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(Circulation Research. 2008;102:914.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Bone Morphogenetic Protein-9 Is a Circulating Vascular Quiescence Factor

Laurent David, Christine Mallet, Michelle Keramidas, Noël Lamandé, Jean-Marie Gasc, Sophie Dupuis-Girod, Henri Plauchu, Jean-Jacques Feige, Sabine Bailly

From the Institut National de la Santé et de la Recherche Médicale (L.D., C.M., M.K., J.-J.F., S.B.), U878, Grenoble; Commissariat à l’Energie Atomique (L.D., C.M., M.K., J.-J.F., S.B.), Institut de Recherches en Technologies et Sciences pour le Vivant/Laboratoire Angiogenèse et Physiopathologie Vasculaire, Grenoble; Université Joseph Fourier (L.D., C.M., M.K., J.-J.F., S.B.), Grenoble; Institut National de la Santé et de la Recherche Médicale (N.L., J.-M.G.), Unité 833, Collège de France, Paris; Hospices Civils de Lyon (S.D.-G., H.P.); Hôpital Hôtel-Dieu (S.D.-G., H.P.), Lyon; and Département de Génétique Clinique and Centre National de Réference pour la maladie de Rendu-Osler (S.D.-G., H.P.), Lyon, France.

Correspondence to Sabine Bailly, U878, iRTSV/LAPV, 17 rue des Martyrs, 38054 Grenoble, France. E-mail sbailly{at}cea.fr

	Abstract

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Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. ALK1 (activin receptor like-kinase 1) is an endothelial-specific type 1 receptor of the transforming growth factor-β receptor family. Heterozygotes with mutations in the ALK1 gene develop hereditary hemorrhagic telangiectasia type 2 (HHT2). Recently, we reported that bone morphogenetic protein (BMP)9 and BMP10 are specific ligands for ALK1 that potently inhibit microvascular endothelial cell migration and growth. These data lead us to suggest that these factors may play a role in the control of vascular quiescence. To test this hypothesis, we checked their presence in human serum. We found that human serum induced Smad1/5 phosphorylation. To identify the active factor, we tested neutralizing antibodies against BMP members and found that only the anti-BMP9 inhibited serum-induced Smad1/5 phosphorylation. The concentration of circulating BMP9 was found to vary between 2 and 12 ng/mL in sera and plasma from healthy humans, a value well above its EC₅₀ (50 pg/mL). These data indicated that BMP9 is circulating at a biologically active concentration. We then tested the effects of BMP9 in 2 in vivo angiogenic assays. We found that BMP9 strongly inhibited sprouting angiogenesis in the mouse sponge angiogenesis assay and that BMP9 could inhibit blood circulation in the chicken chorioallantoic membrane assay. Taken together, our results demonstrate that BMP9, circulating under a biologically active form, is a potent antiangiogenic factor that is likely to play a physiological role in the control of adult blood vessel quiescence.

Key Words: BMP9 • ALK1 • HHT • angiogenesis

	Introduction

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Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor (TGF)β superfamily, were originally identified as inducers of ectopic bone growth and cartilage formation. Since then, there has been substantial progress in our knowledge of the multiple functions of these growth factors.¹ BMPs regulate cell growth, differentiation, and apoptosis of various cell types, and they are critically important in the morphogenesis and differentiation of tissues and organs. BMP9, also known as growth differentiation factor-2, is expressed in the adult liver by nonparenchymal cells (ie, endothelial, stellate, and Kupffer cells)² and in the septum and spinal cord of mouse embryos.³ BMP9 has been described as a hematopoietic, hepatogenic, osteogenic, and chondrogenic factor. It has also been identified as a regulator of glucose metabolism, capable of reducing glycemia in diabetic mice and as a differentiation factor for cholinergic neurons in the central nervous system.³ More recently, it was shown to induce the expression of hepcidin, a hormone that plays a key role in iron homeostasis.⁴

ALK1 (activin receptor like-kinase 1) is an endothelial-specific type I receptor of the TGFβ receptor family that is implicated in the pathogenesis of hereditary hemorrhagic telangiectasia type 2 (HHT2), also known as the Rendu–Osler disease type 2.⁵ The disease is an autosomal dominant vascular disorder characterized by recurrent nosebleeds, cutaneous telangiectases, and arteriovenous malformations in the lungs, brain, liver, and gastrointestinal tract.⁶ The majority of cases are caused by mutations in either endoglin (ENG) or ALK1 (ACVRL1) genes, thus defining HHT1 and HHT2, respectively. Mutations in SMAD4 are seen in patients with the combined syndrome of juvenile polyposis and HHT (JP-HHT).⁷ Despite the identification of these mutations as the causative factor in HHT, the mechanism by which these mutations cause the HHT phenotype remain unclear.

ALK1 is 1 of 7 known type I receptors for TGFβ family members.⁸ Signaling through the TGFβ receptor family occurs via ligand binding to heteromeric complexes of type I and type II serine/threonine kinase receptors.⁹ The type I receptor determines signal specificity in the receptor complexes. Activation of ALK1 induces phosphorylation of receptor-regulated Smad1, -5, and -8,¹⁰ which assemble into heteromeric complexes with the common partner Smad4. These heteromeric complexes translocate to the nucleus, where they regulate the transcription of target genes.

