lrg1 promotes angiogenesis by modulating endothelial tgf-b signalling

ARTICLEdoi:10.1038/nature12345

LRG1 promotes angiogenesis bymodulating endothelial TGF-b signallingXiaomeng Wang1, Sabu Abraham1, Jenny A. G. McKenzie1, Natasha Jeffs1, Matthew Swire1, Vineeta B. Tripathi1,Ulrich F. O. Luhmann2, Clemens A. K. Lange2,3,4, Zhenhua Zhai5, Helen M. Arthur5, James W. B. Bainbridge2,3, Stephen E. Moss1*& John Greenwood1*

Aberrant neovascularization contributes to diseases such as cancer, blindness and atherosclerosis, and is the consequenceof inappropriate angiogenic signalling. Although many regulators of pathogenic angiogenesis have been identified, ourunderstanding of this process is incomplete. Here we explore the transcriptome of retinal microvessels isolated frommouse models of retinal disease that exhibit vascular pathology, and uncover an upregulated gene, leucine-richalpha-2-glycoprotein 1 (Lrg1), of previously unknown function. We show that in the presence of transforming growthfactor-b1 (TGF-b1), LRG1 is mitogenic to endothelial cells and promotes angiogenesis. Mice lacking Lrg1 develop a mildretinal vascular phenotype but exhibit a significant reduction in pathological ocular angiogenesis. LRG1 binds directly tothe TGF-b accessory receptor endoglin, which, in the presence of TGF-b1, results in promotion of the pro-angiogenicSmad1/5/8 signalling pathway. LRG1 antibody blockade inhibits this switch and attenuates angiogenesis. These studiesreveal a new regulator of angiogenesis that mediates its effect by modulating TGF-b signalling.

The formation of new blood vessels by angiogenesis is a key feature ofseveral diseases including age-related macular degeneration, prolifera-tive diabetic retinopathy (PDR), atherosclerosis, rheumatoid arthritisand cancer. The factors that promote neovascularization have beenthe subject of extensive research, with the vascular endothelial growthfactors (VEGFs) and their receptors emerging as master regulators1–3.Despite the prominent role of VEGF, other factors contribute to neoan-giogenesis through coordinated crosstalk that is often highly context-dependent4–6. Such complexity is exemplified in TGF-b1 signalling,which can switch from being mostly angiostatic to pro-angiogenic7.What regulates this switch is not fully understood, but activation ofthe pro-angiogenic pathway involves TGF-b type II receptor (TbRII)recruitment of the predominantly endothelial TGF-b type I receptoractivin receptor-like kinase-1 (ALK1), which in turn initiates activa-tion of the transcription factors Smad1, 5 and 8, resulting in a pro-angiogenic phenotype7–10. The regulation of this differential signallingis contingent on several factors including the concentration of TGF-b,its bioavailability and the presence or absence of other regulatory fac-tors such as bone morphogenic proteins and accessory receptors suchas endoglin (ENG) and betaglycan (also known as TGF-b type IIIreceptor)11.

Our incomplete understanding of the role of the fine-tuning ofangiogenesis suggests that additional modulators have yet to be iden-tified. Our objective in this study, therefore, was to identify new reg-ulators of pathogenic angiogenesis that may lead to the developmentof more effective treatment strategies.

Retinal vascular expression of LRG1To identify new regulators of neovascularization we exploited three mousemutants that exhibit marked remodelling of the retinal vasculature (Sup-plementary Fig. 1 and Supplementary Videos 1–4). Genome-wide trans-criptome analysis of retinal microvessel fragments isolated from the retinaldegeneration 1 (rd1) mouse, the very low density lipoprotein receptor

(VLDLR) knockout mouse (Vldlr2/2), the Grhl3ct/J curly tail mouse(Jackson Laboratory) and appropriate wild-type control mice yielded 62genes that were differentially regulated but common to all three retinaldisease models (Supplementary Table 1). When ranked according to foldchange, a gene encoding a secreted glycoprotein of unknown function,namely Lrg1, emerged as the most significantly upregulated. LRG1 is ahighly conserved member of the leucine-rich repeat family of proteins,many of which are involved in protein–protein interactions, signalling andcell adhesion (Supplementary Fig. 2a, b).

Validation of the microarray data revealed that in the retina, LRG1 isrestricted almost exclusively to the vasculature, is expressed undernormal conditions and is upregulated during retinal vascular remodel-ling in the three mouse models of retinal disease (Fig. 1a–d andSupplementary Fig. 3). However, LRG1 expression was not restrictedto the retina, as we also observed LRG1 staining in the choriocapillarisof the mouse eye (Supplementary Fig. 4a). Consistent with the dataobtained in the mouse, we observed low levels of constitutive LRG1expression in normal adult human retinal vessels and weakly, but notexclusively, in vessels in other human tissues including breast, skin andintestine (Supplementary Fig. 4b).

We next investigated whether the Lrg1 transcript is also increasedin the retinae of models of choroidal and retinal neovascularization.Choroidal neovascularization (CNV) was induced in wild-type mice,and 1 week after laser injury we observed a significant increase in Lrg1transcript levels in both the retina and retinal pigment epithelium(RPE)/choroid (Fig. 1e, f). We then examined intra-retinal/pre-retinalneovascularization in the mouse model of oxygen-induced retino-pathy (OIR), which displays hypoxia-driven retinal angiogenesis. Atpostnatal day (P) 17, during the ischaemic proliferative phase of OIRwhen neovascularization is most prevalent, Lrg1 transcript levels werealso upregulated (Fig. 1g). However, at the end of the hyperoxic phase(P12), Lrg1 messenger RNA was significantly reduced. Indeed, thepattern of Lrg1 expression at the two time points observed mirrored

*These authors contributed equally to this work.

1Departmentof Cell Biology,UCL Institute of Ophthalmology, London EC1V 9EL, UK. 2Department of Genetics,UCL Institute of Ophthalmology, London EC1V 9EL, UK. 3NIHR BiomedicalResearch Centre forOphthalmology, Moorfields Eye Hospital, London EC1V 2PD, UK. 4University Eye Hospital Freiburg, Freiburg 79106, Germany. 5Institute of Genetic Medicine, Newcastle University, Newcastle NE1 3BZ, UK.

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the expression of the hypoxia-responsive genes Vegfa, Apln (apelin)and its receptor Aplnr (Supplementary Fig. 5). To determine whetherLRG1 is upregulated in human retinal disease in which there is neo-vascular pathology, vitreous samples from human subjects with PDRwere analysed by western blot, which revealed increased LRG1expression compared to control vitreous (Fig. 1h and Supplemen-tary Fig. 6). It is unclear, however, whether this increase is the con-sequence of increased local production, leakage from the systemiccirculation or a combination of both.

These data show that in the retina, LRG1 expression is predomi-nantly vascular, is constitutive, and is increased during neovasculargrowth.

LRG1 and angiogenesisTo investigate the function of LRG1 we used cultured endothelial cellassays and in vitro and ex vivo models of angiogenesis. We observedthat overexpression of human LRG1 in endothelial cells increasedproliferation, whereas knockdown of mouse Lrg1 decreased prolifera-tion (Supplementary Fig. 7). In addition, endothelial cell migrationwas inhibited by an anti-LRG1 polyclonal antibody (SupplementaryFigs 7 and 8). In the Matrigel human umbilical vein endothelial cell(HUVEC) tube-formation assay, the supplementation of media withrecombinant human LRG1 (Supplementary Fig. 8) caused a signifi-cant increase in tube formation and branching, whereas an anti-LRG1antibody significantly blocked tube formation (Fig. 2a and Supplemen-tary Figs 8 and 9). Consistent with the latter observation, LRG1 was

found to be present in the conditioned media of these assays (Sup-plementary Fig. 10). We next investigated whether LRG1 promotesblood vessel growth in two ex vivo models of angiogenesis. Mousemetatarsals (embryonic day (E) 16.5) and aortic rings (P7) were pre-pared using tissues from wild-type mice. Vessel outgrowth and branch-ing from explanted metatarsals (Supplementary Fig. 11) or aortic ringsin the absence of other added growth factors were significantlyincreased after the addition of exogenous LRG1, and inhibited in thepresence of the anti-LRG1 polyclonal antibody (Fig. 2b). Again, con-ditioned media from both assays was found to contain LRG1 protein(Supplementary Fig. 10).

Having demonstrated that LRG1 influences vascular growth invitro and ex vivo we then investigated the retinal vasculature of theLrg1 knockout mouse (Supplementary Fig. 12). Lrg12/2 mice wereviable but exhibited a delay in the development of the deep vascularplexus at P10–P12 and the intermediate vessels between P17 and P25that had resolved by P35 (Supplementary Fig. 13). In addition, thehyaloid vessels failed to regress fully, with vessel persistence beyond

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Figure 1 | LRG1 is overexpressed in pathogenic retinal vasculature.a, b, Quantification of Lrg1 mRNA (a) and LRG1 protein expression(b), showing upregulation in the retinae of mice exhibiting retinal vascularchanges. c, Lrg1 in situ hybridization at P21. Scale bar, 50mm.d, Immunohistochemical detection of CD31 (red) and LRG1 (green) at P10,showing LRG1 expression in the retinal vasculature. e, f, Upregulation of Lrg1mRNA in the retina (e) and RPE/choroid (f) in CNV mice. g, Reduced Lrg1transcript levels in OIR at P12 and increased levels at P17. h, Increase in LRG1protein in the vitreous of patients with PDR. All images shown arerepresentative and data are mean 6 s.e.m. of n $ 3 independent experimentalgroups. *P , 0.05; **P , 0.01; ***P , 0.001 (Student’s t-test).

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Figure 2 | LRG1 promotes angiogenesis. a, Increased HUVEC tube andbranch formation after the addition of LRG1, and inhibition by a LRG1neutralizing antibody (LRG1Ab). Scale bar, 160mm. b, Vessel outgrowth in themetatarsal (top) and aortic ring (bottom) assay is enhanced by LRG1 andattenuated by a LRG1 neutralizing antibody. Scale bar, 1,500mm.c, Comparison of vessel growth from metatarsals and aortic rings isolated fromwild-type and Lrg12/2 mice shows reduced angiogenesis in the latter that couldbe rescued by the addition of LRG1. All images shown are representative andvalues are expressed as mean 6 s.e.m. of n $ 3 independent experimentalgroups. *P , 0.05; **P , 0.01; ***P , 0.001 (Student’s t-test).

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P35 and integration into the inner retina (Supplementary Fig. 14).Defective retinal vascular development and persistent hyaloid vesselswere also reported in mice with deletions in Ndp (Norrie disease (pseu-doglioma), also known as norrin), Fzd4 (frizzled homolog 4), Lrp5 andAngpt2 (angiopoietin 2), which also contribute to angiogenesis12–14. Wealso observed an increase in the incidence of crossover of the radialarteries and veins and of their side branches, with occasional smallvessels forming arteriovenous anastomosis (Supplementary Fig. 15).Arteriovenous crossing has been reported in the retina of the hypo-morphic Vegfa mouse15 and is associated with susceptibility to branchedvein occlusion in the human retina16,17. In this context it was interestingto note that Vegfa gene expression in the Lrg12/2 mouse retina is sig-nificantly lower than in control mice in contrast to Plgf, which isunchanged (Supplementary Fig. 16). Aside from these mild defects,the retinal vasculature of the Lrg12/2 mice exhibited similar pericyte

coverage (Supplementary Fig. 17) and barrier properties (Supplemen-tary Fig. 18) to wild-type controls.

As we had observed that LRG1 inhibition or supplementation had asignificant effect on vessel formation in the metatarsal and aortic ringassays, we proposed that Lrg1 knockout would lead to reduced angio-genesis in these models. Indeed, vessel formation was significantlyreduced in Lrg12/2 mice in both the metatarsal and aortic ring assay(Fig. 2c and Supplementary Fig. 19), and could be rescued by theaddition of exogenous LRG1. Together, these data support the hypo-thesis that LRG1 contributes to, and is necessary for, robust vasculargrowth.

LRG1 and pathogenic neovascularizationAs our data thus far had demonstrated increased Lrg1 transcriptexpression in CNV and OIR in wild-type mice, we investigatedwhether neovascularization in these models is attenuated in Lrg12/2

mice. CNV was induced in wild-type and Lrg12/2 mice, and at 7 dayspost-laser fundus fluorescein angiography at 90 s revealed a dimi-nished neovascular response in the Lrg12/2 mice compared to controls(Fig. 3a; P , 0.01). Concomitant with this was an equivalent reductionin fluorescein leakage at 7 min after injection. This effect was con-firmed in a group of animals in which the neovascular lesion wasvisualized in posterior eyecup whole mounts, quantitative analysisof which showed that mean lesion volume was about 70% smaller inLrg12/2 than wild-type mice (Supplementary Fig. 20). The reductionin lesion size in the Lrg12/2 mouse was similar to that reported inPlgf2/2 (ref. 18) and Ccr32/2 (ref. 19) mice, two other pro-angiogenicfactors, and could not be explained by changes in macrophage recruit-ment (Supplementary Fig. 20) or pericyte coverage (SupplementaryFig. 21). We next investigated intra-retinal/pre-retinal neovasculariza-tion, as observed in PDR, in the OIR model of angiogenesis. After the5-day hyperoxia phase, the size of the avascular region at P12 was notsignificantly different between the Lrg12/2 and wild-type animals(Fig. 3b and Supplementary Fig. 22). Furthermore, after 5 days in nor-moxia, revascularization of the avascular region with ordered vesselswas similar between the two groups, demonstrating that hyperoxia-induced regression and hypoxia-induced physiological revasculariza-tion are not affected by the loss of LRG1. However, the area occupiedby disordered neovascular growth (tufts) was significantly reducedin the absence of LRG1 (Fig. 3b; P , 0.01; Supplementary Fig. 22),demonstrating that LRG1 is specifically required for robust pathogenicangiogenesis.

