Selective BMP-9 Inhibition Partially Protects Against Experimental Pulmonary Hypertension
- This article is commented on by the following:
- is related to
- Other version(s) of this article
You are viewing the most recent version of this article. Previous versions:
Abstract
Rationale:
Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized.
Objective:
To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension.
Method and Results:
The absence of BMP9 in Bmp9−/− mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9−/− mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9+/+ littermates.
Conclusions:
Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.
Pulmonary endothelial dysfunction associated to pulmonary arterial hypertension (PAH) is a key pathogenetic mechanism that could be detrimental for disease susceptibility and development of pulmonary vascular remodeling.1–3 Despite recent progress in the treatment of PAH,4 most patients still die from the disease or fail to respond adequately to medical therapy with a 5-year survival of 59%.5 Current treatments can relieve some PAH symptoms and slow the progress of the disease in some patients, but they have a limited impact on the progressive pulmonary vascular remodeling that eventually culminates in right heart failure. Since 2000, heterozygous germline mutations in the Bone MorphogeneticProtein Receptor type 2 (BMPR2) gene have been identified as critical genetic factors predisposing to pulmonary vascular remodeling and PAH development with low penetrance.6–8 Still, the specific contribution of the long-term loss of signal transduction triggered by this signaling pathway remains poorly characterized.
Editorial, see p 822
Meet the First Author, see p 818
Among BMPs, BMP9 and BMP10 are 2 high-affinity ligands for ALK1 (Activin receptor-like kinase 1) and BMPR2 present in a heterotetrameric complex on pulmonary endothelial cells (ECs),9 and thereby are key actors of vascular development and homeostasis.10 Perturbation in the BMP9/BMP10 signaling pathway has emerged as essential in endothelial (dys)function and vascular remodeling, in particular in PAH, and hereditary hemorrhagic telangiectasia.11,12 In line with this notion, several works have highlighted a potential role for this pathway in the regulation of vascular tone13 and in the modulation of ET-1 (endothelin-1) 14,15 and apelin, 2 potent vasoreactive mediators for the pulmonary vasculature.16,17 Even if Bmp9-KO mice are viable and present no obvious defects, these mice exhibit an altered lymphatic maturation that leads to drainage deficiency.18 Although new biomedical therapeutics targeting BMPs or the BMP signaling pathway hold promise in future treatment strategies for PAH and other life-threatening diseases, our current understanding of the molecular signaling pathways activated by BMPs and their exact pathogenic roles remains limited.11
Therefore, we hypothesized that long-term loss of endothelial BMP9 signaling in rodents could alter pulmonary vascular tone and remodeling, thereby reducing their susceptibility to pulmonary hypertension.
Methods
The authors declare that all supporting data are available within the article and its Online Data Supplement.
Because of space limitations, a detailed description of the Materials and Methods is presented in the Online Data Supplement.
Results
Mice Deficient in BMP9 (Bmp9−/−) are Less Susceptible to Chronic Hypoxia-Induced Pulmonary Hypertension
To gain information about the role of BMP9 in the susceptibility of mice to develop pulmonary hypertension (PH), we used mice congenitally deficient in the expression of the BMP9 gene (Gdf2). The loss of active circulating BMP9 levels in Bmp9−/− mice was confirmed using a BMP responsive luciferase reporter assay (Figure 1A). Consistent with these findings, a 2-fold decrease in phosphorylated (p)-Smad1/5/8 and p-P38 were found in lungs of Bmp9−/− versus Bmp9+/+ littermates and no compensatory upregulation of other BMP family members was present in these mice deficient in BMP9 (Online Figure I). Bmp9−/− mice in the C57BL/6 background are viable and present no obvious defects except a defect in lymphatic maturation.17,18 Our present findings indicate that Bmp9−/− mice are less susceptible to pulmonary vascular remodeling than Bmp9+/+ littermates when exposed to chronic hypoxia. Indeed, the elevations in right ventricular systolic pressure (Figure 1B) and right ventricular hypertrophy right ventricle (RV/left ventricle+septum weight ratio) (Figure 1C) following chronic hypoxia were lower in the Bmp9−/− than the Bmp9+/+ mice. No significant differences were observed in heart rate or systemic pressures between Bmp9−/− and Bmp9+/+ mice (heart rate: 320±34 versus 324 ±44 bpm, respectively, NS; systemic blood pressure: 110±20 versus 105±18, n=10, respectively, NS). Similar findings have also been observed with intravenous administration of the active monocrotaline metabolite (monocrotaline pyrrole) to Bmp9−/− and Bmp9+/+ mice (Online Figure II). Following chronic hypoxia, the less pronounced right ventricular systolic pressure elevation in the Bmp9−/− was also associated with a decreased number and thickness of muscularized distal pulmonary arteries versus Bmp9+/+ mice (Figure 1D). Consistent with these observations, we did not observe a significant increase in the number of PCNA+ (proliferating cell nuclear antigen positive) cells or an accumulation of F4/80+ cells (monocytes/macrophages) in the perivascular area of muscularized vessels in Bmp9−/− mice under normoxic versus hypoxic conditions in contrast to Bmp9+/+ mice (Figure 1E).