ALK1 has long been known as an orphan type I receptor of the TGFβ family predominantly present on endothelial cells. Subsequently, TGFβ1 and -3, primarily known as ligands for ALK5, were also shown to bind ALK1, albeit only in the presence of ALK5.¹¹ In 2005, a publication describing the crystal structure of BMP9 reported that BMP9 specifically binds biosensor-immobilized recombinant ALK1 and BMPRII extracellular domains.¹² More recently, we demonstrated that BMP9 and BMP10 are potent ligands for ALK1 on human dermal microvascular endothelial cells,¹³ and this has since been confirmed by another group.¹⁴ BMP9 is very potent (EC₅₀=2 pmol/L) and, in contrast to TGFβ1 or -3,¹¹ induces a very stable Smad1/5/8 phosphorylation over time.¹³ Interestingly, another ALK1 ligand, distinct from TGFβ1 and TGFβ3 and that could signal in the absence of ALK5 or TGFβRII, had been described previously in human serum but had not been identified.¹⁵ The aim of the present work was to identify this circulating ALK1 ligand. Here, we demonstrate that BMP9 is indeed the ALK1 ligand present in human serum. BMP9 circulates in a biologically active form at a concentration of 2 to 12 ng/mL. Furthermore, we report that BMP9 is a potent inhibitor of angiogenesis and a regulator of vascular tone.

	Materials and Methods

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An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.

DNA Transfection and Dual Luciferase Activity Assay
NIH-3T3 cells were transfected as previously described.¹³ Firefly and renilla luciferase activities were measured sequentially with the Dual-Luciferase reporter assay (Promega). Results are expressed as ratios of firefly luciferase activity over renilla luciferase activity. (See the online data supplement).

Purification of the ALK1 Ligand From Human Serum
Two hundred fifty milliliters of human serum (pool of human sera from 250 different individuals; Cambrex) were diluted with 250 mL PBS (0.15 mol/L, pH 7.4) and purified through 5 different steps, as detailed in the online data supplement.

Western Blot Analysis
Western blots were performed as previously described.¹³ (See the online data supplement).

Blood Donors
Between December 2006 and July 2007, blood samples (7 mL) were taken from 20 patients (8 women, 12 men; mean age, 44±12 years) with clinical features of HHT (13 with ACVRL1 mutations, 2 with ENG mutations, and 5 with unidentified mutations) and 20 healthy volunteers (8 women, 12 men; mean age, 44±10 years) from which serum or plasma (K3E tubes, Becton Dickinson, Pont de Claix, France) were obtained. Serum and plasma aliquots were frozen at –20°C. Informed consent was obtained from all blood donors. The investigation conformed to the principles outlined in the Helsinki declaration. The donors were randomly assigned a number. Patients were considered to be affected by HHT if they had at least 3 out of the 4 Curaçao consensus criteria¹⁶: epistaxis, telangiectases, visceral lesions, and family history of HHT disease.

Chorioallantoic Membrane Assay
The effect of BMP9 on vascularization in the chick chorioallantoic membrane (CAM) was studied as described in the online data supplement.

Mouse Subcutaneous Sponge Angiogenesis Assay
The effect of BMP9 on neovascularization in the mouse sponge assay in response to fibroblast growth factor (FGF)-2 was studied as described in the online data supplement.

Statistics
Statistical analysis was performed using a Mann Whitney test (**P<0.01; *P<0.05).

	Results

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Presence of an ALK1 Ligand in Human Serum That Differs From TGFβ
The luciferase reporter construct (BMP-responsive element [BRE]), which contains repeated sequences from the Id1 promoter, has been developed to specifically measure activation of the Smad1/5/8 pathway.¹⁷ This plasmid, together with an ALK1-expression plasmid, was transfected in NIH-3T3 cells to check for the presence of an ALK1 ligand in human serum. Treatment of these cells with 2% human serum strongly stimulated luciferase activity (9-fold; Figure 1A). To determine whether this activity was attributable to TGFβ, a pan-specific TGFβ-neutralizing antibody was added to serum. As shown in Figure 1A, addition of the neutralizing antibody did not affect serum activity. Furthermore, the addition of recombinant TGFβ1 (0.5 ng/mL) did not activate this reporter gene and actually decreased basal luciferase expression (Figure 1A). Heat treatment, to activate the latent TGFβ present in serum, did not result in BRE activation (Figure 1A). We next examined whether human serum could activate the CAGA promoter, which is known to be specifically activated by the Smad2/3 pathway in response to TGFβ. We found that human serum caused a small induction of the CAGA promoter (4-fold), whereas heat-treated serum and recombinant TGFβ1 strongly activated it (17- and 42-fold, respectively, Figure 1B). These activations were inhibited by the addition of the pan-specific TGFβ-neutralizing antibody (Figure 1B). The BRE promoter is specific for the Smad1/5/8 pathway and, therefore, can be activated by all of the type I receptors known to phosphorylate these Smads, namely ALK1, ALK2, ALK3, and ALK6. Therefore, to confirm that the activation of BRE by human serum was actually attributable to ALK1 activation, we tested the ability of the recombinant extracellular domains of these receptors to interfere with the human serum response. As shown in Figure 1C, addition of ALK1ecd very strongly inhibited the human serum response. ALK3ecd and ALK6ecd only slightly inhibited this response, whereas ALK2ecd had no effect. Interestingly, we could also demonstrate that soluble endoglin inhibited this biological response (Figure 1C). Taken together, these findings demonstrate that an ALK1-stimulating ligand, distinct from TGFβ1, -2, or -3, is present in human serum.

