis, excessive showed retinas DMSO DMSO 500 DMSO 100 100 100 20 mice showed morphologically distinguishable distinguishable morphologically showed mice a µ µ ). A higher dose of thalidomide (150 mg per per mg (150 thalidomide of dose higher A ). µ µ µ m e m m m m a 2 ). A low dose of thalidomide (75 mg per kg kg per mg (75 thalidomide of dose ). low A Eng Eng Eng 4 a a Isolectin B4–positive . As in wild-type littermates, postnatal postnatal littermates, wild-type in As . Thalidomide v +/ +/ area per field +/ – – (pixels) – 10,000 15,000 20,000 25,000 v 5,00 v 150 mgperkg 150 mgperkg 0 0 Thalidomid Thalidomid 75 mgperkg a 75 mgperkg Thalidomid Thalidomide +/+ – – * NS +/– 500 100 100 100 Eng +/– *** 75 µ µ µ µ Eng e e m m m m +/− e Supplementary Fig. 4 Fig. Supplementary 150 +/– Thalidomid Thalidomid Eng mice ( mice e +/− +

Thalidomide NG2 cells per Acta1 mRNA Thalidomide +/

mice compared to compared mice vessel length Vessel branch s e l c i t r a – relative expression 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045 0.0050 0.0055 NG2/isolectin B4

0.5 1.0 1.5 2.0 2.5 3.0 3.5 points per field 25 50 75 100 200 300 400 500 600 0 0 Fig. 1 Fig. e e 0 0 150 mgperkg +/+ +/+ Thalidomid – – – – +/+ NS – – NS 7 – * * a +/– +/– NS Fig. 1 Fig. +/– Fig. 1 Fig. ** ). Thus, ). Thus, 500 *** 5 ** *** +/– 75 150 +/– 150 +/– 75 µ e 150 +/– m b a ). ), ).  - -

© 2010 Nature America, Inc. All rights reserved. cytes ( cytes significantly increased cells, ( cycling of marker a Ki67, expressing ( mice transgenic NG2DsRedBAC- P7 of plexus vascular the to recruitment pericyte stimulated strongly weight) body kg per mg 150 and (75 doses both ( points branch capillary at cells of thalidomide and STI-571. The mice shown at the bottom are examples on P7 of (from left to right) control wild-type mice and mice treated on P4 with and treated mice mice wild-type on P7 control to of left are right) at examples (from The shown the mice bottom STI-571. and thalidomide of a combination or STI-571 thalidomide, control), (vehicle DMSO with P4 at treatment after P7 ( (bottom). field per points branch vessel of number and (top) density area surface positive B4– isolectin of quantification Right, front. vascular the of edge the indicates image white black-and- a in cells endothelial of staining Isolectin-B4 P7. at retinas mouse wild-type on ( weight) body kg per mg (50 with STI-571 combination ( weight) body kg per mg (150 thalidomide with P4 on treatment of effects the showing imaging image. each in indicated is (blue) capillaries B4–positive isolectin of that to (red) pericytes positive NG2- of area total the of ratio The green. cells muscle smooth vascular marks staining and red pericytes marks staining NG2 blue, cells endothelial marks staining B4 Isolectin gradient). a form not does PDGF-B in and normally) matrix extracellular the with associated becomes and secreted is PDGF which (in mice in P4 on intraperitoneally injected thalidomide or control) (carrier DMSO of effects ( per group. thalidomide. to exposed mice in and mice control in cells tip PDGF-B–expressing in protrusions cell characteristic indicate enlargements in Arrows right. at shown are boxes in cells tip endothelial the of images Higher-magnification thalidomide. weight body kg per mg 150 or control) (carrier DMSO with P4 on injected mice in P7 at (green) immunostaining cell) (endothelial B4 isolectin with combination in (blue) PDGF-B ( thalidomide. with Eng In PCR. quantitative by determined as P7, at expression on P4 on injected thalidomide ( PCR. quantitative by determined as P7, at retina the on P4 on injected thalidomide weight body kg per mg 150 of ( function. pericyte regulating by 2 Figure com arteries pletely and the the veins ( partially covering cells, endothelial to apposition close in DsRed promoter NG2) fluorescent protein (DsRed) under the control of the ( labeling B4 isolectin to NG2 of by ratio the assessed as measuring vasculature, the to recruitment pericyte stimulated A  means as given are Values and STI-571. or thalidomide both STI-571 thalidomide, f P

) Quantification of mouse body weight at at weight body mouse of Quantification )

We next used NG2DsRedBAC-transgenic mice, which express the red ≤ s e l c i t r +/−