Having demonstrated that an antibody against LRG1 inhibitsangiogenesis in vitro, we investigated whether this antibody wouldalso reduce CNV lesion size. After the laser burn, animals receivedintravitreal injections of the anti-LRG1 polyclonal antibody or a pre-immune IgG as a control, and 5 days later lesion sizes were measured.In the anti-LRG1 antibody-treated eyes, a dose-dependent reductionin CNV lesion volume (Fig. 3c, d) and area (Supplementary Fig. 23)was observed compared to control-antibody-treated eyes. Indeed, the58% reduction of CNV volume (Fig. 3d) and 46% reduction in area ata dose of 10mg (Supplementary Fig. 23) was of similar magnitude asthat achieved with blockade of the VEGF/PLGF signalling axis20 or thechemokine receptor CCR3 (ref. 19).

As antibody blockade of LRG1 reduces CNV lesion size, we inves-tigated the effects of combination therapy with a VEGF receptor2 (VEGFR2)-blocking antibody20. Antibody blockade of LRG1,VEGFR2, or both LRG1 and VEGFR2 together inhibited CNV lesionvolume (Fig. 3e and Supplementary Fig. 23), with the combined therapygiving the most significant inhibition. The effect of combinatorial treat-ment was also evaluated in OIR. Under the treatment conditions used,in which the individual antibodies elicited no significant effect, animalstreated with the antibody combination exhibited a significant inhibitionof both patterned revascularization and the formation of pathogenicvascular tufts (Supplementary Fig. 24). These data provide compellingevidence that inhibition of LRG1 is effective in preventing pathological

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Figure 3 | LRG1 contributes to pathogenic neovascularization.a, Representative images of wild-type (WT) and Lrg12/2 mouse laser-burnlesions by infrared (IR) fundus imaging. At 7 days after the laser, early- and late-phase fundus fluorescein angiography (FFA) revealed a reduction in CNVlesion size and a decrease in fluorescein leakage, respectively, in Lrg12/2 mice.Representative images of isolectin B4 stained (red) CNV in choroidal/RPE flat-mount 7 days after induction, confirming decreased lesion size in Lrg12/2

mice. Scale bar, 100mm. b, In OIR, Lrg1 deletion does not affect the size of theavascular region at P12 (delineated by white boundary line) or the organizednormal revascularization at P17, but does decrease the formation of pathologicalneovascular (NV) tufts (highlighted in red and delineated in higher power bywhite boundary line). Scale bars, 1,000mm (P12 and P17 lower magnification)and 50mm (P17 higher magnification). c, Volume-rendered examples ofPECAM-1 stained CNV lesions in wild-type mice after intravitreal injectionof irrelevant IgG or LRG1 neutralizing antibody. Scale bar, 100mm. d, Dose-dependent anti-LRG1 antibody reduction of CNV lesion volume. e, Combinationof anti-LRG1 and DC101 (anti-VEGFR2) in CNV in wild-type mice resulted inenhanced reduction of lesion volume compared to single treatments. Data aremean 6 s.e.m. of n $ 10 for each group. *P , 0.05; **P , 0.01; ***P , 0.001(Student’s t-test (a, b), and one-way analysis of variance (ANOVA)(d, e)).

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angiogenesis, and suggest that LRG1 has potential as a therapeutic targeton its own or in combination with other anti-angiogenic therapies.

LRG1 and TGF-b signallingAlthough little is known about the biology of LRG1, concomitantincreases in the expression levels of TGF-b1, TbRII and LRG1 havebeen reported in cancer cells21 and hydrocephalus22, and LRG1 has beenshown to bind to TGF-b1 in high endothelial venules23. Consistent withthis, and with earlier proteomic and transcriptome analyses24,25, we haveshown here that in vitreous samples from the eyes of human subjectswith PDR, both LRG1 (Fig. 1h and Supplementary Fig. 6) and TGF-b1protein levels (Supplementary Fig. 25) are significantly increased.Furthermore, alongside increased Lrg1 gene expression, Tgfb1 trans-cript levels were also significantly upregulated in the retinae of laser-induced CNV mice and in OIR mice during the ischaemic proliferativephase (Supplementary Fig. 5). These data prompted us to investigatewhether LRG1 acts as a modulator of TGF-b signalling.

To determine whether LRG1 associates with components of theTGF-b receptor complex, we performed co-immunoprecipitationexperiments. In primary brain endothelial cells, LRG1 was presentin immunoprecipitates of TbRII, ALK1, ALK5 and the auxiliaryreceptor ENG (Fig. 4a). Conversely, immunoprecipitates of LRG1from HUVECs were found to contain TbRII, ALK1, ALK5 andENG (Supplementary Fig. 26a). These observations suggested thatLRG1 might be involved in regulating TGF-b signalling throughfine-tuning the stoichiometry of the TGF-b receptor complex. TGF-b1 signals in endothelial cells by TbRII recruitment of either theubiquitous ALK5 receptor, or the predominantly endothelial ALK1receptor together with ALK5. Stimulation of the TbRII–ALK5 signal-ling complex results in phosphorylation and activation of the trans-cription factors Smad2 and 3, which increases extracellular matrixdeposition, inhibits endothelial cell proliferation and migration, andpromotes cell homeostasis, whereas signalling via the TbRII–ALK5/ALK1 complex (possibly in association with ENG) activates Smad1, 5and 8, resulting in a pro-angiogenic state7,8. We therefore investigated thedirect one-to-one binding of LRG1 to recombinant extracellular domainsof individual TGF-b receptors in serum-free conditioned media fromtransfected HEK293T cells (Supplementary Fig. 26b). Immunoprecipita-tion of the receptor ectodomain revealed co-immunoprecipitation ofLRG1 with ALK5, TbRII and ENG, indicating a direct interaction ofLRG1 with these individual receptors (Fig. 4b). This occurred in theabsence of TGF-b1, which was not present in HEK293T-cell-conditionedmedium (Supplementary Fig. 10). Addition of conditioned mediumcontaining non-tagged ENG, ALK5 or TbRII out-competed taggedreceptor binding to LRG1 (Fig. 4c), confirming the specificity of theseprotein–protein interactions. The observation that ENG seems to beone of the receptors for LRG1 is germane, given its proposed role inswitching TGF-b signalling towards the pro-angiogenic Smad1/5/8pathway26. A potential functional relationship between LRG1 andENG was additionally strengthened by our observation that Eng isupregulated in CNV and OIR (Supplementary Fig. 27). To define theLRG1–ENG interaction further we undertook surface plasmon res-onance analysis (Biacore) and obtained an affinity rate constant (KD)of 2.9mM (Supplementary Fig. 28) for binding of the ENG ectodomainto LRG1.

These data raise the possibility that LRG1 facilitates a receptorconfiguration conducive to the pro-angiogenic signalling pathway.To investigate this, LRG1 was incubated with conditioned mediacontaining the extracellular domains of either ALK1 or ALK5 in thepresence or absence of different combinations of TGF-b1, and theextracellular domains of ENG and TbRII. These studies revealed thatLRG1 only associated with ALK1 in the presence of ENG, to whichit bound, and this was enhanced by the addition of TGF-b1 (Fig. 4d).Conversely, ALK5 binds LRG1 in the absence of ENG but in its pres-ence this interaction is attenuated, suggesting competition betweenENG and ALK5 for LRG1, whereas the addition of TGF-b1 resultsin complete loss of the LRG1–ALK5 association. Moreover, neitherTGF-b1 nor TbRII on their own, nor a combination of both, affectsALK1–LRG1 or ALK5–LRG1 association in the absence of ENG.However, in the presence of ENG, TbRII is able to form a complexwith ALK1–LRG1 or ALK5–LRG1, with the former association beingenhanced and the latter being further inhibited by TGF-b1. In accord-ance with previously suggested models8, these data indicate that LRG1may be able to form an intermediate complex with ALK5, ALK1, TbRIIand ENG, but that in the presence of TGF-b1 the LRG1–ALK1–TbRII–ENG complex predominates. An association between LRG1and TGF-b1 may therefore lead to more efficient ALK1–TbRII–ENG receptor complex formation, and consequently to the promotionof pro-angiogenic Smad1/5 signalling.

To test this hypothesis, we treated mouse brain endothelial cellswith TGF-b1 (5 ng ml21) and showed that both Smad2/3 and Smad1/5 phosphorylation are induced in wild-type cells, but in Lrg1-null cellsonly Smad2/3 is activated (Fig. 5a). The addition of LRG1 alone did

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Figure 4 | LRG1 modifies the TGF-b receptor complex.a, Immunoprecipitation (IP) of TbRII, ALK1, ALK5 and ENG with anti-receptor antibodies (RecAb) from wild-type mouse brain endothelial celllysates co-precipitates LRG1. Control IgG in wild-type endothelial cells or anti-receptor antibodies in Lrg12/2 endothelial cells did not co-precipitate LRG1.WB, western blot. b, Immunoprecipitation of peptide-tagged extracellulardomains of ALK5 (haemagglutinin (HA)-tagged), TbRII (Myc-tagged) or ENG(V5-tagged) added individually to histidine (His)-tagged LRG1 resulted in co-precipitation of LRG1, indicating direct interactions with these receptors.Immunoprecipitation of ALK1 (HA-tagged) in the presence of LRG1 did notco-precipitate the latter. c, Addition of appropriate soluble non-taggedextracellular domains of ENG, ALK5 and TbRII out-competed peptide-taggedreceptor binding to LRG1. d, LRG1 was incubated in vitro with differentcombinations of TGF-b receptor extracellular domains and TGF-b1 (Tb1). Inthe presence of ENG, binding between LRG1 and ALK5 is diminished, and iscompletely lost with the further addition of TGF-b1. Conversely, ENGfacilitates the association between LRG1 and ALK1, which is enhanced in thepresence of TGF-b1. Although TbRII has no effect on LRG1–ALK1 or LRG1–ALK5 interactions, it is recruited to the complex in the presence of ENG. Alldata are representative western blots of n $ 3 for each experiment.

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not activate Smad2/3 or Smad1/5, but in combination with TGF-b1there was a marked induction of Smad1/5 phosphorylation, showingthat LRG1 requires the presence of TGF-b1 to stimulate the pro-angiogenic TbRII–ALK1–Smad1/5/8 pathway (Fig. 5a). As TGF-b1is an endothelial cell mitogen7 we also investigated whether LRG1augmented TGF-b1-mediated cell proliferation. Brain endothelialcells from Lrg12/2 mice proliferated more slowly than those fromwild-type animals (Fig. 5b). Addition of TGF-b1 significantlyenhanced endothelial cell proliferation from wild-type animals butinhibited the growth of cells from Lrg12/2 mice, presumably throughenhanced ALK5–Smad2/3 signalling in the absence of activation ofthe ALK1–Smad1/5/8 arm. The addition of LRG1 on its own had noeffect, but TGF-b1 and LRG1 in combination increased proliferationsignificantly in both wild-type and Lrg1-null endothelial cells (Fig. 5b).Moreover, in the metatarsal angiogenesis assay, the combined additionof LRG1 and TGF-b1 led to a substantial increase in vessel formation(Fig. 5c). The observation that LRG1 alone induced a small increase invessel formation indicates that TGF-b1 is produced constitutively bythe metatarsal tissue, which was confirmed by western blotting of con-ditioned medium (Supplementary Fig. 10).

To confirm that the pro-angiogenic effect of LRG1 was mediatedthrough the ALK1–Smad1/5/8 pathway, ALK1 was knocked downwith short interfering RNA (siRNA) (Fig. 5d and SupplementaryFig. 29) or inhibited by LDN193189 (Supplementary Fig. 29), result-ing in prevention of LRG1-induced Smad1/5 phosphorylation with-out affecting Smad2 phosphorylation. As predicted, in the Matrigelassay ALK1 inhibition led to a significant decrease in HUVEC tubeand branch formation, and blocked the angiogenic activity of LRG1 inthis assay (Fig. 5f and Supplementary Fig. 29). Conversely, knock-down of ALK5 with siRNA (Fig. 5d, f) or inhibition with SB43152

(Supplementary Fig. 29), which inhibited constitutive Smad2 phos-phorylation, did not prevent LRG1-induced HUVEC tube formation.siRNA knockdown of TbRII or ENG also resulted in the abrogation ofLRG1-induced Smad1/5 phosphorylation (Fig. 5e and SupplementaryFig. 29) and HUVEC tube and branch formation (Fig. 5g). To corrob-orate the involvement of ENG in LRG1-mediated signalling further,lung endothelial cells derived from Rosa26-CreERT:Engfl/fl mice27 weretreated with 4OH-tamoxifen to deplete ENG (MLEC;Eng2/2, in whichMLECs denotes mouse lung endothelial cells) (Fig. 5h). Unlike controlcells, treatment of MLEC;Eng2/2 with a combination of TGF-b1 andLRG1 failed to induce Smad1/5 phosphorylation (Fig. 5h). Consistentwith this, similar treatment resulted in a significant increase in celldivision of control MLECs, whereas the cell division of MLEC;Eng2/2

was significantly reduced and refractive to treatment with TGF-b1 6 LRG1 (Fig. 5i). In addition, we used an endothelial-specific con-ditional knockout approach in which metatarsals were collected fromEng-floxed mice (Cdh5(PAC)-CreERT2;Engfl/fl)28 and treated with 4OH-tamoxifen (to generate Eng-iKOe metatarsals). This resulted in a loss ofvascular ENG expression, compared with Engfl/fl controls (Supplemen-tary Fig. 30), and a 51% reduction in LRG1-induced vessel growth(Fig. 5j; P , 0.01) and a 53% reduction in vessel branching (Fig. 5k;P , 0.05). In agreement with these data, there were fewer cells expressingphosphorylated Smad1/5/8 in CNV lesions in Lrg12/2 mice comparedto wild-type animals (Supplementary Fig. 31). Moreover, in both CNVand OIR (during the neovascular phase) the Smad1/5 mediated pro-mitogenic gene inhibitor of DNA binding 1 (Id1) was significantly upre-gulated (Supplementary Fig. 5).