View larger version (16K): [in this window] [in a new window]   Figure 1. Presence of an ALK1 ligand in human serum that differs from TGFβ. NIH-3T3 cells were transiently transfected with pALK1 and pRL-TK-luc and either pGL3(BRE)-luc (A) or pGL3(CAGA)12-luc (B). Transfected cells were then treated with either human serum (2%) or TGFβ1 (0.5 ng/mL) or heat-activated human serum (2%) with or without pan-specific neutralizing TGFβ antibody (1 µg/mL). C, NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc, pALK1, and pRL-TK-luc. Transfected cells were then treated with human serum (2%) in presence or absence of either ALK1ecd, ALK2ecd, ALK3ecd, ALK6ecd, or soluble endoglin (200 ng/mL). The luciferase activities were then measured as described in Materials and Methods. Data shown in A, B, and C are expressed as means±SD from 1 representative experiment of 3.

Purification and Molecular Weight Estimation of the ALK1 Ligand From Human Serum
We next attempted to purify the activating factor from human serum. The factor was purified 100-fold from 250 mL of human serum, following the purification scheme shown in Figure 2A. After 5 purification steps, the fractions eluting from the Pro-RPC column were analyzed by SDS-PAGE under nonreducing conditions. The gel lanes containing the active fractions (23 and 24) were then cut into 6 bands, and the proteins in each band were electroeluted, renatured, and tested for ALK1-stimulating activity. The activity was detected in band 5, which corresponded to an apparent molecular weight comprised between 17 and 28 kDa (Figure 2B).

View larger version (17K): [in this window] [in a new window]   Figure 2. Purification and estimation of the molecular mass (MW) of the ALK1 ligand from the human serum. A, Scheme of purification of ALK1 ligand from 250 mL of a pool of human sera. The proteins present in the active fractions (23 and 24) of the Pro-RPC column and the 2 surrounding fractions (22 and 25), as determined with the BRE reporter gene assay (see Material and Methods), were then separated by 12% SDS-PAGE. After the migration, the gel (fractions 23 and 24) was sliced into 6 parts, as indicated by the dotted lines, and the proteins were electroeluted. B, NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc, pALK1, and pRL-TK-luc. Transfected cells were then treated with 100 µL of either the active fractions (fraction 23 and 24) or 100 µL of the proteins eluted from each gel slice. The luciferase activities were then measured as described in Materials and Methods. Data are expressed as means±SD from 1 representative experiment of 3.

The ALK1-Stimulating Activity of Human Serum Is Attributable to BMP9
In a recent work, we have demonstrated that BMP9 is an activating ligand for ALK1,¹³ and this has since been confirmed by another group.¹⁴ Because the apparent molecular mass of the ALK1-stimulating activity present in human serum appears to lie between 17 and 28 kDa, we hypothesized that this activity could be attributable to circulating BMP9 (molecular mass, 22 kDa). To test this hypothesis, we used a BMP9-neutralizing antibody. This antibody was highly specific because it completely abolished the BRE-luciferase response to BMP9, whereas it had no effect on the BMP10-induced response (BMP10 has the highest sequence homology with BMP9) or on the BMP2-induced response (Figure 3A). We then tested this antibody on the ALK1-stimulating activity in serum and observed nearly complete inhibition of BRE-stimulating activity (Figure 3B). This was also the case for the purified active fractions (fractions 23 and 24 from Figure 2A) from human serum (Figure 3B). To further confirm that BMP9 is the only active circulating member of the TGFβ family present in serum capable of activating the BRE promoter in ALK1-expressing NIH-3T3 cells, we tested neutralizing antibodies for other BMPs. Neutralizing BMP2/4 and BMP7 antibodies had no effect on human serum activity (Figure 3C), whereas both inhibited the BRE response to either recombinant BMP2 or BMP7 (Figure 3D). We also evaluated whether the circulating BMP antagonist noggin inhibits human serum ALK1-stimulating activity. We observed that the addition of noggin did not inhibit the ALK1-stimulating activity from human serum (Figure 3C), whereas it inhibited BMP2 or BMP7 activity (Figure 3D). We could also demonstrate, for the first time, that noggin did not inhibit the induction of BRE activity by recombinant BMP9 (Figure 3D). Finally, we tested the effects of the neutralizing BMP9 antibody on serum activation of Smad1/5 phosphorylation in human microvascular endothelial cells. As shown in Figure 3E, human serum induced rapid and strong Smad1/5 phosphorylation that could be inhibited in a dose-dependent manner by the addition of neutralizing anti-BMP9 antibody or by the addition of ALK1ecd. Taken together, these data lead to the conclusion that the ALK1-stimulating activity of human serum is attributable to BMP9.