0.01 b pups and pups Supplementary Fig. 5 Fig. Supplementary + ) Effect of 150 mg per kg body weight weight body kg per mg 150 of Effect ) a

cells were distributed widely throughout the vascular plexus plexus vascular the throughout widely were distributed cells n n ) and Thalidomide mediates vessel coverage coverage vessel mediates Thalidomide = 10) or with STI-571 alone ( alone STI-571 with or 10) = ( DMSO or 12) = , indicating that thalidomide induced cell cycle entry of peri d ) Confocal images showing the the showing images Confocal ) n = 3 ( per group. b , , n n Pdgfb 2 = 6 = 6 wild-type pups, pups, = 6 wild-type 5 Vegfa c , to quantify the number of pericytes in the retina. retina. the in pericytes of number the , quantify to Fig. 1 Fig. ) ) In Eng ret/ret

mRNA expression in in expression mRNA situ c +/− ). In addition, the proportion of pericytes pericytes of proportion the addition, In ). n mice (in which which (in mice hybridization for for hybridization = 10) alone or in pups treated treated pups e Pdgfb ). Higher doses of thalidomide resulted resulted thalidomide of doses Higher ). ) Left, confocal confocal Left, ) Fig. 1 a ) Effect Effect ) Pdgfb Fig. 1 Fig. mRNA mRNA n c = 3 α n ). We also found DsRedsolitary = 13 -SMA -SMA n ret/+ = 6) 6) = c

). Thalidomide treatment at at treatment Thalidomide ).

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100 20

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≤ 500 500 100 0.05; ** ** 0.05; 20 20 µ µ µ µ µ m m m m m Pdgfb Isolectin B4–positive Thalidomid Thalidomide P

area (pixels) in vivo

58.37 Pdgfb

≤ Vessel branch STI-571 10,000 20,000 30,000 mRNA expression ( expression mRNA STI-571 0.01; *** *** 0.01; points per field 800 100 200 300 400 500 700 600 Flt1 ret/ ± 0 0 promotes e 0.12 + Thalidomide b or or – – – – – *** *** Thalidomide 150mgperkg Thalidomide 150mgperkg + + + – – NS NS

Pdgfb mRNA Kdr ** ** P + + + – Fig. 1 Fig. Eng 100 relative expression 20 ≤ + + 3 0 2 1 0.001. (

µ retinal µ +/+ +/− Supplementary Fig. 6 Fig. Supplementary m m – – Thalidomid e NS ). Thus, thalidomide thalidomide Thus, ).

and wild-type litter and wild-type 50 Pdgfb 4.90 +/–

STI-571 expression of of expression nature medicine nature f µ *** Fig. 2 Fig.

Weight (g) m vessel maturation 150 ± +/– ret/re 2 4 6 5 0 1 3 2.91 e t – – b ** ). PDGF-B PDGF-B ). + – 100 * + – 20 20 Acta1 + + µ µ µ m m m ) ­ -

© 2010 Nature America, Inc. All rights reserved. in the number of pericytes observed in thalidomide-treated mice mice thalidomide-treated in ( observed pericytes of number the in increase the inhibit not did STI-571 of administration Concurrent behavior cell mural affects directly Thalidomide effects. thalidomide’s of a mediator as key PDGF establishing vessels, blood the of cells lial to endothe apposition and on pericyte on angiogenesis thalidomide ( ( development vascular retinal postnatal activity kinase tyrosine receptor PDGF of inhibitor potent a Gleevec), or mesylate STI-571 imatinib as with known (also and thalidomide of dose high a with mice wild-type Moreover, recruitment. pericyte postnatal of treatment simultaneous thalidomide-mediated for required is PDGF-B that idea the porting postnatal in defect recruitment cyte ( defects of surface abluminal vascular to retinal leads the formation gradient of lack to this microvessels; pericytes of adhesion tight to stimulate required gradient the form not does so and matrix extracellular the by (ret) retained not is but secreted is PDGF-B which in model mechanism. paracrine a by endothelial recruitment pericyte enhance of upregulation ( mice thalidomide-mediated thalidomide-treated and both in control pericytes by not and cells tip endothelial by expressed was factor growth ­receptor- platelet-derived expressing pericytes migrating for s.e.m. represent bars error All experiments. independent DsRed ( experiments. independent six of representative are Results shown. images the for cell per protrusions of number the of quantification Bottom, STI-571. and thalidomide of 50 control), (vehicle DMSO of addition the 3 h after taken were Images gels. fibrinogen three-dimensional in pups NG2DsRedBAC-transgenic P7 of from cells DsRed FACS-isolated cultured on thalidomide by induced protrusions on STI-571 of effect the ( STI-571. and thalidomide of a or combination alone weight) body kg per mg (50 STI-571 alone, weight) body kg per mg (150 thalidomide control), (vehicle DMSO with treatment after P7 on area surface isolectin-B4–stained per pericytes of number the of quantification Bottom, pups. STI-571–treated in cells endothelial the from detached partially processes dendritic with pericytes indicate arrows White arrowheads. white by indicated are conditions treated thalidomide- and control both in surface endothelial abluminal the with intimately associate that pericytes by extended processes ( weight) body kg per mg (50 STI-571 with combination in thalidomide or ( alone weight) body kg per mg (150 ( injection intraperitoneal on P4 with DMSO mice. Retinas were from P7 mice after NG2DsRedBAC-transgenic of retinas the in red, in marked pericytes, and staining, B4 isolectin by blue in marked plexus, vascular the of cells endothelial between association the on STI-571 by signaling PDGF of inhibition and thalidomide ( 3 Figure nature medicine nature only were pericytes However, PDGF-B. of independently directly, n a Fig. Fig. 2 Fig. ) Top, confocal imaging showing the effects of effects the showing ) Top,imaging confocal = 4), STI-571 alone ( alone = 4), STI-571 To test this hypothesis, we took advantage of Tothe of we advantage took hypothesis, test this

3 + a f cells cultured for 3 d in presence of 0.1% FBS, epidermal growth factor and basic fibroblast growth factor. Results are representative of three three of representative are factor. Results growth fibroblast basic and factor growth epidermal FBS, 0.1% of 3 d presence for in cultured cells ). Thus, blocking PDGF signaling counteracts the effects of of effects the counteracts signaling PDGF blocking Thus, ).