Figure 1. Susceptibility of mice deficient in BMP9 (bone morphogenetic protein; Bmp9−/−) to the development of chronic hypoxia–induced pulmonary hypertension (PH). A, Analyses of the luciferase reporter activity under the control of the BMP response element (BRE)-containing promoter in the sera of Bmp9+/+ and Bmp9 knockout mice (Bmp9−/−; n=5–6). The relative firefly luciferase activity was normalized to renilla luciferase activity. B, Right ventricular systolic pressures (RVSP) and (C) right ventricular hypertrophy expressed by the Fulton index in Bmp9−/− and Bmp9+/+ mice (n=7–13). D, Representative images of hematoxylin and eosin (H&E), α-SMA (alpha-smooth muscle actin) staining, and quantification of the percentage of muscularized distal pulmonary arteries (intraalveolar vessels <200 μm) and of wall thickness in lungs from Bmp9−/− and Bmp9+/+ mice (n=3–6). E, Representative images of PCNA (proliferating cell nuclear antigen) and F4/80 immunostaining, and quantification of the percentage of PCNA and F4/80 positive cells (arrow heads) per vessels in lungs from Bmp9−/− and Bmp9+/+ mice. Scale bars: 50 μm. Data are presented as mean±SEM (n=4–5). Comparisons were made using 1-way ANOVA with Tukey post hoc tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Bmp9+/+ mice under normoxia; #P<0.05, ##P<0.01, vs Bmp9+/+ mice under chronic hypoxia. AU indicates arbitrary unit; LV, left ventricle; RV, right ventricle; and S, septum.
Administration of Neutralizing Anti-BMP9 Antibodies in Mice Prevents Chronic Hypoxia-Induced Pulmonary Hypertension
To further demonstrate the role of BMP9 in the susceptibility of mice to develop PH, we study the effect of suppressing BMP9 action in adult C57BL/6 mice (wild type) with neutralizing anti-BMP9 antibodies (kindly obtained from Dr M. Yan). Weekly administration of neutralizing anti-BMP9 antibodies (5 mg/kg, IP) after hypoxia exposure completely inhibited BMP circulating ALK1-dependent activity (Figure 2A). Consistent with our hypothesis and our findings obtained in Bmp9 deficient mice, we found that C57BL/6 mice chronically exposed to hypoxia and treated with neutralizing anti-BMP9 antibodies were less susceptible to the development of chronic hypoxia–induced PH than C57BL/6 mice treated with IgG. The severity of the disease was assessed by right ventricular systolic pressure measurement (Figure 2B), by right ventricular hypertrophy (Figure 2C), and by pulmonary distal arterial muscularization (Figure 2D). We also noted that cell proliferation and the degree of inflammatory cell infiltration were significantly decreased in lungs from C57BL/6 mice chronically exposed to hypoxia and treated with neutralizing anti-BMP9 antibodies when compared with lungs of C57BL/6 mice chronically exposed to hypoxia and treated with IgG (Figure 2E).