View larger version (21K): [in this window] [in a new window]   Figure 3. The ALK1 activity of the human serum is attributable to BMP9. A through D, NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc, pRL-TK-luc, and pALK1. A, Transfected cells were then treated with BMP9 (0.1 ng/mL) or BMP10 (20 ng/mL) or BMP2 (100 ng/mL) in the presence or the absence of a neutralizing BMP9 antibody (1 µg/mL) or an isotype-matched control antibody (1 µg/mL). B, Transfected cells were then treated with human serum (1%) or 100 µL of active fraction (fractions 23 and 24 of Figure 2A). C, Transfected cells were treated with 2% human serum in the presence or the absence of neutralizing antibodies (anti-BMP9 [2 µg/mL], anti-BMP2/4 [10 µg/mL], or anti-BMP7 [10 µg/mL]) or with recombinant noggin (1 µg/mL). D, Transfected cells were treated with either BMP9 (0.05 ng/mL), BMP2 (50 ng/mL), or BMP7 (100 ng/mL) in the presence or the absence of neutralizing antibodies (anti-BMP9 [2 µg/mL], anti-BMP2/4 [10 µg/mL], or anti-BMP7 [10 µg/mL]) or with recombinant noggin (1 µg/mL). The luciferase activities were then measured as described in Materials and Methods. Data shown in A through D are expressed as means±SD from 1 representative experiment of 3. E, Human microvascular endothelial cells were serum-starved for 1 hour and were then treated with 2% human serum for 1 hour in the presence or absence of neutralizing BMP9 antibody (1 or 10 µg/mL) or ALK1ecd (100 ng/mL). Cell lysates (20-µg proteins) were resolved by 10% SDS-PAGE and immunoblotted with antibodies against phospho-Smad1/5/8 or against -tubulin.

Determination of BMP9 Concentration in Human Serum
Having demonstrated that BMP9 is present in human serum, we also evaluated its presence in human plasma. We measured BMP9 levels in the sera and the plasma of 4 healthy individuals and found similar levels of BRE activity in both biological fluids (Figure 4A). Using the BRE luciferase reporter assay and recombinant mature BMP9 (R&D Systems) as a standard for calibration, we determined that the BMP9 concentration in a pool of human sera was 7.5±0.6 ng/mL (Figure 4B). BMP9 binds ALK1 and endoglin,¹³ which are 2 receptors whose genes are mutated in HHT. This prompted us to evaluate the serum levels of BMP9 in HHT patients versus a normal population. Twenty patients with clinical features of HHT were enrolled in this study. The 2 populations were matched for gender ratio (8 were female and 12 were male) and age (mean, 44 years). The study of BMP9 levels in the healthy population demonstrated a mean level of circulating BMP9 very close to the one found in the pooled human sera (6.2±0.6 ng/mL) with a range of variation between 2 and 12 ng/mL (Figure 4C). As shown in Figure 4C, no statistically significant difference in the serum level of BMP9 could be detected between healthy individuals and HHT patients (6.2±0.6 ng/mL versus 5.0±0.7 ng/mL, respectively, n=20). Similar data were obtained using plasma (data not shown). Sera that had high levels of BMP9 (>8 ng/mL) were tested again in the presence of the neutralizing anti-BMP9 antibody to confirm that all of this activity was attributable to BMP9. The antibody totally neutralized the activity in all samples (data not shown).

View larger version (8K): [in this window] [in a new window]   Figure 4. Determination of BMP9 concentration in human serum. A, NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc, pRL-TK-luc, or pALK1. Transfected cells were treated with 0.5% of human serum or plasma of 4 different healthy donors. B, Linear regression for the determination of BMP9 serum concentration. NIH-3T3 cells were transiently transfected with pGL3(BRE)-luc, pRL-TK-luc, or pALK1. Transfected cells were then treated with 0.1% or 0.3% of a pool of human sera. The luciferase activities were then measured as described in Materials and Methods. Data shown in A and B are expressed as means±SD from 1 representative experiment of 3. C, BMP9 serum levels measured in 20 patients with HHT and 20 healthy donors. The line indicates the mean value. The difference was not statistically significant.