), indicating that thalidomide may influence pericyte number ), may that thalidomide pericyte influence indicating Fig. 2 Fig. Effects of thalidomide on pericytes. pericytes. on thalidomide of Effects β (PDGFR- b d ) Top, confocal imaging showing showing ) Top,imaging confocal ) 3 0 . Injection of thalidomide did not rescue the peri the rescue not did thalidomide of . Injection advance online publication online advance β n ) = 7), thalidomide thalidomide = 7), 26–29 n = 8). Dendritic Dendritic = 8). . In In situ n = 7) hybridization showed that that showed hybridization Pdgfb

31,32 Fig. Fig. 2 + i. 2 Fig.

ret/ret , was sufficient to restore restore to sufficient ,was e ) and growth of pups pups of growth ) and mice ( mice µ c a g g ml ), indicating that that indicating ),

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−1 < 0.05, ** < 0.05, Fig. 2 Fig. of thalidomide, 10 10 thalidomide, of Pdgfb ret/ret d might might mouse mouse ), sup ), c Pdgfb ) The effect of 50 50 of effect ) The

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vessel length STl-571 0.0005 0.0010 0.0015 0.0020 0.0025 0.0030 0.0035 0.0040 0.0045 20 20 the number and location of lesions are highly variable and depend depend and variable highly are lesions of location and number the HHT, although of hallmark pathological the are vasculature neous the and mucosal cuta in wall throughout the vascular Abnormalities in defects wall vessel rescues Thalidomide behavior. cell mural of regulator a as ( cultures control)-treated (vehicle DMSO to compared cells BrdU-labeled number of the in increase twofold a showed cultures pericyte/VSMC Thalidomide-treated h. 3 for BrdU to cultures exposed we eration, or guiding devices. as exploratory function could suggest we that extensions dendritic resembled protrusions membrane rod-like These signaling. receptor PDGF of independent is thalidomide by protrusions membrane of induction the that indicating STI-571, of presence additional the by ( h 12 at pronounced ( extensions branched highly and long that became from pericytes the cultured protrusions membrane for 3 of h led to thalidomide the formation with cultures cell these of for the purified DsRed DsRed the cultured then and pups NG2DsRedBAC-transgenic P7 of lungs DsRed sorted we cells, mural affects directly indeed thalidomide whether Totest PDGF-B. of independent cells ( mice STI-571–treated in compared with control mice cells and mice treated with alone thalidomide endothelial with associated loosely Fig. 3 Fig. µ µ µ 0 m m To test whether thalidomide directly controls mural cell prolif cell mural controls directly thalidomide whether test To e M STI-571 or a combination a or combination M STI-571 α STI-571 Thalidomid Thalidomid -SMA, desmin and PDGFR- – – – ** + a NS + + cells on fibrin. We confirmed the pericyte/VSMC identity of of identity We on pericyte/VSMC fibrin. the cells confirmed ). These results indicate that there may be effects on mural mural on effects be may there that indicate results These ). µ ** g g ml P Fig. Fig. 3 + + – < 0.001, results from unpaired unpaired from results < 0.001, * e e −1 c thalidomide on BrdU labeling of FACS-isolated P7 lung lung P7 FACS-isolated of labeling BrdU on thalidomide ), providing evidence for a direct role of thalidomide for a thalidomide role of direct ),evidence providing + cell population by mRNA expression and staining Supplementary Fig. 8 Fig. Supplementary 20 20 µ µ m m b c NG2 β STI-571 DMSO ( Fig. 3 Fig. Thalidomide Thalidomid Supplementary Supplementary Fig. 7 BrdU-positive

cells (%) STI-571 Number of b 10.0 12.5 15.0 17.5 2.5 5.0 7.5 t protrusions ). This effect was even more more even was effect ). This test. 6 1 3 0 0 2 4 5 e 10 10 – – – ) and was not affected affected not was and ) NS *** + µ µ Eng cells by FACS from from FACS by cells m m – – 50 NS ** 50 – s e l c i t r a STI-571 Thalidomid Thalidomid ( +/− µ 10 g ml – *** mice 10 50 –1 ). Treatment ( ( ) µ µ e e M) g ml –1 10 10 ) µ µ m m  - -