Figure 2. Efficacy of chronic treatment with the neutralizing anti-BMP9 (bone morphogenetic protein 9) antibodies in the mouse model of hypoxia-induced pulmonary hypertension. A, Analyses of the luciferase reporter activity under the control of the BMP response element (BRE)-containing promoter in the sera of C57BL/6 mice treated with either the neutralizing anti-BMP9 or IgG antibodies (n=6–7). The relative firefly luciferase activity was normalized to renilla luciferase activity. B, Right ventricular systolic pressures (RVSP) and (C) right ventricular hypertrophy expressed by the Fulton index in C57BL/6 mice treated with either the neutralizing anti-BMP9 or IgG antibodies (n=5–7). D, Representative images of hematoxylin and eosin (H&E), α-SMA (alpha-smooth muscle actin) staining and quantification of the percentage of muscularized distal pulmonary arteries (intraalveolar vessels <200 μm) and of wall thickness in lungs from C57BL/6 mice treated with either the neutralizing anti-BMP9 or IgG antibodies (n=4–5). E, Representative images of PCNA (proliferating cell nuclear antigen) and F4/80 immunostaining, and quantification of the percentage of PCNA and F4/80 positive cells (arrow heads) per vessels in lungs from C57BL/6 mice treated with either the neutralizing anti-BMP9 or IgG antibodies. Scale bars: 50 μm. Data are presented as mean±SEM (3–5). Comparisons were made using 1-way ANOVA with Tukey post hoc tests. **P<0.01, ***P<0.001, ****P<0.0001 vs C57BL/6 mice treated with IgG antibodies under normoxia; #P<0.05, ##P<0.01, ###P<0.001 vs C57BL/6 mice treated with IgG antibodies under chronic hypoxia. AU indicates arbitrary unit; LV, left ventricle; RV, right ventricle; and S, septum.
Efficacy of Treatment With the Soluble Extracellular ALK1 Domain (ALK1ECD) on the Progression of Established PH in 2 Complementary and Well-Established Models of Severe PH in Rats
Next, these results prompted us to test the efficacy of the soluble extracellular ALK1 domain (ALK1ECD), a ligand trap targeting ALK1’s ligands thus both BMP9 and BMP10,19 on the development of monocrotaline (MCT)-induced PH in Wistar rats. Treatment with ALK1ECD (4 mg/kg, IP) was started 1 week after a subcutaneous MCT injection and was repeated 1 week later (3 mg/kg, IP; Figure 3A). We first validated that the weekly ALK1ECD administration completely inhibited BMP circulating ALK1-dependent activity (Figure 3B). On day 21, in MCT-injected rats treated with human IgG, a marked increase in mean pulmonary arterial pressure, total pulmonary vascular resistance, RV/(left ventricle+septum) ratio (Figure 3C), percentage medial wall thickness, and numbers of muscularized distal pulmonary arteries (Figure 3D) were found compared with controls. However, MCT-injected rats receiving ALK1ECD exhibited reduced mean pulmonary arterial pressure, total pulmonary vascular resistance, and RV/(left ventricle+septum) ratio as compared with MCT-injected rats receiving IgG (Figure 3C). Consistent with these results, the percentages of medial wall thickness and of muscularized distal pulmonary arteries were substantially decreased in MCT-injected rats receiving ALK1ECD when compared with MCT-injected rats receiving IgG (Figure 3D). In addition, we found a substantial decrease in collagen deposition in the RV myocardium of MCT-injected rats receiving ALK1ECD when compared with MCT-injected rats receiving IgG, an observation that is consistent with the more preserved cardiac function observed in this ALK1ECD-treated rats (Online Figure IIIA-B). Interestingly, no significant differences were found in the number of PCNA+ and CD68+ (monocyte/macrophages) cells in pulmonary vessel walls in lungs of MCT-injected rats receiving ALK1ECD as compared to lungs of control rats receiving IgG (Figure 3E).