ConclusionsTGF-b signalling has an important role in determining endothelialcell function during both development and vascular pathology7,8,29,30,

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Figure 5 | LRG1 promotes angiogenesis via a switch in TGF-b signalling.a, In wild-type brain endothelial cells (EC), TGF-b1 stimulates Smad2/3phosphorylation (pSmad2/3) and low levels of Smad1/5 phosphorylation(pSmad1/5), but in Lrg12/2 cells only Smad2/3 is phosphorylated. LRG1addition has no effect on Smad phosphorylation in wild-type or Lrg1-null cells,but co-treatment with TGF-b1 and LRG1 enhances Smad1/5 phosphorylationwithout affecting Smad2/3 phosphorylation (n $ 3). Ctrl, control.b, Proliferation of brain endothelial cells isolated from wild-type control andLrg12/2 mice after exogenous TGF-b1 and/or LRG1 treatment normalized tocontrol (n $ 3). Non-treated Lrg12/2 cells are less proliferative than wild-typecells. TGF-b1 addition to wild-type cells results in enhanced proliferation butreduces proliferation in Lrg12/2 cells, whereas TGF-b1 and LRG1 co-treatmentresults in enhanced proliferation in wild-type and Lrg12/2 cells. c, Addition ofexogenous TGF-b1 and LRG1, compared to LRG1 alone or denatured(D)LRG1, enhances microvessel formation in the mouse metatarsalangiogenesis assay (n 5 3 independent experiments, n $ 30 metatarsals pertreatment). d, siRNA knockdown of ALK1 or ALK5 in HUVECs results inreduced Smad1/5 or Smad2 phosphorylation, respectively. ALK1, but notALK5, knockdown results in prevention of LRG1-induced Smad1/5phosphorylation. e, siRNA knockdown of TbRII or ENG inhibits LRG1-induced Smad1/5 phosphorylation. Histograms in d and e show semi-quantification of Smad phosphorylation relative to GAPDH (n $ 3).f, g, Knockdown of ALK1, TbRII or ENG, but not ALK5, reduces LRG1-mediated HUVEC Matrigel tube formation (n 5 3 independent groups for eachassay). h, Treatment of lung endothelial cells isolated from Rosa26-CreERT:Engfl/fl mice (MLEC;Engfl/fl) with a combination of TGF-b1 and LRG1results in Smad1/5 phosphorylation; this response is lost after pre-treatmentwith 4OH-tamoxifen to delete ENG (MLEC;Eng2/2). i, Treatment of controlMLEC;Engfl/fl with TGF-b1 and LRG1 stimulates cell division. InMLEC;Eng2/2 cells, cell division is reduced and refractive to treatment withTGF-b1 6 LRG1 (n 5 3 independent experiments). j, k, 4OH-tamoxifentreatment of metatarsals isolated from Engfl/fl (control) and Cdh5(PAC)-CreERT2;Engfl/fl (Eng-iKOe) mice results in loss of ENG expression in the latter(Supplementary Fig. 30), and decreases LRG1-induced metatarsal vessel length(j) and branching (k) (metatarsals from five independent litters). Data aremean 6 s.e.m. *P , 0.05; **P , 0.01; ***P , 0.001 (Student’s t-test (a–i) andtwo-way ANOVA (j and k)).

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and its activity is regulated at several levels from gene expression tocontrol of extracellular bioavailability. The multiplicity of regulatorymechanisms together with variable combinations of receptors/co-receptors creates complex patterns of TGF-b activity that define itscontext-dependent effects. In particular, the balance between theALK5 and ALK1 signalling pathways is considered to be central indetermining the angiogenic switch, with ENG being proposed as akey regulatory molecule in promoting signalling through the ALK1pathway26,30,31. In searching for mediators of vascular remodelling inthe diseased/damaged retina we have discovered a new regulator ofTGF-b signalling. The data presented here support a hypothesis thatLRG1 activates the TGF-b angiogenic switch by binding to the access-ory receptor ENG and, in the presence of TGF-b1, promotes signallingvia the TbRII–ALK1–Smad1/5/8 pathway (Supplementary Fig. 32).Moreover, our evidence suggests that LRG1 may have a more domi-nant role in disorganized pathological rather than developmental/physiological angiogenesis. Although in the retina this is clearly sup-ported by our in vivo data, the ex vivo and in vitro studies indicatethat LRG1 angiogenic activity is not restricted to the eye. The modu-lating effect of LRG1 on TGF-b1 signalling is the first demonstration,to our knowledge, of a definitive function for LRG1 and raises theintriguing possibility that it may influence other major biological pro-cesses in which TGF-b has a role, such as neoplasia32 and the immuneresponse33. Inhibition of LRG1, which we show here causes a shift awayfrom angiogenic signalling, could prevent pathogenic activation ofthis pathway, while leaving homeostatic TGF-b signalling unperturbed.From these studies we suggest, therefore, that LRG1 is a highly pro-mising therapeutic target for controlling pathogenic angiogenesis inocular disease, and potentially in other diseases such as cancer andatherosclerosis.

METHODS SUMMARYMicrovessel global gene expression analysis was undertaken using Affymetrixmouse 430.2 gene arrays. CNV and OIR were induced in mice as described inthe Methods. All other methods are described in the Methods.

Full Methods and any associated references are available in the online version ofthe paper.

Received 26 August 2011; accepted 3 June 2013.

1. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V. & Ferrara, N. Vascularendothelial growth factor is a secreted angiogenic mitogen. Science 246,1306–1309 (1989).

2. Carmeliet, P. et al. Abnormal blood vessel development and lethality in embryoslacking a single VEGF allele. Nature 380, 435–439 (1996).

3. Ferrara, N. et al. Heterozygous embryonic lethality induced by targetedinactivation of the VEGF gene. Nature 380, 439–442 (1996).

4. Holderfield, M. T. & Hughes, C. C. Crosstalk between vascular endothelial growthfactor, notch, and transforming growth factor-b in vascular morphogenesis. Circ.Res. 102, 637–652 (2008).

5. Chung, A. S. & Ferrara, N. Developmental and pathological angiogenesis. Annu.Rev. Cell Dev. Biol. 27, 563–584 (2011).

6. Carmeliet, P. & Jain, R. K. Molecular mechanisms and clinical applications ofangiogenesis. Nature 473, 298–307 (2011).

7. Pardali, E., Goumans,M. J.& ten Dijke,P. Signaling bymembers of the TGF-b familyin vascular morphogenesis and disease. Trends Cell Biol. 20, 556–567 (2010).

8. Goumans, M. J., Liu, Z. & ten Dijke, P. TGF-b signaling in vascular biology anddysfunction. Cell Res. 19, 116–127 (2009).

9. Cunha, S. I. et al. Genetic and pharmacological targeting of activin receptor-likekinase1 impairs tumorgrowthandangiogenesis. J. Exp.Med.207,85–100(2010).

10. Cunha, S. I. & Pietras, K. ALK1 as an emerging target for antiangiogenic therapy ofcancer. Blood 117, 6999–7006 (2011).

11. ten Dijke, P. & Arthur, H. M. Extracellular control of TGFb signalling in vasculardevelopment and disease. Nature Rev. Mol. Cell Biol. 8, 857–869 (2007).

12. Xu, Q. et al.Vasculardevelopment in the retinaand innerear: controlby Norrin andFrizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895 (2004).

13. Ye, X. et al. Norrin, Frizzled-4, and Lrp5 signaling in endothelial cells controls agenetic program for retinal vascularization. Cell 139, 285–298 (2009).

14. Hackett, S. F., Wiegand, S., Yancopoulos, G. & Campochiaro, P. A. Angiopoietin-2playsan important role in retinal angiogenesis.J.Cell. Physiol.192,182–187 (2002).

15. Haigh, J. J. et al. Cortical and retinal defects caused by dosage-dependentreductions in VEGF-A paracrine signaling. Dev. Biol. 262, 225–241 (2003).

16. Zhao, J., Sastry, S. M., Sperduto, R. D., Chew, E. Y. & Remaley, N. A. Arteriovenouscrossing patterns in branch retinal vein occlusion. The Eye Disease Case-ControlStudy Group. Ophthalmology 100, 423–428 (1993).

17. Kumar, B. et al. The distribution of angioarchitectural changes within the vicinity ofthe arteriovenous crossing in branch retinal vein occlusion. Ophthalmology 105,424–427 (1998).

18. Rakic, J. M. et al. Placental growth factor, a member of the VEGF family, contributesto the development of choroidalneovascularization. Invest. Ophthalmol. Vis. Sci. 44,3186–3193 (2003).

19. Takeda, A. et al. CCR3 is a target for age-related macular degeneration diagnosisand therapy. Nature 460, 225–230 (2009).

20. VandeVeire, S.et al. Furtherpharmacological and genetic evidence for the efficacyof PIGF inhibition in cancer and eye disease. Cell 141, 178–190 (2010).

21. Sun, D., Kar, S. & Carr, B. I. Differentially expressed genes in TGF-b1 sensitive andresistant human hepatoma cells. Cancer Lett. 89, 73–79 (1995).

22. Li, X., Miyajima, M., Jiang, C. & Arai, H. Expression of TGF-bs and TGF-b type IIreceptor in cerebrospinal fluid of patients with idiopathic normal pressurehydrocephalus. Neurosci. Lett. 413, 141–144 (2007).

23. Saito, K. et al. Gene expression profiling of mucosal addressin cell adhesionmolecule-11 high endothelial venule cells (HEV) and identification of a leucine-rich HEV glycoprotein as a HEV marker. J. Immunol. 168, 1050–1059 (2002).

24. Spirin, K. S. et al. Basement membrane and growth factor gene expression innormal and diabetic human retinas. Curr. Eye Res. 18, 490–499 (1999).

25. Gao, B. B., Chen, X., Timothy, N., Aiello, L. P. & Feener, E. P. Characterization of thevitreous proteome in diabetes without diabetic retinopathy and diabetes withproliferative diabetic retinopathy. J. Proteome Res. 7, 2516–2525 (2008).

26. Lebrin, F. et al. Endoglin promotes endothelial cell proliferation and TGF-b/ALK1signal transduction. EMBO J. 23, 4018–4028 (2004).

27. Anderberg, C. et al. Deficiency for endoglin in tumor vasculature weakens theendothelial barrier tometastatic dissemination. J. Exp.Med. 210, 563–579 (2013).

28. Mahmoud,M.et al. Pathogenesisof arteriovenousmalformations in theabsenceofendoglin. Circ. Res. 106, 1425–1433 (2010).

29. Bobik, A. Transforming growth factor-betas and vascular disorders. Arterioscler.Thromb. Vasc. Biol. 26, 1712–1720 (2006).

30. ten Dijke, P., Goumans, M. J. & Pardali, E. Endoglin in angiogenesis and vasculardiseases. Angiogenesis 11, 79–89 (2008).

31. Ray, B. N., Lee, N. Y., How, T. & Blobe, G. C. ALK5 phosphorylation of the endoglincytoplasmic domain regulates Smad1/5/8 signaling and endothelial cellmigration. Carcinogenesis 31, 435–441 (2010).

32. Lynch, J. et al. MiRNA-335 suppresses neuroblastoma cell invasiveness by directtargeting of multiple genes from the non-canonical TGF-b signalling pathway.Carcinogenesis 33, 976–985 (2012).

33. Gregory, A. D., Capoccia, B. J., Woloszynek, J. R. & Link, D. C. Systemic levels ofG-CSF and interleukin-6 determine the angiogenic potential of bone marrowresident monocytes. J. Leukoc. Biol. 88, 123–131 (2010).

Supplementary Information is available in the online version of the paper.

Acknowledgements This project was supported by grants from the Lowy MedicalResearch Foundation, the Medical Research Council, The Wellcome Trust, UCLBusiness (Proof of Concept Grant) and the Rosetrees Trust. J.W.B.B. is supported by aNIHRResearchProfessorship.H.M.A. is supported byaBritish Heart FoundationSeniorFellowship. We would also like to thank M. Gillies for his role in initiating the originalproject, P. Luthert and C. Thaung for human tissue samples and advice on humanpathology specimens, S. Perkins and R. Nan for assistance with the surface plasmonresonance analysis, and P. ten Dijke for discussions and advice.

Author Contributions The project was conceived by J.G., S.E.M. and X.W. Experimentsweredesigned by J.G., S.E.M., X.W. andS.A. Microarrays wereperformed byJ.A.G.M. andqPCR reactions by X.W. X.W. and S.A. characterized the Lrg1 knockout mice and LRG1antibody. X.W. performed all the metatarsal assays (except in Fig. 5j, k), aortic ringassays and Matrigel assays, carried out all the biochemical and molecular biology workand analysed the data. S.A. and X.W. undertook the immunohistochemistry andgenerated the OIR mouse model. U.F.O.L., C.A.K.L., S.A., X.W. andJ.W.B.B. performed theCNV experiments, and S.A. and X.W. analysed the data. J.W.B.B. provided human vitrealsamples. Z.Z. and H.M.A. generated MLEC;Engfl/fl cells and X.W. performed proliferationassay and biochemical analysis. Z.Z., S.A. and H.M.A. carried out the metatarsal assayson Eng knockout mice. V.B.T. performed the Biacore experiments. N.J. and M.S.provided assistance and technique support. X.W., S.A., J.G. and S.E.M. produced thefigures, and J.G. and S.E.M. wrote the text, with all authors contributing to the finalmanuscript. J.G. and S.E.M. provided leadership throughout the project.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondenceand requests for materials should be addressed to J.G. ([email protected]) orS.E.M. ([email protected]).