© 2010 Nature America, Inc. All rights reserved. is quiescent. Analysis of dorsal skin sections of of sections skin dorsal of Analysis quiescent. is vasculature the which in old), weeks (60–70 mice adult aged in mide growth. vessel affecting without coverage cell stimulate mural can thalidomide that suggesting treatment, thalidomide nor patterning and morphology ( coating cell muscle smooth regular in of defects coverage dose arterial the rescue to a at twice sufficient at P21, was once at P7 and once weight, administered kg body 75 mg per normal show to Thalidomide failed coverage. arteries VSMC the of of areas direction the large to but flow, blood perpendicular being cells the with mice, type wild- in as aligned circumferentially still were Where VSMCs the size. present, normal muscle of were smooth themselves by arteries the coverage although cells, irregular and loose showed of ducibly skin ear the in arteries However, vessel normal coverage. showed vessels lymphatic and Veins points. of branch number or and density area vessel in differences morphology no with littermates vessel overall the in that patterning showed staining and This PECAM-1 for ears mouse to 1-month-old of fashion simple a in the of growth the accommodate longitudinally expands ear of the of skin ear tecture the in month and postnatal Eng growth first vessel the during examined we ­maturation phenotype, HHT the of vessel’ aspect ‘thin-walled this rescue could thalidomide whether Totest Eng muscle cells have in of the skin been of reported adult the vasculature age on epidermis. ep, v, veins; arteries; * s.e.m. represent bars error All cells). flat of layer thin a very as appear cells endothelial (red; ( thalidomide of mg 100 with treatment daily after subject one in and HHT with subjects control three in nose the in vessels of coverage cell muscle smooth vascular on thalidomide of mg 100 with treatment daily ( quantified. was coverage vessel which in areas the indicate micrographs the in boxes outlined The weeks). of area total of ratio the on injection thalidomide of effect untreated the in VSMCs by medium the of coating ( thalidomide of weight body kg per mg 75 with 8 weeks for weekly twice treated mice in ( ( wild-type aged of (green) to antibody and (red) PECAM-1 to antibody with stained skin dorsal the of area ( shown. mounts whole the for software Metamorph by measured arteries the of intensity (VSMC) green relative the and branches vessel of number density, area surface PECAM-1–positive of of ears the in defective is arteries the of coverage VSMC where indicate arrowheads VSMCs), (green, and cells) endothelial (red, PECAM-1 ( ( wild-type DMSO-treated of ears the in coverage vessel on P21 and P7 at weight) body kg per mg (75 injection thalidomide intraperitoneal of effects the showing imaging coverage defects in 4 Figure A  and PECAM-1–specific with n n n

= 6). The white arrowhead indicates irregular irregular indicates arrowhead white The = 6). and thalidomide of absence the in mice = 5) for stained mounts whole On P28. at = 9) Next, we explored the effects of prolonged treatment of thalido of treatment prolonged of effects the explored we Next, s e l c i t r +/− +/− mice; this results in fragile vessels that mice; vessels are this in results prone fragile to bleeding ie Drn ti pro, h vsua fne-ie archi finger-like vascular the period, this During mice. 4

b . As in individuals with HHT, irregular layers of smooth smooth of layers irregular HHT, with individuals in As . Thalidomide normalizes vessel vessel normalizes Thalidomide ) Confocal images of the subcutis subcutis the of images ) Confocal Eng Eng Eng +/− +/– Eng mice ( mice mice. Bottom, quantification quantification Bottom, mice. +/− Eng mice. Bottom right, the the right, Bottom mice. +/− +/− mice was very similar to that in wild-type wild-type in that to similar very was mice n n mice. ( = 18) compared to compared = 18) n = 9) and and = 9) = 5) and and = 5) a α ) Top, confocal α -SMA–specific antibodies revealed revealed antibodies -SMA–specific -SMA -SMA

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a Supplementary Fig. 1 Fig. Supplementary

points per field v +/ 1,000 1,500 2,000 2,500 – response to thalidomide treatment. thalidomide to response in ( individual treated the of vessels blood the around layers cell muscle Table of prevention of terms in epistaxis, he treatment withdrew from the study because of neuropathy) the ( from benefit individual to this seemed (although group treatment the for withdrawn had but thalidomide of dose 100-mg a daily received had who HHT with untreated three from isolated ­individuals with HHT and in sections isolated sections from the one individual in staining compared We and endoglin for biopsies mucosal nasal human of sections Westained humans in coverage vessel stimulates Thalidomide ( mice untreated and tracheal of between density vessels the in similarities by evidenced as vasculature, on the thalidomide of effects systemic any did not long-term observe ( arterioles with points branch their near especially cells, muscle smooth of layers by regular covered were found, be normally few only where dermis, the in capillaries mice, ( unchanged covered by vessels PECAM of ratio the measuring by assessed as area, subcutis the in at least 2 months rescued the mural cell coverage of defects the vessels for weekly twice administered weight body kg per mg 75 of a dose at mice were irregularly coated by VSMCs ( compared to micewild-type ( with sels Wewalls. ves fewer vessel in the observed abnormalities widespread 50 Fig. 0 0 Eng + – – /+ α 0.5 mm NS

4 +/ -SMA as markers of endothelial cells and and respectively. VSMCs, cells endothelial of markers as -SMA +/– 1 c NS + – and and ), a 75 mice may be relevant to humans and may explain the clinical v

/– α suggesting that mechanisms similar to those we uncovered we uncovered to those similar mechanisms that suggesting 75 mgkg Thalidomid T -SMA Eng v a halidomide Supplementary Fig. 1 Fig. Supplementary Fig. 4 Fig. Coverage of arteries +/ P –

advance online publication online advance (arbitrary units) < 0.05 and ** and < 0.05 Eng 100 25 50 75 ). VSMCs are stained for for stained are VSMCs ). + –1 0 α associated cells in the subcutis area of of area subcutis the in cells associated b e +/+ – – -SMA +/− 0.5 mm ). Moreover, in the thalidomide-treated thalidomide-treated the in Moreover, ). Supplementary Fig. 9 Fig. Supplementary ** +/– a mice treated for 6 months with thalidomide thalidomide with months 6 for treated mice v ** +/ 75 + – b c cells, although the vessel area density was area density the vessel cells, although c