Figure 3. Efficacy of chronic treatment with the BMP9/BMP10 (bone morphogenetic protein) ligand trap targeting ALK1’s ligands in the rat model of monocrotaline-induced pulmonary hypertension. A, Experimental design. B, Analyses of the luciferase reporter activity under the control of the BMP response element (BRE)-containing promoter in the sera of control+IgG and monocrotaline (MCT)-injected rats receiving either ALK1ECD or IgG (n=4–6). The relative firefly luciferase activity was normalized to renilla luciferase activity. C, Mean pulmonary arterial pressures (mPAP), total pulmonary vascular resistance (TPVR), and right ventricular hypertrophy expressed by the Fulton index in control+IgG and MCT-injected rats receiving either ALK1ECD or IgG (n=8–11). D, Representative images of hematoxylin and eosin (H&E), α-SMA (alpha-smooth muscle actin) staining, and quantification of the percentage of muscularized distal pulmonary arteries (intraalveolar vessels <200 μm) and of wall thickness in lungs from control+IgG and MCT-injected rats receiving either ALK1ECD or IgG (n=5–7). E, Representative images of PCNA (proliferating cell nuclear antigen) and CD68 immunostainings, and quantification of the percentage of PCNA and CD68 positive cells (arrow heads) per vessel in lungs from control+IgG and MCT-injected rats receiving either ALK1ECD or IgG. Scale bars: 50 μm. Data are presented as mean±SEM (n=6). Comparisons were made using 1-way ANOVA with Tukey post hoc tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control rats treated with IgG antibodies; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 vs MCT rats treated with IgG antibodies. ALK1 indicates activin receptor-like kinase 1; AU, arbitrary unit; ECD, extracellular domain; LV, left ventricle; RV, right ventricle; and S, septum.
To validate our findings obtained in the MCT rat model, weekly treatments of Sugen-hypoxia (SuHx) rats with ALK1ECD or IgG were next performed (Figure 4). Treatment with ALK1ECD (4 mg/kg, IP) was started 5 weeks post-SU5416 injection and was repeated 1 and 2 weeks later (3 mg/kg, IP; Figure 4A). We first validated that the weekly ALK1ECD administration completely inhibited BMP circulating ALK1-dependent activity (Figure 4B). Eight weeks post-SU5416 injection, SuHx rats develop severe experimental PH, as reflected by a marked increase in values of mean pulmonary arterial pressure total pulmonary vascular resistance, and Fulton index (Figure 4C). Although only a trend towards lower mean pulmonary arterial pressure values was found, SuHx rats treated with ALK1ECD exhibit less severe PH when compared with SuHx rat-treated with IgG, as reflected by significantly lower values of total pulmonary vascular resistance and Fulton index (Figure 4C). Consistent with these findings and those obtained in the MCT rat model, we also noted a beneficial effect of ALK1ECD treatments on the pulmonary arterial muscularization (Figure 4D), numbers of PCNA+ and CD68+ cells per vessel (Figure 4E), as well as on RV function and collagen deposition in the RV myocardium (Online Figure IIIC-D) when compared with SuHx rat-treated with IgG.

Figure 4. Efficacy of chronic treatment with the BMP9/BMP10 (bone morphogenetic protein 9/10) ligand trap ALK1ECD in the Sugen-hypoxia (SuHx) rat model of severe pulmonary hypertension. A, Experimental design. B, Analyses of the luciferase reporter activity under the control of the BMP response element (BRE)-containing promoter in the sera of Control+immunoglobulin G (IgG) and SuHx rats receiving either ALK1ECD or IgG (n=4–7). The relative firefly luciferase activity was normalized to renilla luciferase activity. C, Mean pulmonary arterial pressures (mPAP), total pulmonary vascular resistance (TPVR), and right ventricular hypertrophy expressed by the Fulton index in control+IgG and SuHx rats receiving either ALK1ECD or IgG (n=4–7). D, Representative images of hematoxylin and eosin (H&E), α-SMA (alpha-smooth muscle actin) staining, and quantification of the percentage of muscularized distal pulmonary arteries (intraalveolar vessels <200 μm) and of wall thickness in lungs from Control+IgG and SuHx rats receiving either ALK1ECD or IgG (n=4–6). E, Representative images of proliferating cell nuclear antigen (PCNA) and CD68 immunostainings, and quantification of the percentage of PCNA and CD68 positive cells (arrow heads) per vessel in lungs from Control+IgG and SuHx rats receiving either ALK1ECD or IgG (n=3–7). Scale bars: 50 μm. Data are presented as mean±SEM. Comparisons were made using 1-way ANOVA with Tukey post hoc tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control rats treated with IgG antibodies; #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 vs MCT rats treated with IgG antibodies. ALK1, activin receptor-like kinase 1; AU, arbitrary unit; ECD, extracellular domain; LV, left ventricle; RV, right ventricle; and S, septum.