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METHODSAnimals. C75BL/6J mice were purchased from Harlan Laboratories. rd1 (ref. 34),Vldlr2/2 (ref. 35) and Grhl3ct/J curly tail mice were purchased from the JacksonLaboratory. Lrg12/2 mice were generated by the University of California Daviesknockout mouse project (KOMP) repository (http://www.komp.org/ and Supplemen-tary Fig. 12). Rosa26-CreERT;Engfl/fl and Cdh5(PAC)-CreERT2;Engfl/fl mice have beenpreviously described27,28. All procedures were performed in accordance with the UKAnimals (Scientific Procedures) Act and with the Association for Research in Visionand Ophthalmology Statement for the Use of Animals in Ophthalmic and VisionResearch and the Animal Welfare and the Ethical Review Bodies of the UCL Instituteof Ophthalmology and Newcastle University.Vessel isolation and gene expression analysis. Mouse retinal vessels from wild-type C57BL/6J (15 weeks), Vldlr2/2 (16 weeks), rd1 (18 weeks) and Grhl3ct/J curlytail (13 weeks) mice were isolated as described elsewhere36. RNA was extractedfrom the enriched microvascular preparations and processed for whole-genomemicroarray analysis as previously described36. Twelve mice were used per strainper RNA extraction. This was repeated four times, providing RNA for four chipsper animal model.Quantitative PCR (qPCR). RNA was extracted using Trizol (Invitrogen) followedby an RNeasy clean-up (QIAGEN). RNA was reverse transcribed using theQuantiTect Reverse Transcription Kit (QIAGEN) and PCR was conducted withQuantiTect PowerSybr Green (Applied Biosystems) using a 7900HT Fast Real-TimePCR System (Applied Biosystems); samples were normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Primers used in this study are listed inSupplementary Table 2. Student’s t-test was performed to determine statistical sig-nificance between test groups.SDS–PAGE and western blotting. Proteins were separated by SDS–PAGE. Gelswere either stained using Coomassie-blue or transferred onto a Hybond-P PVDFmembrane (GE Healthcare). Blots were probed with phospho-Smad1/5 antibody(rabbit monoclonal antibody, NEB), phospho-Smad2 antibody (rabbit monoclo-nal antibody, NEB), TGF-b1 antibody (mouse monoclonal, R&D systems), TbRIIantibody (mouse monoclonal, R&D systems), ALK1 antibody (rabbit polyclonal,Santa Cruz Biotechnology), ALK5 antibody (rabbit polyclonal, Abcam), endoglinantibody (mouse monoclonal, R&D Systems), LRG1 antibody (HPA001888,rabbit polyclonal, Sigma) or GAPDH antibody (mouse monoclonal, Novus),followed by horseradish peroxidase (HRP)-conjugated secondary antibodies(GE Healthcare) or HRP-conjugated Protein A (GE Healthcare). Densitometrywas performed using ImageJ software (National Institutes of Health). Student’st-test was performed to determine statistical significance between test groups.RNA in situ hybridization. Eyes were fixed in 2% (w/v) paraformaldehyde (PFA)in PBS for 2 min and dissected in 23 PBS. Retinae were flattened and fixed in100% ice-cold methanol overnight at 220 uC. After recovery from methanol,retinae were re-fixed for 10 min in 4% PFA and washed in PBS before digestionfor 10 min in proteinase K (80mg ml21 in PBS). Retinae were re-fixed for 5 min in4% PFA and 0.2% glutaraldehyde in PBS. After a brief wash in PBS, retinae werepre-incubated in hybridization buffer (50% formamide, 53 SSC, 50 mg ml21

transfer RNA, 1% SDS, 50 mg ml21 heparin) for 1 h at 65 uC. Denatured RNAprobes were incubated with retinae at 65 uC overnight. Primers used to generateRNA probes by PCR are listed in Supplementary Table 2. Probes were labelledwith digoxigenin (DIG)-UTP using a DIG RNA labelling kit (Roche Diagnostics).Signal was developed with alkaline phosphatase-conjugated anti-digoxigenin Fabfragments, according to the manufacturer’s instructions.Immunohistochemistry. Retinal/RPE whole-mounts were fixed and stained aspreviously described37, and incubated overnight with antibodies against humanLRG1 (HPA001888), mouse PECAM-1 (rat monoclonal, BD Biosciences), mousecollagen IV (rabbit polyclonal, AbD Serotec), human collagen IV (goat polyclonal,Millipore), rat NG2 (rabbit polyclonal, Millipore), mouse F4/80 (rat monoclonal,AbD Serotec), mouse endoglin (rat monoclonal, Santa Cruz Biotechnology) orhuman VE-cadherin (mouse monoclonal, Santa Cruz Biotechnology), and iden-tified with Alexa 488, Alexa 594 or Alexa 647 secondary antibodies (Invitrogen) orFITC-GSL (DyLight 594) isolectin B4 (Vector Labs). Retinae were flat-mounted inMowiol and examined by epifluorescence (Leica DM IRB inverted research micro-scope or Olympus SZX16 research stereo zoom microscope) or confocal (CarlZeiss LSM 510 or 710) microscopy. For quantifying the retinal vascular area,raw image data were processed with Photoshop CS4.3. Three-dimensional ren-dering of confocal Z-stacks was carried out using Imaris 7.5 software (BitplaneAG). The retinal vasculature was analysed through automatic surface renderingaided by manual threshold adjustment so that only blood vessels were included foranalysis. Imaris Key Frame Animation was used for movie generation.Human tissue. Vitreous and plasma samples were collected from patients havingsurgery for PDR or epiretinal membrane. Human tissue arrays were obtainedfrom Pantomics and stained with rabbit anti-LRG1 antibody (Sigma). The study

followed the ethical guidelines of the Declaration of Helsinki. Institutional ReviewBoards granted approval for allocation and biochemical analysis of specimens.Cells and cell culture. Pooled HUVECs were purchased from Lonza and culturedaccording to suppliers instructions. HEK293T cells were purchased fromInvitrogen and cultured as recommended. Mouse primary brain endothelial cellswere isolated, purified and cultured as previously described for rat38. The immor-talized Lewis rat brain microvascular endothelial cell line GPNT was grown aspreviously described39. MLECs were isolated from Rosa26-CreERT;Engfl/fl micecarrying the Immortomouse transgene, and were collected and cultured as previ-ously described27. Cells were pre-treated with 1 mM 4OH-tamoxifen for 48 h inculture to generate ENG-depleted cells (MLEC;Eng2/2) and untreated cellsserved as controls (MLEC;Engfl/fl).Generation of LRG1 polyclonal antibody. Rabbits were immunized with puri-fied full-length His-tagged human LRG1 protein (Covalab). Pre-immune serawere collected to produce control IgG. Antisera were collected after 3 months andantibody was purified by HiTrap Protein G FF column (GE Healthcare) andconcentrated and desalted using HiPrep 26/10 Desalting (GE Healthcare).Matrigel HUVEC tube formation assay. HUVECs were grown on growth factor-reduced Matrigel (BD Biosciences) as described elsewhere40. The 96-well plateswere coated with Matrigel-containing diluent (control) or LRG1 (20mg ml21),rabbit polyclonal antibody against LRG1 (C10-54, 100 nM), rabbit IgG (100 nM),ALK1 inhibitor (LDN 193189, Axon Medchem BV, 100 nM) or ALK5 inhibitor(SB43152, Sigma, 10mM), and allowed to polymerize in the incubator at 37 uC for45 min. Tube formation was visualized using an Olympus SZX16 Research stereo-microscope and analysed by counting the number of branch points and total tubelength per well using ImageJ. Three independent experiments were carried out andeach was performed in triplicate. Student’s t-test was performed to determinestatistical significance between test groups.Metatarsal angiogenesis assay. The metatarsal angiogenesis assay was carriedout as described41. Metatarsal bones were isolated from E16.5 wild-type control orLrg12/2 littermate mice and treated with TGF-b1 (5 ng ml21, R&D systems),LRG1 (20mg ml21), anti-LRG1 polyclonal antibody (100 nM) or rabbit IgG(100 nM) as indicated. Medium was replaced every 2 days. At day 10 of culture,the explants were fixed and stained for PECAM-1 (rat monoclonal, BDBiosciences) and visualized under an Olympus SZX16 Research stereo-zoommicroscope. After image processing in Photoshop CS4 to mask the cartilage,the length of PECAM-1-positive tubular structures and the number of branchpoints were determined by Imaris 7.5 software (Bitplane) using automatic fila-ment tracing with manual threshold corrections. Statistical data were importedinto Excel (Microsoft) for calculating total vessel length and the number of branchpoints. A least three independent experiments were carried out, comprising aminimum of 30 metatarsals for each treatment. Student’s t-test was performed todetermine statistical significance between test groups.

To investigate the effect of ENG depletion on the pro-angiogenic effect ofLRG1, metatarsals from Cdh5(PAC)-CreERT2;Engfl/fl mice28 were used to gene-rate endothelial-specific depletion of ENG after addition of 1 mM 4OH-tamoxifen3 days after metatarsal bone isolation, when neovessels began to emerge. Onday 4, LRG1 was added to a final concentration of 20mg ml21 and the media(including LRG1 and 4OH-tamoxifen supplements) was refreshed every otherday until day 12. ENG depletion was confirmed using an anti-ENG antibody(E-Bioscience). Separate experiments using control metatarsals confirmed that1 mM 4OH-tamoxifen per se did not affect neovessel formation in the metatarsalangiogenesis assay. Analysis of angiogenesis was carried out as described above.Metatarsals from five independent litters was used and two-way ANOVA wasperformed to determine statistical significance between test groups.Aortic ring angiogenesis assay. The aortic ring angiogenesis assay was performedusing a modified method described previously42. Diameter rings (1 mm) weresliced from aortae of P7 wild-type control or Lrg12/2 littermate mice. Aortic ringswere then placed in a 96-well plate coated with a rat tail collagen I gel (BD Bio-sciences) containing LRG1 (20mg ml21), anti-LRG1 polyclonal antibody (100 nM)or rabbit IgG (100 nM) as indicated, and cultured in DMEM supplemented with2.5% FBS containing relevant compounds. Medium was replaced every 2 days. Atday 10 of culture, the explants were fixed, stained for GSL isolectin IB4 (VectorLabs) and visualized under an Olympus SZX16 Research stereo-zoom microscope.The number of sprouts was counted manually. Three independent experimentswere carried out with a mean of $15 aortic rings being analysed for each treatment.Student’s t-test was performed to determine statistical significance between testgroups.Mouse model of CNV. CNV was induced as described elsewhere36,43. In the anti-LRG1 antibody blockade, study animals received an intravitreal injection ofimmediately after the laser burn. Antibody at a concentration of 1, 2.5, 5 or10 mg (each in 1 ml) of the anti-LRG1 polyclonal antibody was delivered to oneeye and a pre-immune IgG, serving as control, delivered to the contralateral eye.

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http://www.komp.org

Five (in the case of antibody treatment) or seven days after injury, CNV lesionswere imaged as described before36,43,44. Mice were then killed for retina and RPEflat-mount preparation and mRNA extraction. The CNV lesions were visualizedafter FITC-conjugated GSL isolectin B4 (Vector Labs) and mouse PECAM-1 (ratpolyclonal, BD Biosciences) staining using an Olympus SZX16 Research stereo-zoom microscope and a Zeiss LSM 710 confocal microscope. Student’s t-test wasperformed to determine the statistical significance between wild-type and Lrg1knockout mice. One-way ANOVA was used to test statistical significancebetween antibody treatment groups.Mouse model of OIR. Nursing mothers and neonatal mice were placed in a 75%oxygen supply chamber from P7 to P12 and exposed to a standard 12 h light–darkcycle as previously described45. The extent of vaso-obliteration was determined inretinal flat-mounts at P12, and the extent of normal vessel regrowth and neovascu-larization were evaluated at P17 as previously described46. Retinae were also recov-ered for mRNA extraction and analysis at P12 and P17. The effect of antibodyblockade on retinal revascularisation and neovascular tuft formation was carriedout by delivering anti-LRG1 blocking antibody (50 mg kg21 intraperitoneal in100ml at P13 and P15), anti-VEGFR2 blocking antibody (DC101, 12.5 mg kg21

intraperitoneal at P13 and P15) or a combination of the two, followed by assessmentof the vasculature at P17. Student’s t-test was performed to determine the statisticalsignificance between wild-type and Lrg1 knockout mice. One-way ANOVA wasused to test statistical significance between antibody treatment groups.Co-immunoprecipitation. Primary mouse brain endothelial cells from wild-typeor Lrg12/2 littermate mice, GPNT cells or HUVECs were lysed in RIPA buffer(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholateand 1% Nonidet P-40). Soluble peptide-tagged extracellular domains of TbRII(Myc-tagged), ALK1 (HA-tagged), ALK5 (HA-tagged) and ENG (V5-tagged), aswell as full-length LRG1 (His-tagged), were generated in separate cultures oftransfected HEK293T cells, and serum-free media containing the individual pro-teins was collected after 5 days. Non-tagged secreted extracellular domains of theTGF-b receptors were also generated in an identical manner. Media containingindividual extracellular domains of a TGF-b receptor were incubated with mediacontaining LRG1 in the presence or absence of TGF-b1 at 4 uC with rotationbefore immunoprecipitation. After pre-clearing, cell lysates or recombinant pro-tein mixtures were incubated with TGF-b receptor antibodies or anti-LRG1antibody-conjugated protein G beads at 4 uC overnight and then fractionatedby SDS–PAGE and blotted. The membranes were probed with antisera asdescribed earlier.Proliferation assay. Mouse primary brain endothelial cells from Lrg12/2 andwild-type mice were cultured in EGM2 media supplemented with puromycin(5 mg ml21) until sub-confluent, followed by 48 h serum starvation in EBM2medium. Cells were stimulated with TGF-b1 (5 ng ml21), LRG1 (20mg ml21)or TGF-b1 plus LRG1 in EBM2 medium at 37 uC. After 3 h, cells were fixed andstained with an antibody to Ki67 (mouse monoclonal antibody, Dako) to detectproliferating cells. The proliferation rate was evaluated as the percentage of Ki67-positive cells of the total endothelial cell number per well. Student’s t-test wasperformed to determine the statistical significance between treatment groups.Molecular biological methods. The coding sequence of human LRG1(NM_052972) carrying a 63His tag or HA tag at the 39 end and Kozak consensussequence at the 59 end was cloned into pcDNA3.1 (Invitrogen) at the HindIII/XhoIsites to form pcDNA-LRG1-His or pcDNA-LRG1-HA (Supplementary Fig. 8).The coding sequence of human LRG1 was cloned into a pEGX4T1 GST expressionvector (GE Healthcare) at the BamHI/SalI site to form glutathione S-transferase(GST)–ENG. The extracellular domain of human TbRII (NM_001024847.2) car-rying a Myc tag, ALK1 (NM_000020.2) carrying a HA tag, ALK5 (NM_004612.2)carrying a HA tag, ENG (NM_001114753.1) carrying a V5 tag at the 39 end andKozak consensus sequence at the 59 end were cloned into pcDNA3.1 at HindIII/EcoRI sites. The recombinant human proteins were expressed in HEK293T cells(Invitrogen). siRNA oligonucleotides (SASI_Rn01_00111211 (Sigma)) were usedfor Lrg1 gene knockdown in GPNT cells, and siRNA oligonucleotides (ON-TARGETplus SMARTpools, Thermo Scientific) were used for knockdown inHUVECs of ALK1 (L-005302-00-0005), ALK5 (L-003929-00-0005), ENG (L-011026-00-0005) and TBRII (L-001000-00-50), and control siRNA (D-001810-10-05) was used as a negative control for knockdown in HUVECs.