) Confocal imaging showing the effect of effect the showing imaging ) Confocal Endoglin α-SMA PECAM α-SMA Hoescht Fig. 4 75 mgperkg Thalidomide DMSO Subject 10 Subject Eng P Eng < 0.01, results from unpaired unpaired from results < 0.01, +/ +/ – + b ). There were many more smooth smooth more many were There ). 8 ). Moreover, the arteries of ep Fig. 4 α ep -SMA (green) and endoglin endoglin and (green) -SMA 100 100 50 ). 50 b µ − µ µ µ ). Of note, thalidomide m m m m

α Thalidomid Thalidomid Subject Subject DMSO Eng -SMA nature medicine nature Vessel coverage +/

(%) – 7 9 10 15 20 25 30 35 40 45 50 55 ep + 0 5 e Fig. Fig. 4 cells would would cells e +/+ – – Eng t * * test. a, test. +/– 100 100 Fig. 4 Eng Eng b 50 +/− +/– * 75 ). We µ µ µ m m m +/− +/− as as c + - ,

© 2010 Nature America, Inc. All rights reserved. mural cell-endothelial cell contact as well as contact-independent contact-independent mechanisms. as well as contact cell cell-endothelial mural migration and tion prolifera cell endothelial suppress to shown been have cells Mural stabilization vessel mediating in cells mural of function crucial a and cell cell–mural endothelial contact have demonstrated previously ponents of the signaling pathways that regulate mural cell recruitment com individual of Targeted deletions around vessels. blood layer small-diameter discontinuous, often single, a form or solitary either are that layers and pericytes concentric in and veins multiple arteries vessels ( that in all cases thalidomide enhanced mural cell found recruitment to we blood here, investigated angiogenesis of models the HHT. In of hallmark and prone thus to become fragile the pathological bleeding, of skin variable the in diameter capillary abnormally an and endothelial with coincide stabilization vessel and recruitment TGF- TGF- of activation TGF- defective process. for this in role signaling PDGF-B key a uncovered also We stabilization. vessel to leads ence that by infer formation on protrusion protrusions effect This vessels. blood form embrace to ability their and proliferation their both increasing cells, mural of activation the modulates thalidomide that We found vasculature. the of coverage cell mural increased to due is rather but migration, and proliferation cell endothelial of inhibition direct of result the not is thalidomide of property antihemorrhagic the that Moreover, showed we hemorrhage. reduced of consequence a be to infer we with which rose, also subjects of concentrations HHT.Hemoglobin group and small a in nosebleeds of requirements transfusion duration blood the and frequency the both reduced challenge anomalies, clinical a remained vascular has surfaces, mucosal these entire over vascular of disseminated treatment underlying shed HHT, in have mechanisms years ­malformations cellular few on last light the over considerable conducted studies Although DISCUSSION growth and maturation. v, . artery; a, tip cells and mural cells directly to control vessel indicate that thalidomide targets both endothelial maturity, and inhibits angiogenesis. Black arrows mural cells expressing dose of thalidomide enhances the number of stimulation of mural cell proliferation. ( which attracts comigrating mural cells, and direct PDGF-B expression from the endothelial tip cells, induced vessel maturation include increased cell recruitment. Mechanisms of thalidomide- and excessive angiogenesis by stimulating mural thalidomide normalizes vessel coverage defects irregular capillary diameter. ( and vessels show endothelial hyperplasia and Smooth coverage of arteries is irregular, in endothelial cells reduces pericyte recruitment. HHT. ( thalidomide normalizes vascular malformations in Figure 5 nature medicine nature and thalidomide, of doses low of administration by normalized was We found that excessive angiogenesis in the retinas of of retinas in the We angiogenesis excessive that found of consequence important an that indicated work previous Our β a ) Reduced expression of endoglin or ALK-1 from endothelial cells is thus reduced, affecting mural cell cell mural affecting reduced, thus is cells endothelial from

Fig. 5 Schematic illustration of how 1 0 . Here, b ). Mural cells are subdivided into VSMCs that surround β signaling in HHT is dysfunctional processing and and processing dysfunctional is HHT in signaling

we

β in vitro in α by endothelial cells endothelial by advance online publication online advance