BMP9 Regulates In Vitro and In Vivo the Balance of Key Vascular Tone Regulators
BMP9 has been described as a potential vasoconstriction factor in the chick chorioallantoic membrane assay13 and as a BMP ligand that can down-regulate the expression of the vasodilator factor apelin16,17 and increase the vasoconstrictor peptide ET-1.14,15 An in silico reanalysis of the microarray data (accession number E-MTAB-2495) obtained from Long et al20 in which, human pulmonary ECs were stimulated with 1 ng/mL of recombinant BMP9 for 4 hours, revealed 2 key pathways that are susceptible to regulate vascular contraction, namely the cGMP-PKG signaling pathway” and the “Vascular smooth muscle contraction pathway. Based on these sets of genes and their known effectors, we set-up a list of genes that allowed us to identify 23 genes that were significantly regulated by BMP9 (Online Figure IV), including apelin and ADM (adrenomedullin), 2 potent vasodilator factors. We thus investigated whether recombinant human BMP9 could alter the balance between pulmonary endothelium-derived relaxing factors and contracting factors in vitro using freshly isolated pulmonary ECs established from lung tissue obtained from control and PAH patients (both idiopathic and BMPR2 mutation carriers). Interestingly, we found that recombinant human BMP9 induced a dose-dependent production of ET-1, and a dose-dependent decrease in the production of apelin and ADM in control pulmonary ECs (Figure 5A). A similar profile was observed in pulmonary ECs derived from PAH patients except for ADM level that was already significantly reduced at the basal level in comparison to control cells (Figure 5A). Based on these in vitro findings, we then assessed the levels of the synthesis of potent vasoreactive factors in lungs of Bmp9−/− and Bmp9+/+ mice under normoxic and hypoxic conditions. Consistent with our findings showing that Bmp9−/− mice are less susceptible to chronic hypoxia-induced PH, we found a decreased ET-1 mRNA level and an increased apelin and ADM mRNA levels in Bmp9−/− mice chronically exposed to hypoxia compared with Bmp9+/+ mice chronically exposed to hypoxia (Figure 5B). Since these mediators can regulate the function and the thickness of the underlying smooth muscle cells and because myosin light chain is a major regulatory molecule for smooth muscle contraction, we next studied its expression in lungs of Bmp9−/− mice under normoxic and hypoxic conditions. Interestingly, we found that myosin light chain protein expression is decreased in lungs of Bmp9−/− mice chronically exposed to hypoxia compared with Bmp9+/+ mice chronically exposed to hypoxia (Figure 5C and 5D), a phenomenon that was associated with decreased activity of the ROCK (Rho/Rho-associated protein kinase) signaling pathway (Online Figure V). Consistent with these findings obtained in lungs of Bmp9−/− mice, a substantial decrease in myosin light chain protein expression was found in lungs of MCT- and SuHx rats receiving ALK1ECD when compared with lungs of MCT- and SuHx rats receiving IgG (Figure 5E and 5F).

Figure 5. Effects of BMP9 (bone morphogenetic protein 9) on the expression of endothelin-1 (ET-1), apelin, and adrenomedullin (ADM). Gene expression analysis of ET-1, apelin and ADM (A) in freshly isolated pulmonary endothelial cells derived from lung tissue obtained from control (n=5) and pulmonary arterial hypertension (PAH) patients (n=7) exposed to increasing doses of recombinant human (rh) BMP9 and (B) in lungs of Bmp9−/− and Bmp9+/+ mice under normoxic and hypoxic conditions (n=7–11). C, Representative Western blots and quantification of the myosin light chain (MLC)/GAPDH ratio in lungs of Bmp9−/− and Bmp9+/+ mice under normoxic and hypoxic conditions (n=5–11). D–F, Confocal immunofluorescence analyses of MLC, SM22 or α-SMA (alpha-smooth muscle actin), and 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) in lungs from (D) Bmp9−/− and Bmp9+/+ mice under normoxic and hypoxic conditions (n=5) and from (E) MCT (monocrotaline) injected rats receiving either ALK1ECD or IgG (n=5) and from (F) SuHx rats receiving either ALK1ECD or IgG (n=4). Scale bars: 50 μm. Data are presented as mean±SEM. Comparisons were made using 1-way ANOVA with Tukey post hoc tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs cells exposed to 0 ng/mL of rhBMP9 or Bmp9+/+ mice under normoxia; #P<0.05, ##P<0.01, ####P<0.0001 vs Bmp9+/+ mice under chronic hypoxia. ALK1 indicator activin receptor-like kinase 1; AU, arbitrary unit; ECD, extracellular domain; and L, lumen.