Lipofectamine 2000 transfection reagent (Invitrogen) was used for transfectionof mammalian cells. Oligofectamine 2000 transfection reagent (Invitrogen) wasused for siRNA knockdown in GPNT cells. GeneFECTOR transfection reagent

(VennNova) was used for siRNA knockdown in HUVECs. PCR and qPCRprimer sequences are shown in Supplementary Table 2.Purification of recombinant proteins. LRG1–His was expressed in HEK293Tcells and purified using HisPrep FF16/10 column (GE Healthcare) and bufferexchanged into PBS using HiPrep 26/10 Desalting (GE Healthcare) according tomanufacturer’s instruction. GST–ENG was expressed in BL21-competent cellsand purified using glutathione Sepharose 4B (GE Healthcare) and eluted inelution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) accordingto the manufacturer’s instruction. Denatured LRG1–His protein was generatedby boiling at 100 uC for 15 min.Surface plasmon resonance. All surface plasmon resonance experiments werecarried out on a BiacoreT200 instrument (GE Healthcare). LRG1 was covalentlyimmobilized via primary amino groups on a CM5 sensor chip as per manufac-turer’s instructions (specific contact time 20 s at a flow rate of 10 ml min21; LRG1concentration at 25 mg ml21 diluted using 10 mM sodium acetate, pH 5.0). Theamount of immobilized LRG1 corresponded to 2,000 response units in flow cell 2.Flow cell 1 on the same sensor chip, reserved for control runs, was treatedidentically but without LRG1 immobilization. For all SPR measurements, GST-tagged ENG was diluted in running buffer (13 PBS, pH 7.2). The association wasmonitored by injecting different concentrations (1–50 nM) of the analyte (ENG)into channels 1 and 2, starting with the lowest analyte concentration. All experi-ments were conducted in triplicates at 25 uC at a flow rate of 30 ml min21. Theassociation time for ligand–analyte steady state binding was optimised to 180 sand a subsequent 300 s were allowed for dissociation. Between injections thesensor chip surface was regenerated with glycine-HCl, pH 2.0, at a flow rate of30 ml min21 for 30 s. All curves were corrected for nonspecific binding by sub-traction of control curves obtained from injection of the analyte through theblank flow cell 1. The affinity and dissociation constants were calculated fromthe plots of the steady-state binding as a function of protein concentration, usingthe Biacore T200 evaluation software and a homogenous 1:1 Langmuir bindingkinetic model. The analysis provided values for the dissociation affinity constant(KD), the association rate constant (Ka) and the dissociation rate constant (Kd).Statistical analyses. Data are represented as mean 6 s.e.m. Statistical analyseswere performed by Student’s t-test or one-way ANOVA followed by Tukey/Bonferroni post-test analysis or two-way ANOVA as appropriate, using Prism5 (GraphPAD Software Inc.). *P , 0.05; **P , 0.01; ***P , 0.001. Each repre-sents significant statistical comparisons among the listed (x axis) experimentalgroups.

34. Blanks, J. C. & Johnson, L. V. Vascular atrophy in the retinal degenerative rd mouse.J. Comp. Neurol. 254, 543–553 (1986).

35. Heckenlively, J. R. et al. Mouse model of subretinal neovascularization withchoroidal anastomosis. Retina 23, 518–522 (2003).

36. McKenzie, J. A. et al. Apelin is required for non-neovascular remodelling in theretina. Am. J. Pathol. 108, 399–409 (2012).

37. Fruttiger, M. Development of the mouse retinal vasculature: angiogenesis versusvasculogenesis. Invest. Ophthalmol. Vis. Sci. 43, 522–527 (2002).

38. Abbott, N. J., Hughes, C. C., Revest, P. A. & Greenwood, J. Development andcharacterisation of a rat brain capillary endothelial culture: towards an in vitroblood–brain barrier. J. Cell Sci. 103, 23–37 (1992).

39. Romero, I. A. et al. Changes in cytoskeletal and tight junctional proteins correlatewith decreased permeability induced by dexamethasone in cultured rat brainendothelial cells. Neurosci. Lett. 344, 112–116 (2003).

40. Arnaoutova, I. & Kleinman, H. K. In vitro angiogenesis: endothelial cell tubeformation on gelled basement membrane extract. Nature Protocols 5, 628–635(2010).

41. Deckers, M. et al. Effect of angiogenic and antiangiogenic compounds on theoutgrowth of capillary structures from fetal mouse bone explants. Lab. Invest. 81,5–15 (2001).

42. Nicosia, R. F. & Ottinetti, A. Growth of microvessels in serum-free matrix culture ofrat aorta. A quantitative assay of angiogenesis in vitro. Lab. Invest. 63, 115–122(1990).

43. Balaggan, K. S. et al. EIAV vector-mediated delivery of endostatin or angiostatininhibits angiogenesis and vascular hyperpermeability in experimental CNV. GeneTher. 13, 1153–1165 (2006).

44. Toma, H. S., Barnett, J. M., Penn, J. S. & Kim, S. J. Improved assessment of laser-induced choroidal neovascularization. Microvasc. Res. 80, 295–302 (2010).

45. Smith, L. E. et al. Oxygen-induced retinopathy in the mouse. Invest. Ophthalmol. Vis.Sci. 35, 101–111 (1994).

46. Connor, K. M. et al. Quantification of oxygen-induced retinopathy in the mouse: amodel of vessel loss, vessel regrowth and pathological angiogenesis. NatureProtocols 4, 1565–1573 (2009).

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Supplementary Figure 1. Retinal vasculature of C57/BL6 wild type mouse and threemouse models of retinal disease. Imaris rendered representative 3D images of PECAM-1 stained retinal vasculature (red) of control C57/BL6 mouse and of the vascular abnormalities(highlighted by arrow heads) in Vldlr-/-, Curly tail (Grhl3ct/J) and RD1 mice. In each image asingle X-Y section stained for nuclei (blue) is incorporated to show the relative positioning ofthe nuclear layers (see also Supplementary movies 1-4).

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Supplementary Figure 2. Predicted structure and species homology of LRG1. a, Predicted ribbon structure (left) and space-filling model (right) of mouse LRG1 (342 amino acids) obtained through the Robetta full-chain protein structure prediction server(http://robetta.bakerlab.org). The leucine-rich C-terminal (LRC) contains mainly β strands and is connected to the leucine-rich repeats(LRR) by loops. Each LRR contains a β strand and an α helix with the structural units arranged in parallel with a curved β sheet liningthe inner circumference and the α helices flanking the outer circumference. The LRC and LRR domain contain 3 potentialglycosylation sites with a further 4 sites in the linker region between the LRR and the signal peptide. Images generated courtesy ofProfessor C.M. Marson (UCL, London). b, Alignment of the Lrg1 amino-acid sequence between different vertebrate species obtainedthrough the EMBL-EBI European Bioinformatics Institute.


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Supplementary Figure 3. Specificity of Lrg1 probes and antisera. a, Rabbit anti-LRG1 polyclonal antibody western blot of retinal cell lysates from wild type (WT), heterozygous (Lrg1-/+) and homozygous (Lrg1-/-) mice (3 months old) showing loss of LRG1 signal in the latter. b, In situ hybridisation of Lrg1 sense and antisense probes in P35 flat-mounted mouse retinae showing positive signal in antisense image. c, Flat-mounted WT and Lrg1-/- retinae stained for vessels (isolectin B4; green) and with pre-immune rabbit sera, pre-absorbed rabbit anti-LRG1 sera and affinity purified rabbit polyclonal anti-LRG1 antibody. Staining with pre-immune rabbit sera and pre-absorbed rabbit sera in WT mice and affinity purified rabbit polyclonal anti-LRG1 antibody in Lrg1-/- mice give a negative signal. Scale bar 50μm.



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Supplementary Figure 10. LRG1 and TGFß-1 expression in conditioned media. a,Western blot analysis showing the presence of LRG1 and TGFß-1 in conditioned mediaharvested from mouse aortic ring, mouse metatarsal, mouse primary brain endothelial cellcultures, immortalised mouse lung endothelial cell cultures (from Rosa26-CreERT:Engfl/fl

mouse) and HUVEC Matrigel assays. b, Western blot analysis showing the absence of TGFß-1 in HEK 293 cell culture medium and following 5 days of cell conditioning.

W W W. N A T U R E . C O M / N A T U R E | 1 1


PECAM-1 Imaris filament tracing Overlay

Supplementary Figure 11. Metatarsal angiogenesis assay vessel quantification. Example of image rendering for quantification of vascular growth in the metatarsal angiogenesis assay. Metatarsal vessel outgrowth stained with anti-PECAM-1 antibody (red) is captured digitally, and after thresholding vessel configuration quantified (white) using Imaris filament tracing image analysis software (Bitplane AG, Switzerland).



RESEARCH

TUF/TUR

NeoFwd/SD

SU/LacZRev

GAPDH

Wild

type

Lrg1

+/-

Lrg1

-/-

wild ty

pe

Lrg1+

/-

Lrg1-/-

60%

40%

20%

0

Distribution of Lrg1-/- mice

27.45%

48.04%

24.52%

n=102

Forward Primer

Reverse Primer Length WT HET KO

SU LacZRev 367 bp - + +

TUF TUR 76 bp + + -

NeoFwd SD 307 bp - + +

a

b

c

Supplementary Figure 12. Genotyping the Lrg1-/- mouse. a, Strategy for PCR-based genotyping Lrg1 knockout mice (from KOMP). b, The sizes of expected PCR products for wild type (WT), heterozygote (HET) and null mutant (KO). c. Genotyping examples of WT, heterozygous and homozygous mice and their ratio in the progeny of heterozygous breeding pairs.


SUPPLEMENTARY INFORMATIONdoi:10.1038/nature12641

P17

P35

WT Lrg1-/-

P10

P12

COLIV Deep plexusDeep plexus

Deep plexus

Superficial plexus

Superficial plexus

Superficial plexus Intermediate plexus

201510

50

201510

50

201510

50

40302010

0

30

20

10

0

30

20

10

0

30

20

10

0

% v

ascu

lar a

rea *

*

**

Deep plexus

Superficial plexus

Intermediate plexus201510

50

25201510

50

201510

50

**

*

WT Lrg1-/-

% v

ascu

lar a

rea

% v

ascu

lar a

rea

% v

ascu

lar a

rea

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

% v

ascu

lar a

rea

% v

ascu

lar a

rea

% v

ascu

lar a

rea

P25

% v

ascu

lar a

rea

% v

ascu

lar a

rea

% v

ascu

lar a

rea

P25

b

Deep plexus

Superficial plexus

Intermediate plexus

201510

50

30

20

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0

15

10

5

0

% v

ascu

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rea

% v

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rea

% v

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rea

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

Supplementary Figure 13. Retinal vascular development in the Lrg1-/- mouse. Retinal blood vessels were visualized in retinal flat-mounts using an anti-collagen IV antibody followed by FITC-conjugated secondary and imaged using a confocal scanning laser microscope. Z-sections were captured and analysed from the central and peripheral retina (four regions per retina each of 0.4mm2) to reveal the inner, intermediate and deep vascular plexuses of the C57/Bl6 wild type (WT) and Lrg1-/- mouse (n = 6 for each group). Vascularization of the retina is delayed in Lrg1-/- mice compared to that in WT littermate controls at P10, P12, P17 and P25. The P35 retinal vasculature in the Lrg1-/- mice shows no difference compared to that from WT. Significance determined by Student's t-test (*P<0.05; **P<0.01). Scale bar = 40μm.