-SMA, indicating vessel found that oral administration of thalidomide thalidomide of administration oral that found b ) A low dose of 35,36 and and Eng c ) A high +/− in vivo in 8 . These vascular abnormalities abnormalities vascular These . mice 8 . The availability of active active of availability The . 26,28 2 2 ( , both through direct direct through ,both Fig. 5 Fig. a a

a Prolif VSMCs Pe ). Vessels may Eng r icytes erating mural cells +/− mice 3 4 - - - . of NG2, location in the retina and, at this stage and position, lack of of lack position, and stage this at and, retina the in location NG2, of from increasing the number of pericytes (identified by their expression ( contacts tion cell–pericyte and promoted endothelial prolifera pericyte that stimulated low and thalidomide doses of high of pericytes in of pericytes the mouse retina that expressed α ing this effect, however, need further investigation. From our investigation. however, effect, further this ing need ently of effects on PDGF-B The signaling. exact underly mechanisms stimulate their proliferation and ability to form protrusions independ stabilization vessel to pericytes of contribution of deletion PDGF-B have the endothelium-specific demonstrated key PDGFR- express which pericytes, and cells endothelial comigrating for attractant an as serves it where arteries, of remodeling the in as well as vessels formed newly to pericytes of density pericyte normal of <50% with mice in with density vascular organization increased vascular markedly chaotic of regions of formation the to density, pericyte normal of >50% with mice in microaneurysms of presence the and branches capillary an regressing of from number increased ranging retina, the in abnormalities vascular of spectrum a broad develop mice mutant these system. As consequence, nervous extensive have in but variation individual the the throughout central of density pericytes viable are that mice in results endothelium the in PDGF-B of deletion targeted that example, for shown, been It has is number crucial for Pericyte the normal development cells. of a regular retinal tip vessel network. endothelial by PDGF-B of expression vasculature pression of the formation of endothelial sprouts and quiescence of the extend and processes cells cellular numerous endothelial with lamina Indeed, basal the share phenotype. pericytes contractile a of acquisition the possibly, and, numbers in vessels higher blood with cells, mural association their of of retina in the ture gest on that effect of thalidomide the the inhibitory excessive vascula sug strongly results Our contact. cell cell–mural which endothelial affects signaling, PDGF of inhibition were pharmacological effects by these reversed of both thalidomide; of doses high with treated in loss pups and weight angiogenesis of an inhibition with correlated ( phenotype contractile pericyte the of marker v -SMA expression), high doses of thalidomide increased the -SMA increased number expression), doses high thalidomide of We to cells directly mural also target might found that thalidomide stimulated thalidomide that found we level, molecular the At b PDGF- B 26,28,35,36 a 2 9 . PDGF-B also has a crucial role in the recruitment recruitment the in role a crucial has also . PDGF-B Eng ( Fig. 5 Fig. +/− mice is due to the increased proliferation proliferation increased to is due the mice c ). 26,28,35,36 v v PDGF- c . This inte . This Thalidomide B β . Studies in mice with with mice in Studies . α a Fig. 5 Fig. 29,30 r -SMA, an established action leads to sup to leads action s e l c i t r a . c ). This finding finding ). This Fig. 5 b ). Apart in in vitro v  ------

© 2010 Nature America, Inc. All rights reserved. Note: Supplementary information is availableNote: information on the Supplementary of the paper at http://www.nature.com/naturemedicine/. Methods and any associated references are available in the onlineM version up. new opened be may cells, therapy vascular for avenues mural or pericytes target can analogs suggest, its data or our thalidomide as If, malformations. vascular from bleeding on effects beneficial have can cells, endothelial than rather cells, mural targeting a that therapy evidence and first the to provide, knowledge, our maturation vessel stimulating by HHT with subjects in bleeds nose of duration and frequency the reduces thalidomide that strate malformations vascular of etiology the explain to suggested been has which hypothesis, this supporting bilization, desta wall vessel with VEGF, of associated is concentrations high to exposure of as a result observed commonly angiogenesis, early-stage local massive of Activation cells. mural and associated from disengaging destabilizing actively by state growth-arrested its escape must tumor. the into transmigration their and wall to vessel the attachment cell immune affecting thereby haps on might pericytes influence endothelial cell–leukocyte crosstalk, per and T cell activation. We also that speculate the of thalidomide effects maturation vessel both enhancing by growth tumor target thus may and can immunomodulator enhance T cell activation size tumor reduced and tumor the of infiltration T cell increased with associated was maturation vessel ing express pericytes of proportion an coverage, increased with pericyte increased have to shown been recently have signaling, G-protein of Tumormaturation. vessels in mice lacking interfere might with tumor growth, vessel namely through enhancing solid on thalidomide by which mechanism another effects suggest data Our tumors. for trials clinical early-phase in investigated being cancers of models shown experimental in various already activity has Thalidomide cells. endothelial on effects direct density. area vessel or patterning or morphology vessel affect not did thalidomide with treatment long-term and sue, tis the of growth to accommodate expanding period still is it which the during in mice in ear the of plexus vascular the of architecture general the affect not did thalidomide that found We also STI-571. inhibitor signaling PDGF the using by angiogenesis on thalidomide lethal not were angiogenesis sprouting inhibited threshold solubility the reached that thalidomide of doses high only fact, In sprouts. endothelial target to than rather maturation vessel induce to is thalidomide of effect primary the that indicate data our p38 kinase are vessels immature unclear, has in although been CPS49 shown to induce sprouts the stress response endothelial new of formation the cells tip have not which ones, do mature more not but cells) tip by guided cells stalk of of immature, highly angiogenic vessels (which survival the affected severely relevance, more of and, vessels all from outgrowth distal blocked transiently treatment study, CPS49 this In with experiments CSP49, an analog of thalidomide, in chick embryos. of newly formed vessels targeting by thalidomide ( direct exclude cannot we bodies, embryoid in analysis A 