BMP9 Is a Potent Inhibitor of Angiogenesis In Vivo
We and others have previously demonstrated that BMP9 inhibits endothelial cell migration and proliferation.^13,14 In addition, it was further demonstrated that BMP9 inhibited ex vivo endothelial cell sprouting from metatarsals.¹⁴ Similarly, we were able to show that BMP9 inhibited endothelial sprouting from embryoid bodies derived of embryonic stem cells committed to endothelial differentiation (Figure I in the online data supplement). Taken together, these data suggest that BMP9 may act as an inhibitor of angiogenesis. To further characterize the antiangiogenic activity of BMP9, we tested its effect in 2 in vivo angiogenic assays. First, we assessed the effect of BMP9 in the mouse subcutaneous sponge assay. In this study, Balb-C mice received, under the dorsal skin, a cellulose sponge hydrated with FGF-2 or FGF-2 and BMP9. Factors were reinjected into the sponge on days 1, 2, and 4 as described in Materials and Methods. The angiogenic response was then assessed on day 7. As shown in Figure 5, BMP9 treatment clearly inhibited the angiogenic response. This inhibitory effect could be quantitated by measuring the hemoglobin content of the sponges (Figure 5B, 1.23±0.22 mg with FGF-2 versus 0.54±0.06 mg with FGF-2 and BMP9; P<0.05). We then investigated whether addition of BMP9 would also lead to destabilization of already formed vessels. To do this, Balb-C mice received a cellulose sponge hydrated with FGF-2, which was reinjected into the sponge on days 1 and 2. Angiogenesis, as measured by hemoglobin levels, was already strong by day 4 (data not shown). BMP9 was then added on days 4, 5, and 6, and the angiogenic response was assessed on day 7. Interestingly, we found that BMP9 added after the initiation of angiogenesis by FGF-2 still significantly inhibited this process (Figure 5C).

View larger version (75K): [in this window] [in a new window]   Figure 5. Effect of BMP9 on angiogenesis in the mice sponge assay. A and B, Bal-C mice received a subcutaneous cellulose sponge treated with FGF-2 (200 ng) and/or BMP9 (20 ng) under the dorsal skin. Injections in the sponge of FGF-2 and/or BMP9 diluted in PBS were performed on days 1 and 2, and a last injection was performed on day 4 with BMP9 alone. C, Bal-C mice received a subcutaneous cellulose sponge treated with FGF-2 (200 ng) diluted in PBS under the dorsal skin. Injections of FGF-2 were performed on days 1 and 2. BMP9 (20 ng) or PBS was injected on days 4, 5, and 6. Animals were euthanized on day 7, and the sponges were photographed (A). Hemoglobin content was measured in 1 mL of radioimmunoprecipitation assay buffer extract of the sponge and adjacent vascular network (B and C). B, Data are expressed as means±SEM from 1 representative experiment of 3 (5 mice in each group). C, Data are expressed as means±SEM of 2 experiments (9 mice in each group). *P<0.05, **P<0.01.

We also tested the effect of BMP9 in the chick CAM assay, which allows for the study of fetal neoangiogenesis. BMP9 or the vehicle (PBS, BSA 0.1%) was applied for 24 hours side by side onto the same CAM on day 9 of embryo development (Figure 6). Four doses of BMP9 were tested (5.5, 27.5, 55, and 550 ng). BMP9 treatment impaired in a dose-dependent manner CAM angiogenesis as seen on photographs (Figure 6B); this effect was further confirmed by fluorescein isothiocyanate (FITC)–dextran injection (Figure 6C): at low dose (5.5 ng), BMP9 had minimal effect on the vasculature; at 27.5 ng, only the small vessels were affected; and at 55 ng, a complete disappearance of all the vessels is induced. A higher dose of BMP9 (550 ng) produced chick embryo death 4 to 6 hours following its addition (data not shown). Serial cross-sections of the CAM, stained with hematoxylin/eosin, isolectin (endothelial cells), or anti–-smooth muscle actin (pericytes), show that this effect of BMP9 was not attributable to vascular pruning because the number of vessels was not modified (supplemental Figure IIA). These results suggested that vessels were still present but not functional. Indeed, when we follow the effect of BMP9 (550 ng) at earlier time points, we could observe constrictions and/or thrombosis of some vessels, suggesting that BMP9 may regulate vascular tone (supplemental Figure IIB). These irregularities in vessel diameter are also observed on CAM cross-section after a 24-hour treatment with BMP9 (55 ng) visualized after hematoxylin/eosin labeling (supplemental Figure IIC).

View larger version (99K): [in this window] [in a new window]   Figure 6. Effect of BMP9 on vessel formation in the chick CAM assay. On day 9, the CAM received either 25 µL of BMP9 (5.5, 27.5, 55, or 550 ng) or vehicle (Control). The photographs shown were taken before (T 0h) and after treatment (T 24h) and are representative of the results obtained in an additional 5 eggs per group. Low-magnification pictures of CAMs before (A) and after (B) treatment. C, Twenty-four hours after treatment, FITC–dextran was injected in the CAM vessels (fluorescent images). The arrow indicates a vessel that is not affected by BMP9 treatment; arrowhead, a vessel that cannot be seen after BMP9 treatment.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiogenesis is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals. In adulthood, most blood vessels remain quiescent and angiogenesis occurs only in the cycling ovary, in the endometrium, in the placenta during pregnancy, and during wound healing.¹⁸ This implicates that circulating quiescence factors must exist in blood. It has been demonstrated previously that human serum is able to specifically activate the Smad1/5 pathway, suggesting the presence of active BMPs in blood.¹⁵ Here, we report that the Smad1/5-stimulating activity present in human serum is attributable to biologically active BMP9. Furthermore, we demonstrate, with the use of 2 in vivo angiogenic assays, that BMP9 is a potent inhibitor of angiogenesis. These data lead us to propose that the circulating antiangiogenic BMP9 could play a role as a regulator of endothelial quiescence.