P17

P35

WT Lrg1-/-

P10

P12

COLIV Deep plexusDeep plexus

Deep plexus

Superficial plexus

Superficial plexus

Superficial plexus Intermediate plexus

201510

50

201510

50

201510

50

40302010

0

30

20

10

0

30

20

10

0

30

20

10

0

% v

ascu

lar a

rea *

*

**

Deep plexus

Superficial plexus

Intermediate plexus201510

50

25201510

50

201510

50

**

*

WT Lrg1-/-

% v

ascu

lar a

rea

% v

ascu

lar a

rea

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rea

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

% v

ascu

lar a

rea

% v

ascu

lar a

rea

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ascu

lar a

rea

P25

% v

ascu

lar a

rea

% v

ascu

lar a

rea

% v

ascu

lar a

rea

P25

b

Deep plexus

Superficial plexus

Intermediate plexus

201510

50

30

20

10

0

15

10

5

0

% v

ascu

lar a

rea

% v

ascu

lar a

rea

% v

ascu

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rea

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

WT Lrg1-/-

Supplementary Figure 13. Retinal vascular development in the Lrg1-/- mouse. Retinal blood vessels were visualized in retinal flat-mounts using an anti-collagen IV antibody followed by FITC-conjugated secondary and imaged using a confocal scanning laser microscope. Z-sections were captured and analysed from the central and peripheral retina (four regions per retina each of 0.4mm2) to reveal the inner, intermediate and deep vascular plexuses of the C57/Bl6 wild type (WT) and Lrg1-/- mouse (n = 6 for each group). Vascularization of the retina is delayed in Lrg1-/- mice compared to that in WT littermate controls at P10, P12, P17 and P25. The P35 retinal vasculature in the Lrg1-/- mice shows no difference compared to that from WT. Significance determined by Student's t-test (*P<0.05; **P<0.01). Scale bar = 40μm.



RESEARCH

**

Hya

loid

ves

sels

20

10

0

Wild

type

Lrg1-/-

a

b

WT Lrg1-/-

Hyaloid vessels Hyaloid & retinal vessels

Supplementary Figure 14. Hyaloid vessels persist in the Lrg1-/- mouse. a, Wild type (WT) flat mounted retina stained for the vasculature (anti-collagen IV) show regression of the hyaloid vasculature at P35. In the Lrg1-/- mouse retina persistent hyaloid vessels (arrow) at P35 can be observed. Only primary radial hyaloid vessels were enumerated (n=30 for each group). Scale bar = 68μm. b, Following 3D rendering persistent hyaloid vessels (green) are seen integrating into the superficial retina to give rise to anastomosis with the retinal vasculature (n=30 for each group). Significance determined by Student's t-test (**P<0.01).



WT Lrg1-/-

**

A-V

cro

sses

per

retin

a0.6

0.4

0.2

0

Wild

type

Lrg1-/-

a

b WT Lrg1-/-

Supplementary Figure 15. Increased arteriovenous crossing in the Lrg1-/- mouse. a, At P10 the wild type (WT) retina shows a regular alternating pattern of arteries and veins. At the same developmental stage Lrg1-/- mice exhibit an approximate 5-fold increase in the frequency of arteriovenous crossovers within the inner superficial vascular plexus (white arrow). b, 3D Imaris rendering shows close apposition and possible anastomosis between the crossing arteries and veins (n=30 for each group). Significance determined by Student's t-test (**P<0.01). Scale bar 120μm.



RESEARCH

4

3

2

1

0

Wild ty

peLrg

1-/-

Rel

ativ

e P

lgfe

xpre

ssio

n

Wild ty

peLrg

1-/-

Rel

ativ

e V

egfe

xpre

ssio

n 3

2

1

0

*

a

b

Supplementary Figure 16. Plgf and Vegfa gene expression in Lrg1-/- mice. Quantitative RT PCR analysis of a, Plgf mRNA expression in the retina of 3 month old WT and Lrg1-/- mice relative to Gapdh (n=4). b, Vegfa expression in the retina of 3 month old WT and Lrg1-/- mice relative to Gapdh (n=4). Significance determined by Student's t-test (*P<0.05).



20

15

10

5

0Control Lrg1-/-

Imar

is s

pot f

unct

ion

anal

ysis

NG

2M

AC

EP

Lrg1-/-Controla

b

Num

ber o

f per

icyt

es

(mm

of v

esse

l len

gth)

50m

Supplementary Figure 17. Pericyte coverage is identical in WT and Lrg1-/- mice. a,Representative confocal images of retinal vasculature (upper panels) from WT (n=3) and Lrg1-/- (n=3) mice stained for PECAM-1 (red) and NG2 (green). Lower panels depict pericyte detection using the spot function (magenta spots) in Imaris software of white boxed area from upper images. b, Histogram showing average number of pericytes per mm of vessel length. No significant difference was observed (Student's t-test).



RESEARCH

Control Lrg1-/-

anti-

mou

se Ig

GIB

4Fl

uore

scei

n

Supplementary Figure 18. Retinal vascular barrier properties are retained in the Lrg1-/- mouse. Upper panels show staining of representative retinal sections for blood vessels (isolectin B4 - green), mouse IgG (Ab 594 - red) and nuclei (DAPI - blue). IgG is retained within the vascular compartment in control (left) and Lrg1-/- mice (right; n=3 animals for each group) indicating retention of blood-retinal barrier properties. The lower panels show representative fluorescein angiographs from control (left) and Lrg1-/- mice (right; n=3 animals for each group) at late phase (8 minutes) post fluorescein injection showing an absence of fluorescein leakage from the blood and hence maintenance of an intact blood-retinal barrier in the latter.



PBS LRG1

LRG1PBS

PBS

LRG1PBS

LRG1

Wild type

Lrg1-/-

Met

atar

sal

Aor

ta

Met

atar

sal

Aor

ta

b

a

Supplementary Figure 19. LRG1 promotes angiogenesis in WT mice and rescues angiogenesis in Lrg1-/- mice. a, Examples of vessel outgrowth in the metatarsal (top row) and aortic ring (bottom row) assay showing enhanced angiogenesis following LRG1 treatment. b, Examples of vessel outgrowth in metatarsals (top row) and aortic rings (bottom row) from Lrg1-/- mice showing rescue of LRG1 induced angiogenesis. See Figure 2 for quantification. Scale bar = 1,500μm.



RESEARCH

100m

100m

Collagen IV F4/80

lortnoC

1grL-/-

WT Lrg1-/-

CN

V le

sion

volu

me

m )

3

mac

roph

age

cont

ent

(% C

NV

vol

ume)

1x106

2x106

3x106

4x106

0WT Lrg1-/-

*0

10

20

30

40

F4/80

Supplementary Figure 20. Macrophage content of CNV lesions is similar in WT and Lrg1-/- mice. Representative Imaris reconstructed images of CNV lesions from WT control (top row) and Lrg1-/- (bottom row) mice. CNV lesion is stained for collagen IV (green) and macrophages stained for F4/80 (purple). The CNV lesion has been demarcated with a yellow dotted line. Below, histograms showing CNV lesion volume (left) and macrophage content (right) expressed as a percentage of CNV volume in WT and Lrg1-/- mice.



WT CNV Lrg1-/- CNV

PE

CA

M-1

NG

2C

NV

bou

ndar

y

100

80

60

40

20

0WT Lrg1-/-

NG

2 ex

pres

sion

(% o

f PE

CA

M-1

vol

ume)

Supplementary Figure 21. CNV NG2 (pericyte) coverage is similar in WT and Lrg1-/-

mice. Representative Imaris reconstructed images of CNV lesions from WT control (top left) and Lrg1-/- (top right) mice. CNV lesions are stained for PECAM-1 (red) and NG2 (green). Lower panels show delineation of CNV lesion (yellow dotted line). Below, histogram shows NG2 volume as a percentage of PECAM-1 volume in delineated CNV lesions in WT and Lrg1-/- mice. n = 3 per group.



RESEARCH

Retinal avascular region Retinal avascular region delineated

Retinal neovascular tufts Retinal neovascular tufts delineated

Retinal neovascular tufts highlighted Composite

OIR

retin

al fl

atm

out (

P17

)O

IR n

eova

scul

ar tu

fts (P

17)

OIR

retin

al fl

atm

ount

(P17

)

Supplementary Figure 22. Delineation of retinal avascular region and neovascular tufts in OIR for quantitative analysis. Top left shows a representative reconstructed (tile) flat mounted retina stained for PECAM-1 (green) from an OIR-induced mouse at P17. Top right illustrates the manual delineation of the vascular/avascular interface (white line) for area analysis. Middle left shows higher power image of yellow boxed area in top right to reveal the neovascular tufts. These are manually delineated (white line) for area analysis. Bottom left image shows artificially coloured neovascular delineated tufts (red) and the bottom right the composite showing neovascular tufts (red) and avascular region (white line).



DC101 DC101 + LRG1 AbIgG LRG1 Ab

b

Rabbit IgG

LRG1Ab

1g

a

125

100

75

50

25

0

CN

V a

rea

(% o

f IgG

con

trol)

Rabbit

IgG 2.51 5 10

******

Col

lage

n IV

PE

CA

M

100m

100m

Vol

ume

rend

ered

2.5g 5g 10g

FA

LRG1Ab (μg)

***

Supplementary Figure 23. Antibody blockade of LRG1 inhibits CNV lesion formation. a, Representative fluorescein angiographs of laser-induced CNV lesions from WT mice following intravitreal delivery of control IgG or LRG1 polyclonal antibody. The histogram shows a dose-dependent inhibition of CNV lesion area by the anti-LRG1antibody. Data analysed by one-way ANOVA (P = 0.0001) with significance from controls determined by Bonferroni's multiple comparison post-hoc analysis (n ≥ 15 for each group, ***P<0.001). b, Representative confocal images (top) and 3D rendered equivalent reconstructions (bottom) of laser-induced CNV lesions from control, anti-LRG1 and anti-VEGFR2 (DC101) antibody treated WT mice. Whole mounts were stained for collagen IV (green) and PECAM-1 (red) and confocal Z stacks were 3D rendered using Imaris software.



RESEARCH

a

b

IgG

LRG1 A

b

DC101

DC101+

LRG1 A

b0

5

10

15

20

25

Neo

vasc

ular

tuft

area

(% to

tal a

rea)

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Avas

cula

r are

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IgG

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LRG1 A

b

Collagen IV IB4

IgG

LRG

1 A

b +

DC

101

IB4

500m

**NS

NS

*NS

NS

Supplementary Figure 24. Combined antibody blockade of LRG1 and VEGFR2 inhibits angiogenesis in OIR. a, Effect of anti-LRG1 blocking antibody (50mg/kg intraperitoneal in 100μl at P12 and P15), anti-VEGFR2 blocking antibody (DC101, 12.5mg/kg intraperitoneal at P12 and P15) or a combination of the two, on retinal revascularisation and neovascular tuft formation. With these treatment regimens significant inhibition was only observed with combined therapy, reducing both revascularisation (**P ˂0.01, n ≥ 15 for each group) and neovascular tuft formation (*P<0.05; n ≥ 15 for each group). One-way ANOVA (P = 0.0002) with significance from controls determined by Bonferroni's multiple comparison post-hoc analysis. b, Representative images of OIR from IgG control and anti-LRG1/DC101 treated mice. Whole mounts were stained for collagen IV (green) and isolectin B4 (red). Avascular region at day P17 (5 days post hyperoxia) is delineated by the magenta line. Neovascular tufts are more clearly observed as brighter regions on the greyscale images.



a

α-TGFβ1

PDR Ctl PDR Ctl PDRCtl PDR Ctl

1 12 23 34 45 56 67 7 8 8 9 910 10

Rel

ativ

ein

tens

ities

Ctl PDR

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α-TGFβ1

***

Rel

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ities

Ctl PDR

15

10

5

0

20

PDR Ctl

1 12 23 34 45 56 6

PDR CtlPDR Ctl

7 78 8 99 10 10

b

Supplementary Figure 25. TGFß1 is increased in the vitreous of patients with proliferative diabetic retinopathy. a, Western blots of vitreal TGFß1 from ten patients with proliferative diabetic retinopathy (PDR) and ten non-PDR controls (patients with epiretinal membranes). Quantitative analysis shows a significant increase of TGFß1 in PDR patients (***P<0.001). b, No significant difference in plasma TGFß1 protein level was observed between PDR and non-PDR control patients. Total vitreous/plasma protein was normalized before western blotting as shown by Coomassie blue staining of the SDS-PAGE gel in Supplementary figure 5. The TGFß1 protein level was determined by relative intensities against the total protein in vitreous /plasma using Image J. All values represent means ± s.e.m. Significance determined by Student's t-test (***P<0.001).



RESEARCH

Moc

k

ALK

5

ALK

1

EN

G

Moc

k

Moc

k

TβR

IIM

ock

LRG

1M

ock

WB:α-HA

WB:α-HA

WB:α-Myc

WB:α-V5

WB:α-His

LRG

1Ab

Rbt

IgG

IP: LRG1WB: α-TβRII

IP: LRG1WB: α-ALK1

IP: LRG1WB: α-ALK5

IP: LRG1WB: α-ENG

a

b

Supplementary Figure 26. Binding of LRG1 to TGFβ receptors and generation of peptide-tagged LRG1 and TGFβ receptor extracellular domains. a, Western blots showing that immunoprecipitation of LRG1 with polyclonal rabbit antibody from HUVEC lysates co-immunoprecipitates the TGFβ receptors TβRII, ALK1, ALK5 and endoglin (ENG). (Representative of n ≥ 3 for each assay). b, Western blot of peptide-tagged recombinant extracellular domains of TGFβ receptors TβRII (Myc-tagged), ALK1 (HA-tagged), ALK5 (HA-tagged) and ENG (V5-tagged) and His-tagged LRG1 in transfected HEK 293 cell medium. (Representative western blots; n ≥ 3 for each experiment).



CNVControl

3

2

1

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Rel

ativ

e E

ng e

xpre

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*

Control CNV

End

oglin

DA

PII

B4

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End

oglin

Control P17 OIR P17

End

oglin

DA

PII

B4

End

oglin

OIR

End

mRN

A e

xpre

ssio

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atch

ed c

ontr

ol)

P12 P17

300

200

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0

-100 *

***

50 m

a b

c

Supplementary Figure 27. Endoglin expression is upregulated in CNV and OIR neovascularisation. a, Representative immunohistological retinal sections of control (top left hand panel) and CNV lesion (top right hand panel) stained for endoglin (red), isolectin B4 (green) and nuclei (DAPI, blue). Lower panels show endoglin channel only. b,Representative immunohistological retinal sections of P17 control (top left hand panel) and P17 OIR lesion (top right hand panel) stained for endoglin (red), isolectin B4 (green) and nuclei (DAPI, blue). Lower panels show endoglin channel only. c, Histograms showing EngmRNA relative to Gapdh mRNA in control and CNV retina (left) and relative to untreated age-matched WT control mice in OIR at P12 and P17. *P<0.05; ***P<0.001; n = 3 for each group.