Discussion
Our study identifies BMP9, also known as GDF2 (growth and differentiation factor 2), as a major factor for controlling functions of pulmonary vascular cells (Figure 6). Using 3 different approaches of suppressing BMP9 action in rodents (Bmp9−/− mice, administration of neutralizing anti-BMP9 antibodies or of ALK1ECD ligand trap for BMP9 and BMP10 in rats) in different complementary and well-established animal models of PH, we demonstrate that the selective loss of BMP9 partially protects against experimental pulmonary hypertension.

Figure 6. Schematic diagram summarizing our findings. ADM indicates adrenomedullin; ALK1, activin receptor-like kinase 1; BMPR2, bone morphogenetic protein receptor type 2; ECD, extracellular domain; ET-1, endothelin-1; and MLC, myosin light-chain.
These results seem at first glance counter-intuitive, as it would have been expected that less ligands for the BMPR2 receptor should mimic the loss of BMPR2 signaling found in PAH patients. In accordance with this hypothesis, it was recently proposed to enhance this pathway by exogenous BMP9 in PAH.20 Even if there are some arguments that support this notion,21–24 our functional data indicate the opposite and further illustrate that BMP signaling is complex and involves many ligands and many receptor combinations that could explain these unexpected results, especially in the context of PAH. Indeed, it has been found that BMPR2 mutations could alter BMP signaling in a BMP ligand- or receptor-specific manner.25–27 During the writing of this article, it was published that BMP9 could be a sensitive biomarker that segregates portopulmonary hypertension from other forms of PAH.28 Together, these results clearly support that BMP9 signaling pathway is involved in PAH development but also that the molecular mechanisms involved in PAH are complex.
BMP9 is mainly produced by the liver and circulates in blood under a biologically active form and has been proposed to act as a vascular quiescence factor.13 Several works also support a potential role for this pathway in the regulation of vascular tone. Indeed, it was shown by our group and others that BMP9 induces the expression of ET-1, a strong vasoconstrictor,14,15 and decreases the expression of apelin, a potent vasodilator for the pulmonary vasculature.16,17 We also previously showed that addition of BMP9 induced vasoconstriction in the chicken chorionallantoic membrane.13 So, one hypothesis that could explain why the loss of BMP9 protects from PH development is that BMP9 could act as a potent modulator of vasoconstriction in the pulmonary circulation. This hypothesis is consistent with the dilation phenotype observed in Alk1 ± or Eng ± mice29,30 or Alk1 mutants, vbg, in zebrafish.31 This hypothesis is further supported by the fact that we found that Bmp9−/− pups presented a partial reopening of the ductus arteriosus just after birth that could because of a defect in vasoconstriction.32 Interestingly, patent ductus arteriosus is a serious congenital heart disease that can lead to irreversible pulmonary hypertension.33 We confirm here the downregulation of apelin and show, for the first time, the downregulation of adrenomedullin by BMP9, 2 known critical actors that can impair pulmonary endothelial function.34,35
We also confirm here that BMP9 induces a dose-dependent ET-1 expression in human pulmonary ECs and regulate its lung production in rodents. However, although BMP9 and other BMPs have been shown to induce ET-1,14,15 unexpectedly BMPR2 downregulation is also associated with an increase in ET-1 expression,15,36 supporting the notion that BMP9 stimulated ET-1 production by pulmonary ECs is not sufficient alone to lead to PH development and strengthening the evidence for the importance of BMP receptor balance in the control of vascular tone.