We found that BMP9 was present at similar levels in both human serum and plasma, suggesting that circulating BMP9 is derived from plasma rather than from platelets. The circulating concentration of BMP9 is between 2 and 12 ng/mL, as determined with the BRE reporter gene assay using recombinant mature BMP9 as a standard. This concentration is well above its EC₅₀ (50 pg/mL, 2 pmol/L), previously determined in microvascular endothelial cells.¹³ In the present work, we showed that human serum activity could be inhibited by neutralizing BMP9 antibodies and by ALK1 extracellular domain, confirming that this activity is caused by a factor that can bind ALK1. We have shown previously that both BMP9 and BMP10 bind to ALK1.¹³ However, here, we demonstrated that the biological ALK1-stimulating activity in human serum is exclusively attributable to BMP9 and not to BMP10. The absence of BMP10 in blood is likely caused by the pattern of BMP10 expression, which appears to be restricted to the developing and postnatal heart.¹⁹ In contrast, BMP9 expression is high in both embryonic and adult liver,² suggesting that this is the likely source of circulating protein. Other TGFβ family members known to activate the Smad1/5 pathway have been described previously in serum or plasma, specifically BMP7 and BMP4.^20,21 However, their concentrations are lower (100 to 400 pg/mL) and their receptor affinities are also much lower (in the nanomolar range) than the affinity of BMP9 for ALK1 (in the picomolar range), suggesting that they are not circulating at biologically active levels. Furthermore, these factors appear to circulate as inactive complexes associated with antagonists such as noggin.²² In contrast, we found that noggin does not inhibit BMP9- or human serum–induced BRE activity (Figure 3C and 3D). This may be another reason why BMP9 is the only active circulating BMP in healthy human serum under our biological conditions.

HHT is a dominantly inherited genetic disorder (mutations of ACVRL1 or ENG), and haploinsufficiency (reduced amount of functional protein) is likely to be the cause of associated vessel malformations. One could imagine that the organism could compensate this haploinsufficiency by increasing the synthesis of the receptor ligand. However, we observed no significant difference between the serum BMP9 levels of healthy individuals and HHT patients, suggesting that there is no compensation by increased BMP9 in this disease.

BMP9 has been shown previously to be a potent regulator of osteogenesis, chondrogenesis, glucose metabolism, and iron homeostasis⁴ and a differentiation factor for cholinergic neurons.³ In a recent study, we have demonstrated that BMP9 is also a potent inhibitor of endothelial cell proliferation and migration.¹³ This has since been confirmed by another group, who further demonstrated that BMP9 inhibited ex vivo endothelial cell sprouting from metatarsals.¹⁴ In the present work, we confirmed these data in another ex vivo endothelial cell sprouting assay and further demonstrated that BMP9 is an important in vivo regulator of angiogenesis. Using the mouse sponge assay, we could show that BMP9 inhibited neoangiogenesis in response to FGF-2 but also induced destabilization of already formed vessels (Figure 5B and 5C). This latter point suggests that BMP9 could be a useful tool to target tumor angiogenesis. Using the CAM assay, we found that BMP9 treatment inhibited blood circulation in a dose-dependent manner (Figure 6). This was not attributable to a decrease in vessel number but rather to vasoconstrictions and/or thrombosis. This point is interesting because BMP9 signals through BMP receptor (BMPR)II, and mutations in BMPR2 have been found responsible for familial pulmonary hypertension.²³ These data represent the first demonstration of in vivo effects of BMP9 on angiogenesis. Because BMP9 is circulating under a biologically active form in adults, our data prompt us to suggest that BMP9 may be a systemic inhibitor of angiogenesis and a regulator of vascular tone. These data are supported by previous work demonstrating that phosphorylated Smad1, Smad5, and/or Smad8 are detectable in mouse aorta cryosections,²⁴ indicating that, in vivo, these cells constantly receive stimulation by BMPs. The role of BMP family members on vascular development has not been studied extensively. Data are not clear and often show paradoxical effects between in vitro and in vivo assays. Growth differentiation factor-5/BMP14 was among the first BMPs described for its proangiogenic activity in vivo.²⁵ BMP2 was shown to increase angiogenesis in the sponge assay and to induce neovascularization of developing tumors,²⁶ whereas it had no effect in the CAM assay.²⁵ Overall, in vivo data seem to indicate that BMPs acting through ALK3/ALK6 receptors are proangiogenic. Our data demonstrate that, in contrast to these BMPs, BMP9 inhibits angiogenesis via ALK1. This clearly separates BMPs into 2 categories: the proangiogenic BMPs that transduce via ALK3/6 and the antiangiogenic BMPs (BMP9) that transduce via ALK1. Because all of these BMPs activate the Smad1/5 pathway, it is unlikely that this pathway represents the only signaling pathway implicated in these mechanisms. This is highly consistent with our previous work demonstrating that ALK1-mediated inhibition of endothelial proliferation and migration is Smad-independent.²⁷ In accordance with these data, it has recently been described that ALK1 directly phosphorylates endoglin, resulting in inhibition of endothelial cell proliferation.²⁸ BMP9 was also recently reported to inhibit Akt phosphorylation, which is clearly implicated in the migration of endothelial cells.²⁹ The presence of both positive and negative BMP-mediated signaling responses in endothelial cells may provide a useful paradigm for the further dissection of the mechanisms by which BMPs participate in the control of angiogenesis.