RESEARCH

-10

-5

0

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30

0 50 250200150 400350300100

1 nM

50 nM40 nM30 nM20 nM10 nM5 nM

Res

pons

e (R

U)

Time (s)

Supplementary Figure 28. Surface plasmon resonance analysis of LRG1-ENG binding. Real-time binding and dissociation assay was performed to determine binding kinetics of the ectodomain of ENG to LRG1 using Biacore T200. The GST tagged ectodomain domain of ENG (GST-ENG) was passed over LRG1 covalently immobilized to a CM5 sensor chip at increasing concentrations (0-50 nM). An association time of 3 minutes and a dissociation time of 5 minutes were used and the chip was regenerated between each concentration of GST-ENG. The binding curves show RU (Response Unit) as a function of time. The sensograms show a dose-dependent binding of ENG-GST to LRG1 after subtracting the background. GST on its own showed no binding to LRG1. Data provided an affinity rate constant (KD) of 2.9 M, an association rate constant (Ka) of 4.9 x 102 M-1s-1 and a dissociation rate constant (Kd) of 1.4 x 10-3 s-1.



α-pSmad1/5

α-GAPDH

PB

S

LRG

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S

LRG

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S

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DMSO ALK1i ALK5i

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bPBSLRG1

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ALK5 i

+ LR

G1

**

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ALK5 i

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ALK1i

+ LRG1

ALK5 i

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G1

*

*

* Bra

nch

poin

ts(%

cha

nge) **

PBS

ALK1i

LRG1

LRG1 + ALK1i

ALK5i LRG1 + ALK5i

c

control siRNA

ALK1 siRNA

ALK5 siRNA

-pSmad1/5

α-GAPDH

PB

S

LRG

1

PB

S

LRG

1

PB

S

LRG

1

α-pSmad2

control siRNA

TβRII siRNA

PB

S

LRG

1

PB

S

LRG

1

-pSmad1/5

GAPDH

-pSmad2

-

α

α

ENGsiRNA

PB

S

LRG

1

α α

a

Supplementary Figure 29. Inhibition of ALK1, but not ALK5, inhibits LRG1-mediated Smad1/5 phosphorylation and HUVEC tube and branch formation. a, Western blot examples of siRNA knockdown of ALK1, ALK5, TβRII or ENG in HUVEC on LRG1-induced Smad1/5 and Smad2 phosphorylation. Knockdown of ALK1, TβRII and ENG, but not ALK5, results in prevention of LRG1-induced Smad1/5 phosphorylation. See Figure 5 for semi-quantification. (n ≥ 3). b, The ALK1 inhibitor LDN193189 (ALK1i) inhibits LRG1-induced phosphorylation of Smad1/5 but has no effect on Smad2 phosphorylation. Conversely, the ALK5 inhibitor SB43152 (ALK5i) has little effect on LRG1-induced Smad1/5 phosphorylation. Histograms show semi-quantification of Smad phosphorylation relative to GAPDH (n ≥ 3; *P<0.05). c, Representative images of the HUVEC Matrigel assay showing inhibition of LRG1-mediated tube and branch formation with the ALK1 inhibitor LDN193189 (ALK1i) but not the ALK5 inhibitor SB43152 (ALK5i). Histograms show tube length and branch formation under the different conditions. (*P<0.05; **P<0.01; n=3 independent assays). All values represent means ± s.e.m.



RESEARCH

Contro

l

Contro

l + LR

G1

Eng-iK

Oe

Eng-iK

Oe +L

RG1% C

hang

e in

bra

nch

poin

ts

Contro

l

Contro

l + LR

G1

Eng-iK

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Oe +L

RG1

% C

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e in

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sel l

engt

h

0

50

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(vs

cont

rol)

(vs

cont

rol)

a

b

Eng-iKOeControl

PECAM-1 PECAM-1

ENG ENG

0

50

100

150

200

Supplementary Figure 30. Depletion of endothelial ENG expression in metatarsal angiogenesis assay and its effect on vessel length and branching. a, Representative images of PECAM-1 and ENG staining of 4OH-tamoxifen treated metatarsals from Engfl/fl

(control) and Cdh5(PAC)-CreERT2;Engfl/fl (Eng-iKOe) mice showing ENG depletion in the latter. b, Percentage change in metatarsal vessel length and branching following 4OH-tamoxifen treatment of Engfl/fl metatarsals (control) and Cdh5(PAC)-CreERT2;Engfl/fl

metatarsals (Eng-iKOe).



WT Lrg1-/-

DA

PI

pSM

AD

1/5/

8P

EC

AM

-1M

erge

Supplementary Figure 31. pSmad1/5/8 expression in CNV. Representative immunohistological section of CNV lesions in wild type (WT) and Lrg1 knockout (Lrg1-/-) mice stained for PECAM-1 (green) to reveal endothelial cells and phosphorylated Smad1/5/8 (red). The boundary of the CNV lesion has been demarked by a dotted yellow line. Fewer phospho-Smad1/5/8 cells were observed in the CNV lesion in the Lrg1-/- mice. Nuclei were stained with DAPI (blue). Scale bar 50μm.



RESEARCH

TGF-TGF-

p

p

p

p

p

p

p

p

p

p

QUIESCENT BLOOD VESSELS

ANGIOGENIC CONDITIONS

TGF-

TGF-

TGF-TGF-

TRII

LRG1

ALK5 TRII ALK1

Smad2/3Smad2/3

Smad4 Smad4 Smad1/5/8Smad1/5/8

ENDOTHELIAL CELL PROLIFERATION/MIGRATION

LRG1

LRG1

LRG1

LRG1 LRG1

TRIIALK5 TRIIALK1

TRII

ALK5

ENG ALK1ENG

Transient complex?

TGF-

TGF-TGF-

TGF-TGF-

TGF-

Smad2/3Smad2/3

Smad4 Smad4 Smad1/5/8Smad1/5/8

ENDOTHELIAL CELL PROLIFERATION/MIGRATION

LRG1

TGF-

TGF-TGF-TTGTG

TGF-

LRG1 LRG1- TGF-

interaction?

Supplementary Figure 32. Proposed model of LRG1 mediated TGFβ1 endothelial cell signalling. Under resting conditions TGFß1 signalling in endothelial cells is predominantly via the TβRII-ALK5-Smad2/3 pathway which maintains vascular homeostasis. During pathogenic angiogenesis there is an increase in LRG1 and ENG production. The interaction between LRG1 and TGFβ1 may facilitate more efficient ENG/TβRII/ALK1 receptor complex formation resulting in a switch in TGFβ1 signalling to the ALK1-Smad1/5/8 pathway and a pro-angiogenic transcriptional response. LRG1/ENG/TβRII/ALK1/ALK5 may form an intermediate/transient complex to allow the efficient phosphorylation of ALK1 but in the presence of TGFβ1, LRG1/ENG/ALK1/TβRII predominates.



Prob

e ID

(m

ouse

430

2.0

) G

ene

sym

bol

Gen

e na

me

Fold

ch

ange

CT

Adj.P

.Val

C

T Fo

ld

chan

ge

RD

1

Adj.P

.Val

R

D1

Fold

ch

ange

VL

DLR

Adj.P

.Val

VL

DLR

1417

290_

at

Lrg1

le

ucin

e-ric

h al

pha-

2-gl

ycop

rote

in 1

91

.763

6 2.

9E-0

3 14

.092

4 2.

0E-0

2 54

.869

8.

9E-0

3 14

1809

0_at

P

lvap

pl

asm

alem

ma

vesi

cle

asso

ciat

ed p

rote

in

26.6

803

1.7E

-04

4.04

288

1.0E

-02

16.1

691

2.4E

-04

1448

550_

at

Lbp

lipop

olys

acch

arid

e bi

ndin

g pr

otei

n 8.

3072

3 1.

0E-0

2 12

.930

4 2.

2E-0

3 14

.192

6 4.

4E-0

3 14

3865

1_a_

at

Apl

nr

apel

in re

cept

or

8.31

23

8.6E

-04

8.91

962

4.0E

-04

10.7

802

1.4E

-04

1428

909_

at

A13

0040

M12

Rik

R

IKE

N c

DN

A A

1300

40M

12 g

ene

9.92

697

1.5E

-03

6.27

517

2.5E

-03

4.32

157

2.9E

-02

1417

314_

at

Cfb

co

mpl

emen

t fac

tor B

6.

2641

9 5.

4E-0

3 3.

7083

7 1.

4E-0

2 4.

7483

2.

5E-0

2 14

5539

6_at

A

tp8b

1 A

TPas

e, c

lass

I, ty

pe 8

B, m

embe

r 1

4.06

571

3.4E

-02

4.83

276

1.1E

-02

4.81

082

4.8E

-02

1421

813_

a_at

P

sap

pros

apos

in

6.34

634

4.3E

-05

2.39

414

3.0E

-03

2.42

442

8.5E

-03

1424

374_

at

Gim

ap4

GTP

ase,

IMA

P fa

mily

mem

ber 4

2.

8571

8 6.

1E-0

3 3.

1794

7 2.

1E-0

3 3.

1249

2 6.

0E-0

3 14

2911

7_at

Tr

add

TNFR

SF1

A-a

ssoc

iate

d vi

a de

ath

dom

ain

2.16

031

1.0E

-02

2.70

536

1.7E

-03

2.09

701

2.7E

-02

1437

937_

at

Ccb

p2

chem

okin

e bi

ndin

g pr

otei

n 2

2.77

401

3.2E

-03

1.99

412

1.1E

-02

2.12

383

3.0E

-02

1418

133_

at

Bcl

3 B

-cel

l leu

kem

ia/ly

mph

oma

3 2.

7001

2 2.

9E-0

3 1.

6549

7 3.

6E-0

2 2.

2529

2 1.

5E-0

2 14

2308

2_at

D

erl1

D

er1-

like

dom

ain

fam

ily, m

embe

r 1

2.63

764

1.5E

-03

1.80

582

8.7E

-03

1.78

428

4.1E

-02

1420

886_

a_at

Xb

p1

X-bo

x bi

ndin

g pr

otei

n 1

2.03

224

2.4E

-03

2.01

532

1.6E

-03

1.91

993

5.5E

-03

1422

470_

at

Bni

p3

BC

L2/a

deno

viru

s E

1B in

tera

ctin

g pr

otei

n 3

1.61

685

3.5E

-02

2.19

82

1.9E

-03

1.97

173

1.3E

-02

1451

486_

at

Slc

46a3

so

lute

car

rier f

amily

46,

mem

ber 3

2.

0935

5 9.

5E-0

3 1.

7251

6 1.

9E-0

2 1.

9187

2 3.

7E-0

2 14

3114

6_a_

at

Cpn

e8

copi

ne V

III

1.71

393

2.2E

-02

1.76

654

9.1E

-03

2.12

126

7.1E

-03

1451

160_

s_at

P

vr

polio

viru

s re

cept

or

1.63

952

4.0E

-02

1.81

735

9.5E

-03

2.07

993

1.2E

-02

1433

954_

at

4632

419I

22R

ik

RIK

EN

cD

NA

463

2419

I22

gene

1.

6607

2 2.

7E-0

2 1.

8004

7.

2E-0

3 1.

7156

9 4.

8E-0

2 14

1862

1_at

R

ab2a

R

AB

2A, m

embe

r RA

S on

coge

ne fa

mily

1.

5336

5 9.

3E-0

3 1.

8329

9.

5E-0

4 1.

6307

4 7.

2E-0

3 14

2047

5_at

M

tpn

myo

troph

in

1.63

782

6.6E

-03

1.63

484

3.5E

-03

1.63

386

1.2E

-02

1432

271_

a_at

D

cun1

d5

DC

N1,

def

ectiv

e in

cul

lin n

eddy

latio

n 1,

do

mai

n co

ntai

ning

5 (S

. cer

evis

iae)

1.

3827

4 3.

9E-0

2 1.

6943

3 2.

3E-0

3 1.

5318

2 2.

4E-0

2

1428

090_

at

Ptc

d3

pent

atric

opep

tide

repe

at d

omai

n 3

1.45

038

1.1E

-02

1.53

833

2.9E

-03

1.42

394

2.9E

-02

1418

437_

a_at

M

lx

MA

X-lik

e pr

otei

n X

1.31

619

4.4E

-02

1.69

152

1.3E

-03

1.38

383

5.0E

-02

1419

104_

at

Abh

d6

abhy

drol

ase

dom

ain

cont

aini

ng 6

1.

5380

7 3.

5E-0

3 1.

3569

5 1.

0E-0

2 1.

4100

2 2.

3E-0

2 14

6058

5_x_

at

Pis

d ph

osph

atid

ylse

rine

deca

rbox

ylas

e 1.

3893

2 1.

3E-0

2 1.

3505

4 1.

1E-0

2 1.

4525

9 1.

3E-0

2 14

4159

3_at

---

---

-5

.800

6 1.

5E-0

2 -2

0.55

7 6.

7E-0

4 -9

.282

1 8.

1E-0

3 14

2051

1_at

P

rph2

pe

riphe

rin 2

-1

0.88

4 2.

5E-0

2 -1

1.85

5 1.

1E-0

2 -1

1.91

8 4.

8E-0

2 14

4455

2_at

---

---

-1

0.67

9 2.

1E-0

4 -1

7.45

3 6.

6E-0

5 -5

.931

3 1.

1E-0

3 14

4178

9_at

---

---

-8

.173

1.

9E-0

3 -1

4.93

5 2.