The role of BMP9 has recently been studied in a bronchopulmonary dysplasia model induced by chronic exposure to hyperoxia in neonatal rats. Consistent with our present findings, BMP9 treatment has been found to improve aberrant alveolar development and reduced lung inflammation and fibrosis but did not improve aberrant vascularization (arterial medial wall thickness and muscularization) and right ventricular hypertrophy.37 In line with this notion, BMP9 and BMP10 can synergize with TNF-α (tumor necrosis factor α) to induce the upregulation of endothelial selectins and adhesion molecules and the secretion of various key proinflammatory mediators,38,39 and are, therefore, likely to contribute to the proinflammatory signature of the dysfunctional endothelium in PAH.40
In the present study, we provide definitive evidence that the selective BMP9 loss or inhibition partially prevent and protects against experimental pulmonary hypertension. In addition, this study offers important physiopathological insights into the role of BMP9 in the synthesis of potent vasoreactive factors by the pulmonary endothelium in vitro and in vivo, namely ET-1, apelin, and adrenomedullin and may have important implications for human PAH.
ADM | adrenomedullin |
ALK1 | activin receptor-like kinase 1 |
BMPR2 | bone morphogenetic protein receptor type 2 |
EC | endothelial cell |
ET-1 | endothelin-1 |
MCT | monocrotaline |
mPAP | mean pulmonary arterial pressure |
PAs | pulmonary arteries |
PAH | pulmonary arterial hypertension |
PCNA | proliferating cell nuclear antigen |
RV | right ventricle |
ROCK | Rho/Rho-associated protein kinase |
SuHx | sugen/hypoxia |
TPVR | total pulmonary vascular resistance |
Acknowledgments
We thank Dr M. Yan (Genentech Inc, San Francisco) for kindly providing anti-BMP9 and ALK1ECD reagents. We also thank Professor F. Soubrier and Dr M. Eyries (Laboratoire d’Oncogénétique et Angiogénétique Moléculaire, Groupe Hospitalier Pitié-Salpétrière, Paris, France) for the genetic analysis of pulmonary arterial hypertension patients.
Sources of Funding
This research was supported by grants from the French National Institute for Health and Medical Research (INSERM), the University of Grenoble, the University of Paris-Sud and the University Paris-Saclay, the Marie Lannelongue Hospital, the CEA (Commissariat à l’Energie Atomique et aux Energies Alternatives, Direction de la Recherche Fondamentale (DRF)/Institut de Biosciences et Biotechnologies de Grenoble (BIG)/Laboratoire Biologie du Cancer et de l’Infection (BCI)), the French National Agency for Research (ANR) grant no. ANR-17-CE14-0006 (Be9inPH), the Fondation de la Recherche Médicale (FRM) grant no. DEQ20150331712 (Equipe FRM 2015), and in part by the Département Hospitalo-Universitaire Thorax Innovation (TORINO), the Assistance Publique-Hôpitaux de Paris (AP-HP), Service de Pneumologie, Centre de Référence de l’Hypertension Pulmonaire Sévère, the LabEx Laboratoire d’Excellence en Recherche sur le Médicament et l’Innovation Thérapeutique (LERMIT; grant no ANR-10-LABX-0033), the French pulmonary arterial hypertension patient association (HTAP France) and the french Fonds de Dotation Recherche en Santé Respiratoire–(FRSR)–Fondation du Souffle (FdS), the Ligues Départementales contre le Cancer de la Loire et de la Savoie, the association Maladie de Rendu-Osler (AMRO-HHT France), the Association pour la Recherche sur le Cancer (ARC). C. Phan is supported by the FRSR–FdS and J. Bordenave is supported by the FRM.
Disclosures
In the past 3 years, M. Humbert and L. Savale report grants, personal fees, and nonfinancial support from Actelion, Pfizer, Bayer, and GlaxoSmithKline, MSD, outside of the submitted work. The other authors report no conflicts.
Footnotes
References
- 1.
Guignabert C, Dorfmüller P . Pathology and pathobiology of pulmonary hypertension.Semin Respir Crit Care Med. 2017; 38:571–584. doi: 10.1055/s-0037-1606214CrossrefMedlineGoogle Scholar - 2.
Guignabert C, Tu L, Girerd B, Ricard N, Huertas A, Montani D, Humbert M . New molecular targets of pulmonary vascular remodeling in pulmonary arterial hypertension: importance of endothelial communication.Chest. 2015; 147:529–537. doi: 10.1378/chest.14-0862CrossrefMedlineGoogle Scholar - 3.