	Acknowledgments

 
We thank Solène di Prima for helpful participation in this work. We also thank Dr J. LaMarre (University of Guelph, Ontario, Canada) for review of the manuscript and constant enthusiastic support. We thank Drs F. Bono and P. Schaeffer (Sanofi-Aventis, Toulouse, France) for advice about the angiogenesis sponge assay.

Sources of Funding

This work was supported by Institut National de la Santé et de la Recherche Médicale, Commissariat à l’Energie Atomique, Groupement des Entreprises Françaises pour la Lutte contre le Cancer, Grenoble-Dauphiné-Savoie; Programme Hospitalier de Recherche Clinique, Hospices Civils de Lyon (grant 27); and Association des Malades de Rendu-Osler. L.D. was supported by a CFR grant from the Commissariat à l’Energie Atomique and by the Association pour la Recherche sur le Cancer.

Disclosures

None.

	Footnotes

 
Original received October 8, 2007; revision received January 29, 2008; accepted February 18, 2008.

	References

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1. Xiao YT, Xiang LX, Shao JZ. Bone morphogenetic protein. Biochem Biophys Res Commun. 2007; 362: 550–553.[CrossRef][Medline] [Order article via Infotrieve]

2. Miller AF, Harvey SA, Thies RS, Olson MS. Bone morphogenetic protein-9. An autocrine/paracrine cytokine in the liver. J Biol Chem. 2000; 275: 17937–17945.[Abstract/Free Full Text]

3. Lopez-Coviella I, Berse B, Thies RS, Blusztajn JK. Upregulation of acetylcholine synthesis by bone morphogenetic protein 9 in a murine septal cell line. J Physiol Paris. 2002; 96: 53–59.[CrossRef][Medline] [Order article via Infotrieve]

4. Truksa J, Peng H, Lee P, Beutler E. Bone morphogenetic proteins 2, 4, and 9 stimulate murine hepcidin 1 expression independently of Hfe, transferrin receptor 2 (Tfr2), and IL-6. Proc Natl Acad Sci U S A. 2006; 103: 10289–10293.[Abstract/Free Full Text]

5. Johnson DW, Berg JN, Baldwin MA, Gallione CJ, Marondel I, Yoon SJ, Stenzel TT, Speer M, Pericak-Vance MA, Diamond A, Guttmacher AE, Jackson CE, Attisano L, Kucherlapati R, Porteous ME, Marchuk DA. Mutations in the activin receptor–like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat Genet. 1996; 13: 189–195.[CrossRef][Medline] [Order article via Infotrieve]

6. Marchuk DA, Srinivasan S, Squire TL, Zawistowski JS. Vascular morphogenesis: tales of two syndromes. Hum Mol Genet. 2003; 12: R97–R112.[Abstract/Free Full Text]

7. Gallione CJ, Richards JA, Letteboer TG, Rushlow D, Prigoda NL, Leedom TP, Ganguly A, Castells A, Ploos van Amstel JK, Westermann CJ, Pyeritz RE, Marchuk DA. SMAD4 mutations found in unselected HHT patients. J Med Genet. 2006; 43: 793–797.[Abstract/Free Full Text]

8. Attisano L, Carcamo J, Ventura F, Weis FM, Massague J, Wrana JL. Identification of human activin and TGF beta type I receptors that form heteromeric kinase complexes with type II receptors. Cell. 1993; 75: 671–680.[CrossRef][Medline] [Order article via Infotrieve]

9. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003; 113: 685–700.[CrossRef][Medline] [Order article via Infotrieve]

10. Chen YG, Massague J. Smad1 recognition and activation by the ALK1 group of transforming growth factor-beta family receptors. J Biol Chem. 1999; 274: 3672–3677.[Abstract/Free Full Text]

11. Goumans MJ, Valdimarsdottir G, Itoh S, Rosendahl A, Sideras P, ten Dijke P. Balancing the activation state of the endothelium via two distinct TGF- beta type I receptors. EMBOJ. 2002; 21: 1743–1753.[CrossRef][Medline] [Order article via Infotrieve]

