Bone Morphogenetic Protein-9 Is a Circulating Vascular Quiescence Factor
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.
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
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).
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.
Statistical analysis was performed using a Mann Whitney test (**P<0.01; *P<0.05).
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.
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).
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.
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).
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).
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–α-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).
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.
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.
Original received October 8, 2007; revision received January 29, 2008; accepted February 18, 2008.
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