ethods For vascular abnormalities to develop, the quiescent endothelium endothelium quiescent the develop, to abnormalities vascular For to attributed been have properties antiangiogenic Thalidomide’s s e l c i t r α -SMA, indicating increased vessel maturation vessel increased indicating -SMA, α , which can promote endothelial cell death. However, death. cell endothelial promote can which , in vivo in 1 4 , we could counteract the inhibitory effects of of effects inhibitory the counteract could ,we . The mechanisms by which CPS49 inhibits inhibits CPS49 which by mechanisms The . in in vitro 3 7 Fig. 5 . Thalidomide is also a potent potent a also is Thalidomide . , and that for tested all doses N Rgs5, c a ), as recently suggested by t

u are branches composed r e encoding a encoding regulator 1

M 0 . Our data demon data Our . e 2 d 0 3 i and is currently is currently and c 7 2 i . This effect on on effect .This 0 n . Thalidomide e website. ------18. 17. 16. 15. 14. 13. 12. 11. Published Published online at http://www.nature.com/naturemedicine/. The authors declare no competing financial interests. C experiments, interpreted results and wrote the paper. surgery, provided clinical samples and interpreted results; A.E. and C.L.M. designed knockout mice; C.J.J.W., F.D., J.J.M. and R.J.S. directed the clinical study, performed J.-L.T. provided NG2DsRedBAC and T.M. and B.L. gel three-dimensional fibrinogen culture performed experiments; C.B. retina performed experiments; S.v.d.B. stem performed cell experiments; cell culture experiments, interpreted results and the write helped paper; C.F. and immunofluorescence experiments; K.R. FACSperformed and sorting the paper; S.S. and S.M. real-time performed PCR, mouse retina and skin F.L. and experiments,designed performed interpreted results and wrote AUTH Foundation International. program kennisinfrastructuur ‘Dutch Platform for Tissue Engineering’ and HHT Wetenschappelijk Onderzoek Rendu Osler and the Besluit subsidies investeringen the British Foundation, the EU (QLG1-CT-2001-01032), Stichting Recherche sur le Cancer (3980), the Netherlands Heart Foundation (2008B106), pour la Recherche Médicale, Fondation Bettencourt, Association pour la Recherche (Agence Nationale de la Recherche blanc, Neuroscience), Fondation Institut National de la Santé et de la Recherche Médicale, Agence Nationale de la providing NG2DsRedBAC mice. This work was bysupported from grants We thank A. Nishiyama (University Connecticut Stem of Cell Institute) for A 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. reprintsandpermissions/. http://npg.nature.com/ at online available is information permissions and Reprints ck O

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© 2010 Nature America, Inc. All rights reserved. images with a uniform threshold between experiments and analyzed the data data the analyzed and experiments between threshold uniform a with images treated and vehicle control group in four independent experiments). counted the number of protrusions per cell (200 cells analyzed and perHealth) thalidomide-of Institutes National (US software ImageJ with area surface cell contours. cell We the highlight to quantified adjustment threshold manual by followed white and black to Z-stacks compressed converting by (pixels) area vessel length per field as described previously analysis. quantitative and Morphometry described previously microscope (Leica). images with a confocal laser-scanning microscope SP5 (Leica) or with a DMLB and mounted them in DAKO mounting medium (S3023, DAKO). We captured sections for 1 h at 20 °C with secondary antibodies diluted in PBS with 0.2% BSA Methods ( antibodies primary with °C 4 at overnight them incubated ace tone, cold washed them with in PBS, blocked them HHT with blocking reagent (Roche) and with subjects from biopsies nose or skin dorsal mouse of immunohistochemistry. and Immunofluorescence as sections described previously the mice,we killed we processed50- and stained dorsal tissues skin weeks. 6 for thalidomide After of weight body kg per mg 75 with weekly twice them as described previously processed and °C 4 at overnight paraformaldehyde 4% in halves dorsal fixed We leaflets. ventral and dorsal the separating forceps, with ears the dissected and P21 with 75 mg per kg body weight thalidomide,of killed them at P30 and them for immunostaining. processed then and overnight °C 4 at PBS in paraformaldehyde 4% in them 4% formin, °C 10 20 retinas,at the in paraformaldehyde PBS in dissected post-fixed eyes their Wefixed P7. at them killed and both or STI-571 of weight body kg per mg 50 thalidomide, of weight body kg per mg 50–150 with P4 at mice received vehicle only. We injected thalidomide and STI-571 intraperitoneally (volume 20 protocols. all approved France de Ile of Committee Experiments Animal The mice tissues. and Mice the Thalidomide and immunoreagents. described CaCl mM 0.12 with buffer phosphate M 0.1 in paraformaldehyde 0.2% in overnight them fixed of Helsinki. of the Declaration We nose collected biopsies in salt, physiological who provided informed consent. This investigation conforms to the principles Ethics Committee of the St. Antonius Hospital, where we recruited the subjects, biopsies. nasal and Subjects ONLINE METHODS nature medicine nature We determined colocalization of mural cell markers with vessels on similar similar on vessels with markers cell mural Weof colocalization determined as retinas of immunohistochemistry whole-mount performed We For immunofluorescent staining of dorsal skin, we injected 60-week-old mice For cutaneous blood analysis vessels,whole-mount of we treated mice at P7 To analyze postnatal angiogenesis in the mouse retina, we injected pups once Supplementary Methods Supplementary 25,30,39 ) ) in PBS plus 0.2% BSA (A4503, Sigma). After washing, we incubated 3 8 as heterozygotes on a mixed C57BL/6 and CBA genetic background. . We 10- used We maintained maintained We 2 4 and 4% sucrose and then processed them as previously previously as them processed then and sucrose 4% and 1 . µ m sections for staining. for sections m 4 . Clinical studies were approved by the Medical Medical the by approved were studies Clinical 0 . 3 8 . Suppliers and manufacturers are given in Eng +/− We quantified branch points and and points branch quantified We , NG2DsRedBAC and and NG2DsRedBAC , 4 2 . We measured the total cellular We fixed frozen sections sections Wefrozen fixed Supplementary Supplementary µ µ l). Control m sagittal m sagittal Pdgfb ret/+ -