12. Brown MA, Zhao Q, Baker KA, Naik C, Chen C, Pukac L, Singh M, Tsareva T, Parice Y, Mahoney A, Roschke V, Sanyal I, Choe S. Crystal structure of BMP-9 and functional interactions with pro-region and receptors. J Biol Chem. 2005; 280: 25111–25118.[Abstract/Free Full Text]

13. David L, Mallet C, Mazerbourg S, Feige JJ, Bailly S. Identification of BMP9 and BMP10 as functional activators of the orphan activin receptor-like kinase 1 (ALK1) in endothelial cells. Blood. 2007; 109: 1953–1961.[Abstract/Free Full Text]

14. Scharpfenecker M, van Dinther M, Liu Z, van Bezooijen RL, Zhao Q, Pukac L, Lowik CW, ten Dijke P. BMP-9 signals via ALK1 and inhibits bFGF-induced endothelial cell proliferation and VEGF-stimulated angiogenesis. J Cell Sci. 2007; 120: 964–972.[Abstract/Free Full Text]

15. Lux A, Attisano L, Marchuk DA. Assignment of transforming growth factor beta1 and beta3 and a third new ligand to the type I receptor ALK-1. J Biol Chem. 1999; 274: 9984–9992.[Abstract/Free Full Text]

16. Shovlin CL, Guttmacher AE, Buscarini E, Faughnan ME, Hyland RH, Westermann CJ, Kjeldsen AD, Plauchu H. Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome). Am J Med Genet. 2000; 91: 66–67.[CrossRef][Medline] [Order article via Infotrieve]

17. Korchynskyi O, ten Dijke P. Identification and functional characterization of distinct critically important bone morphogenetic protein-specific response elements in the Id1 promoter. J Biol Chem. 2002; 277: 4883–4891.[Abstract/Free Full Text]

18. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005; 438: 932–936.[CrossRef][Medline] [Order article via Infotrieve]

19. Neuhaus H, Rosen V, Thies RS. Heart specific expression of mouse BMP-10 a novel member of the TGF-beta superfamily. Mech Dev. 1999; 80: 181–184.[CrossRef][Medline] [Order article via Infotrieve]

20. Kodaira K, Imada M, Goto M, Tomoyasu A, Fukuda T, Kamijo R, Suda T, Higashio K, Katagiri T. Purification and identification of a BMP-like factor from bovine serum. Biochem Biophys Res Commun. 2006; 345: 1224–1231.[CrossRef][Medline] [Order article via Infotrieve]

21. Sugimoto H, Yang C, LeBleu VS, Soubasakos MA, Giraldo M, Zeisberg M, Kalluri R. BMP-7 functions as a novel hormone to facilitate liver regeneration. Faseb J. 2007; 21: 256–264.[Abstract/Free Full Text]

22. Gazzerro E, Canalis E. Bone morphogenetic proteins and their antagonists. Rev Endocr Metab Disord. 2006; 7: 51–65.[CrossRef][Medline] [Order article via Infotrieve]

23. Trembath RC, Thomson JR, Machado RD, Morgan NV, Atkinson C, Winship I, Simonneau G, Galie N, Loyd JE, Humbert M, Nichols WC, Morrell NW, Berg J, Manes A, McGaughran J, Pauciulo M, Wheeler L. Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med. 2001; 345: 325–334.[Abstract/Free Full Text]

24. Valdimarsdottir G, Goumans MJ, Rosendahl A, Brugman M, Itoh S, Lebrin F, Sideras P, ten Dijke P. Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation. 2002; 106: 2263–2270.[Abstract/Free Full Text]

25. Yamashita H, Shimizu A, Kato M, Nishitoh H, Ichijo H, Hanyu A, Morita I, Kimura M, Makishima F, Miyazono K. Growth/differentiation factor-5 induces angiogenesis in vivo. Exp Cell Res. 1997; 235: 218–226.[CrossRef][Medline] [Order article via Infotrieve]

26. Raida M, Clement JH, Leek RD, Ameri K, Bicknell R, Niederwieser D, Harris AL. Bone morphogenetic protein 2 (BMP-2) and induction of tumor angiogenesis. J Cancer Res Clin Oncol. 2005; 131: 741–750.[CrossRef][Medline] [Order article via Infotrieve]

27. David L, Mallet C, Vailhe B, Lamouille S, Feige JJ, Bailly S. Activin receptor-like kinase 1 inhibits human microvascular endothelial cell migration: Potential roles for JNK and ERK. J Cell Physiol. 2007; 213: 484–489.[CrossRef][Medline] [Order article via Infotrieve]

28. Koleva RI, Conley BA, Romero D, Riley KS, Marto JA, Lux A, Vary CP. Endoglin structure and function: determinants of endoglin phosphorylation by TGFbeta receptors. J Biol Chem. 2006; 281: 25110–25123.[Abstract/Free Full Text]

29. Liu D, Wang J, Kinzel B, Mueller M, Mao X, Valdez R, Liu Y, Li E. Dosage-dependent requirement of BMP type II receptor for maintenance of vascular integrity. Blood. 2007; 110: 1502–1510.[Abstract/Free Full Text]

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