9E-0

4 -5

.557

9 8.

7E-0

3 14

4006

8_at

---

---

-5

.219

9 9.

4E-0

4 -2

.264

6 1.

6E-0

2 -8

.378

8 5.

2E-0

5 14

4661

6_at

---

---

-3

.306

1 6.

6E-0

3 -6

.987

3.

0E-0

4 -3

.829

5.

5E-0

3 14

4167

1_at

---

---

-3

.424

7 9.

4E-0

4 -4

.639

1.

8E-0

4 -4

.847

7 5.

3E-0

5

Supp

lem

enta

ry T

able

1. D

iffer

entia

lly e

xpre

ssed

gen

es c

omm

on to

Grh

l3ct/J

cur

ly ta

il (C

T), R

D1

and

VLD

LR-/-

(VLD

LR)

mou

se, r

anke

d ac

cord

ing

to th

e to

tal f

old

chan

ge.



RESEARCH

Prob

e ID

(m

ouse

430

2.0

) G

ene

sym

bol

Gen

e na

me

Fold

ch

ange

CT

Adj.P

.Val

C

T Fo

ld

chan

ge

RD

1

Adj.P

.Val

R

D1

Fold

ch

ange

VL

DLR

Adj.P

.Val

VL

DLR

1445

746_

at

Eif4

h E

ukar

yotic

tran

slat

ion

initi

atio

n fa

ctor

4H

, m

RN

A (c

DN

A c

lone

MG

C:1

1689

IM

AG

E:39

6210

4)

-3.8

752

1.9E

-03

-4.5

091

6.7E

-04

-4.4

588

7.4E

-04

1458

068_

at

---

---

-3.4

222

1.1E

-02

-4.9

557

1.7E

-03

-3.0

991

3.7E

-02

1447

024_

at

---

---

-3.5

262

5.0E

-03

-4.1

03

1.6E

-03

-3.7

124

6.5E

-03

1457

477_

at

Mbn

l2

mus

cleb

lind-

like

2 -2

.456

1 3.

9E-0

2 -5

.953

4 9.

0E-0

4 -2

.921

8 4.

1E-0

2 14

5968

7_x_

at

---

---

-2.6

253

3.2E

-02

-5.0

14

1.6E

-03

-3.0

497

3.7E

-02

1442

548_

at

---

---

-3.1

449

1.2E

-02

-3.8

378

3.0E

-03

-3.3

423

1.9E

-02

1436

892_

at

Spr

ed2

spro

uty-

rela

ted,

EV

H1

dom

ain

cont

aini

ng 2

-2

.950

3 2.

4E-0

3 -4

.722

6 1.

8E-0

4 -2

.611

7 6.

8E-0

3 14

6056

7_at

R

fx7

regu

lato

ry fa

ctor

X, 7

-2

.818

3 5.

5E-0

3 -3

.544

4 1.

3E-0

3 -3

.880

6 1.

1E-0

3

1442

278_

at

Kdm

5b

Jum

onji,

AT

rich

inte

ract

ive

dom

ain

1B (R

bp2

like)

(Jar

id1b

), m

RN

A

-3.0

173

1.8E

-02

-4.1

552

3.0E

-03

-3.0

153

4.1E

-02

1429

113_

at

2900

092E

17R

ik

/// P

rrt2

RIK

EN

cD

NA

290

0092

E17

gen

e ///

pro

line-

rich

trans

mem

bran

e pr

otei

n 2

-3.3

018

2.6E

-02

-3.1

771

1.6E

-02

-3.5

922

4.4E

-02

1442

411_

at

---

---

-3.1

871

6.6E

-03

-3.2

468

3.2E

-03

-2.5

439

4.1E

-02

1442

735_

at

---

---

-2.7

302

2.0E

-02

-3.1

033

6.0E

-03

-2.7

454

4.6E

-02

1437

965_

at

Hea

tr1

HE

AT

repe

at c

onta

inin

g 1

-2.1

302

3.1E

-02

-3.8

366

1.2E

-03

-2.5

485

2.5E

-02

1458

798_

at

---

---

-2.8

396

2.1E

-04

-2.7

643

1.8E

-04

-2.2

725

7.4E

-04

1444

599_

at

Her

c4

hect

dom

ain

and

RLD

4

-3.2

621

9.4E

-04

-2.1

305

4.6E

-03

-2.3

084

8.6E

-03

1440

755_

at

---

---

-2.1

872

1.1E

-02

-2.8

937

1.6E

-03

-2.1

518

2.7E

-02

1446

954_

at

---

---

-2.1

791

8.6E

-04

-2.5

859

1.8E

-04

-2.4

363

1.1E

-04

1435

984_

at

Zfp4

0 Zi

nc fi

nger

pro

tein

40

(Zfp

40),

mR

NA

-2

.012

5 1.

4E-0

2 -1

.704

5 2.

6E-0

2 -3

.063

7 1.

1E-0

3 14

2628

7_at

A

txn7

at

axin

7

-1.8

399

1.0E

-02

-2.1

381

2.0E

-03

-1.9

469

1.2E

-02

1419

236_

at

Hel

b he

licas

e (D

NA

) B

-1.6

318

1.2E

-02

-2.4

282

3.8E

-04

-1.7

659

1.1E

-02

1428

996_

at

4833

426J

09R

ik

RIK

EN

cD

NA

483

3426

J09

gene

-1

.823

3.

2E-0

2 -1

.605

9 4.

7E-0

2 -2

.361

1.

0E-0

2 14

3297

8_at

90

3060

7L02

Rik

R

IKE

N c

DN

A 9

0306

07L0

2 ge

ne

-2.0

972

3.5E

-03

-1.8

484

4.8E

-03

-1.8

23

2.2E

-02

1442

071_

at

Abc

e1

ATP

-bin

ding

cas

sette

, sub

-fam

ily E

(OAB

P),

mem

ber 1

-1

.710

6 1.

4E-0

2 -2

.335

7 9.

3E-0

4 -1

.640

2 4.

9E-0

2

1445

438_

at

Ddh

d1

DD

HD

dom

ain

cont

aini

ng 1

-1

.798

5 6.

8E-0

3 -2

.130

5 1.

2E-0

3 -1

.695

9 2.

3E-0

2

1445

436_

at

BC

0598

42

PR

ED

ICTE

D: M

us m

uscu

lus

cDN

A s

eque

nce

BC

0598

42 (B

C05

9842

), m

RN

A

-2.0

196

1.8E

-03

-1.9

107

1.6E

-03

-1.6

305

2.3E

-02

1455

611_

at

Pia

s1

prot

ein

inhi

bito

r of a

ctiv

ated

STA

T 1

-1.8

771

1.8E

-03

-1.4

53

1.2E

-02

-1.6

163

1.3E

-02

1440

331_

at

Kds

r 3-

keto

dihy

dros

phin

gosi

ne re

duct

ase

-1.6

376

3.1E

-03

-1.4

473

6.7E

-03

-1.4

862

1.9E

-02

1442

443_

at

---

---

-1.4

444

2.4E

-02

-1.5

212

6.7E

-03

-1.5

421

2.3E

-02

1437

919_

at

Bdp

1 B

dou

ble

prim

e 1,

sub

unit

of R

NA

pol

ymer

ase

III tr

ansc

riptio

n in

itiat

ion

fact

or II

IB

-1.4

457

1.1E

-02

-1.4

95

3.7E

-03

-1.3

805

4.7E

-02

Supp

lem

enta

ry T

able

1 (c

ontin

ued)



Appl

icat

ion

Prim

er n

ame

Prim

er s

eque

nce

Qua

ntita

tive

PCR

prim

ers

mLr

g1_F

or

CC

ATG

TCA

GTG

TGC

AG

ATTC

m

Lrg1

_Rev

A

AG

AG

TGA

GAG

GTG

GA

AGA

G

mTG

Fβ1_Fo

r TT

GC

TTC

AGC

TCC

AC

AG

AG

A

mTG

Fβ1_Rev

TG

GTT

GTA

GA

GG

GC

AA

GG

AC

P

AI1_

For

C

CTC

TTC

CA

CAA

GTC

TGA

TGG

C

PAI

1_R

ev

GC

AG

TTC

CA

CA

AC

GTC

ATA

CTC

G

ID1_

For

TGA

AC

GG

CG

AG

ATC

AGTG

CC

TT

ID1_

Rev

G

TGG

CTG

CG

GTA

GTG

TCTT

TC

mVE

GF_

For

GA

CTT

GTG

TTG

GG

AG

GA

GG

A

m

VEG

F_R

ev

TC

TGG

AA

GTG

AG

CC

AA

TGTG

m

Apl

in_F

or

GG

CC

TTC

TCC

GTC

TTTG

TCG

m

Apl

in_R

ev

CC

CTC

TTG

TGC

TTC

TATC

TCTC

C

mAP

LNR

_For

G

GG

AG

TAAG

TTTG

GG

AAAG

AG

m

APLN

R_R

ev

TGG

AATA

TGTC

TTG

TCC

TTG

G

mP

lGF_

For

TTC

TCA

GG

ATG

TGC

TCTG

TGAA

m

PlG

F_R

ev

CTG

GTT

AC

CTC

CG

GG

AAA

TGAC

G

AP

DH

_For

A

CTG

AG

GA

CC

AGG

TTG

TCTC

C

GA

PD

H_R

ev

CTG

TAG

CC

GTA

TTC

ATTG

TCA

TAC

C

G

enot

ypin

g pr

imer

s TU

F TG

CA

CC

TCTC

GAG

CA

ATC

G

TUR

A

GA

GC

ATT

GC

GG

GTC

AG

ATC

N

eoFw

d TC

ATT

CTC

AG

TATT

GTT

TTG

CC

S

D

GA

CC

CC

TGAA

AC

AGA

CG

TG

SU

TC

CTG

GTG

GG

AG

AGG

AC

TC

LacI

nZR

ev

GTC

TGTC

CTA

GC

TTC

CTC

AC

TG

Pr

imer

s fo

r clo

ning

rhLr

g1

Lrg1

-His

_For

G

TGTA

AG

CTT

GC

CA

CC

ATG

GC

CA

TGTC

CTC

TTG

GA

GC

AG

AC

Lr

g1-H

is_R

ev

GTG

TCTC

GAG

TCAT

CA

CTA

GTG

ATG

ATG

ATG

GTG

ATG

CTG

GG

ACTT

GG

CC

AC

TGC

CA

Pr

imer

s fo

r clo

ning

Lrg

1-H

A

HLR

G1F

or

GTG

TAA

GC

TTG

CC

AC

CA

TGG

CC

ATG

TCC

TCTT

GG

AG

CA

GA

C

HLR

G1R

ev

GTG

TCTC

GAG

CTG

GG

AC

TTG

GC

CA

CTG

CC

A

nu

cleo

tide

sequ

ence

for

3XH

A

GTG

TCTC

GAG

GA

GTA

CC

CAT

ACG

AC

GTA

CC

AG

ATT

AC

GC

TTA

CC

CA

TA

CG

ATG

TTC

CA

GAT

TAC

GC

TTAT

CC

ATA

TGA

CG

TCC

CA

GA

CTA

TGC

CTA

G

TGA

TAA

CTC

GA

GG

TGT

Prim

ers

for g

ener

atin

g R

NA

in

situ

hyb

ridis

atio

n pr

obe

mLr

g1_i

nsitu

_For

C

TGG

TCG

CC

AA

CC

GA

AAC

AAG

A

m

Lrg1

_ins

itu_R

ev

AG

TCA

GC

CTA

GG

AG

CC

GTT

TT

Supp

lem

enta

ry T

able

2. P

rim

er se

quen

ces.



RESEARCH

Appl

icat

ion

Prim

er n

ame

Prim

er s

eque

nce

Prim

ers

for c

onst

ruct

ing

TGFβ

R

ecep

tors

ext

race

llula

r dom

ain

ALK

1_Fo

r G

TGTA

AG

CTT

GC

CA

CC

ATG

GC

CA

TGA

CC

T TG

GG

CTC

CC

C

A

LK1_

Rev

G

TGTG

AA

TTC

CTG

GC

CA

TCTG

TTC

CC

GG

CT

ALK

5_Fo

r G

TGTA

AG

CTT

GC

CA

CC

ATG

GC

CA

TGG

AGG

CG

GC

GG

TCG

CT

ALK

5_R

ev

GTG

TGA

ATT

CC

AG

TTC

CA

CA

GG

AC

CA

AG

GC

C

TβRII_Fo

r G

TGTA

AG

CTT

GC

CA

CC

ATG

GC

CA

TGG

GTC

GG

GG

GC

TGC

T

Tβ

RII_Rev

G

TGTG

AA

TTC

TTA

TCA

CTA

CA

GG

TCC

TCC

TCTG

AG

ATC

AGC

TTC

TGC

TCTT

CTG

AG

AAG

ATG

ATG

TTG

TCA

TTG

E

NG

_For

G

TGTA

AG

CTT

GC

CA

CC

ATG

GG

CA

TGG

AC

CG

CG

GC

AC

GC

TCC

CT

EN

G_R

ev

GTG

TGA

ATT

CTT

ATC

AC

TAC

GTA

GAA

TCG

AG

AC

CG

AG

GAG

AG

GG

TTA

GG

GA

TAG

GC

TTA

CC

GC

CTT

TGC

TTG

TGC

AAC

CA

GA

Prim

ers

for c

loni

ng G

ST-E

NG

E

NG

pGE

XFor

G

TGTG

GA

TCC

ATG

GA

CC

GC

GG

CA

CG

CTC

C

EN

GpG

EXR

ev

GTG

TGTC

GA

CTT

ATC

AC

TAG

CC

TTTG

CTT

GTG

CAA

CC

AG

A

Supp

lem

enta

ry T

able

2. P

rim

er se

quen

ces

(con

tinue

d).

lrg1 promotes angiogenesis by modulating endothelial tgf-b signalling

Documents