42. 41. 40. 50 tured DsRed DMEM supplemented with 20% FBS and then sorted them. After ml sorting, mg 0.2 we cul with them digested lungs, the minced Wemice. DsRedNG2BAC-transgenic DsRed culture. gel fibrinogen three-dimensional and sorting Cell B4 staining. by retinas contralateral the Weanalyzed sample. Actb Vegfa ard procedures ( Quantitative PCR analysis. intensity defined as the sum all pixelsof in the delineated box. calculated and analyzed vessels the eated threshold were excluded this from below analysis. values Forintensity; eachthreshold image at of images the Weset whole Imaging). ear,(Universal we delin it expressed and as a percentage. area pixel overlapping the as colocalization measured We value. threshold set a than greater or to equal channels marker PECAM-1) or intensity in both mural cell (NG2 or with the colocalization function of ImageJ to identify pixels that had fluorescence 39. 38. Methods Additional methods. cally significant. test.Kruskal-Wallis the with subject each for thalidomide) after and during (before, concentrations hemoglobin of Wemedians unknown. compared was values of distribution the which for samples of groups small for treatment, thalidomide during and before HHT Mann-Whitney Student’s unpaired two-tailed, the using (GraphPad) analyses. Statistical incorporation BrdU by factor growth ( fibroblast basic and factor DsRed growth of proliferation the on proliferation. Cell We captured images at 0 h and 12 h by SP5 confocal microscopy (Leica). Supplementary Methods Supplementary We quantified quantified We µ

Hellström, M. Hellström, H. Gerhardt, M. Uutela, Arthur, H.M. mouse the in S. Thorsteinsdottir, & M.J. Duxson, M., Luz, F., Bajanca, during angiogenesis. during filopodia. (2004). pressure, and maturation during angiogenesis. development. (2000). heart in role key a plays and angiogenesis lineage. myotome: developmental changes and differences between the epaxial and hypaxial g ml . We performed two separate , Pdgfb, Pdgfb, Flt1 + cells by FACS-Vantage Flow cytometer (BD) from the lungs of P7 P7 of lungs the from (BD) cytometer Flow FACS-Vantage by cells −1 . −1 of thalidomide, 10 Dev. collagenase (17100-017, Invitrogen), cultured them overnight in in overnight them cultured Invitrogen), (17100-017, collagenase + J. cells on fibrinogen gel (F4753, Sigma) with 20% FBS and DMSO,

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test to compare the number of epistaxis in individuals with with individuals in epistaxis of totest compare number the al.

α al. DFD nue mcohg rcutet icesd interstitial increased recruitment, macrophage induces PDGF-D Biol. Endoglin, an ancillary TGF We determined the effect of 50 50 of effect the determined We

-SMA staining intensity with Metamorph 6.1 software software 6.1 Metamorph with intensity staining -SMA We performed statistical analyses with Prism 4 software software 4 Prism with analyses statistical Weperformed VEGF guides angiogenic sprouting utilizing endothelial tip cell tip endothelial utilizing sprouting angiogenic guides VEGF 231 Detailed methodology is described in the Dll4 signalling through Notch1 regulates formation of tip cells tip of formation regulates Notch1 through signalling Dll4 Kdr Nature

161 , 402–415 (2004). 402–415 , , normalized to a reference pool including ). We isolated RNA from P7 retinas and, using stand , 1163–1177 (2003). 1163–1177 ,

µ 445 M STI-571 or thalidomide and STI-571 together. in in vitro , 776–780 (2007). 776–780 , + cells cultured in 0.1% FBS, epidermal epidermal FBS, 0.1% in cultured cells α P -SMA) -SMA) and endothelial cell values < 0.05 were considered statisti considered were 0.05 < values transcription reactions for every RNA ), analyzed expression of mouse α β -SMA intensity with the integrated integrated the with intensity -SMA receptor, is required for extraembryonic α -SMA, NG2 and isolectin isolectin and NG2 -SMA, t test. We also used the the used We test.also µ Blood Dev. doi:10.1038/nm.2131 ml g

We isolated live Weisolated

Biol. 104 Supplementary −1 thalidomide thalidomide (

, 3198–3204 isolectin isolectin B4 217 Pecam 42–53 , Acta1 and - - - - ,