Humbert M, Guignabert C, Bonnet S, Dorfmüller P, Klinger JR, Nicolls MR, Olschewski AJ, Pullamsetti SS, Schermuly RT, Stenmark KR, Rabinovitch M . Pathology and pathobiology of pulmonary hypertension: state of the art and research perspectives [published online December 13, 2018].Eur Respir J. doi: 10.1183/13993003.01887-2018Google Scholar - 4.
Galiè N, Humbert M, Vachiery JL, . 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: the joint task force for the diagnosis and treatment of pulmonary hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT).Eur Respir J. 2015; 46:903–975. doi: 10.1183/13993003.01032-2015CrossrefMedlineGoogle Scholar - 5.
Boucly A, Weatherald J, Savale L, . Risk assessment, prognosis and guideline implementation in pulmonary arterial hypertension.Eur Respir J. 2017; 50:1700889. doi: 10.1183/13993003.00889-2017CrossrefMedlineGoogle Scholar - 6.
Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, Knowles JA . Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene.Am J Hum Genet. 2000; 67:737–744. doi: 10.1086/303059CrossrefMedlineGoogle Scholar - 7.
Evans JD, Girerd B, Montani D, . BMPR2 mutations and survival in pulmonary arterial hypertension: an individual participant data meta-analysis.Lancet Respir Med. 2016; 4:129–137. doi: 10.1016/S2213-2600(15)00544-5CrossrefMedlineGoogle Scholar - 8.
Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA, Loyd JE, Nichols WC, Trembath RC . Heterozygous germline mutations in BMPR2, encoding a TGF-beta receptor, cause familial primary pulmonary hypertension.Nat Genet. 2000; 26:81–84. doi: 10.1038/79226CrossrefMedlineGoogle Scholar - 9.
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. doi: 10.1182/blood-2006-07-034124CrossrefMedlineGoogle Scholar - 10.
Goumans MJ, Zwijsen A, Ten Dijke P, Bailly S . Bone morphogenetic proteins in vascular homeostasis and disease.Cold Spring Harb Perspect Biol. 2018; 10:a031989. doi: 10.1101/cshperspect.a031989CrossrefMedlineGoogle Scholar - 11.
Guignabert C, Bailly S, Humbert M . Restoring BMPRII functions in pulmonary arterial hypertension: opportunities, challenges and limitations.Expert Opin Ther Targets. 2017; 21:181–190. doi: 10.1080/14728222.2017.1275567CrossrefMedlineGoogle Scholar - 12.
Li W, Salmon RM, Jiang H, Morrell NW . Regulation of the ALK1 ligands, BMP9 and BMP10.Biochem Soc Trans. 2016; 44:1135–1141. doi: 10.1042/BST20160083CrossrefMedlineGoogle Scholar - 13.
David L, Mallet C, Keramidas M, Lamandé N, Gasc JM, Dupuis-Girod S, Plauchu H, Feige JJ, Bailly S . Bone morphogenetic protein-9 is a circulating vascular quiescence factor.Circ Res. 2008; 102:914–922. doi: 10.1161/CIRCRESAHA.107.165530LinkGoogle Scholar - 14.
Star GP, Giovinazzo M, Langleben D . Bone morphogenic protein-9 stimulates endothelin-1 release from human pulmonary microvascular endothelial cells: a potential mechanism for elevated ET-1 levels in pulmonary arterial hypertension.Microvasc Res. 2010; 80:349–354. doi: 10.1016/j.mvr.2010.05.010CrossrefMedlineGoogle Scholar - 15.
Park JE, Shao D, Upton PD, Desouza P, Adcock IM, Davies RJ, Morrell NW, Griffiths MJ, Wort SJ . BMP-9 induced endothelial cell tubule formation and inhibition of migration involves Smad1 driven endothelin-1 production.PLoS One. 2012; 7:e30075. doi: 10.1371/journal.pone.0030075CrossrefMedlineGoogle Scholar - 16.
Poirier O, Ciumas M, Eyries M, Montagne K, Nadaud S, Soubrier F . Inhibition of apelin expression by BMP signaling in endothelial cells.Am J Physiol Cell Physiol. 2012; 303:C1139–C1145. doi: 10.1152/ajpcell.00168.2012CrossrefMedlineGoogle Scholar - 17.
Ricard N, Ciais D, Levet S, Subileau M, Mallet C, Zimmers TA, Lee SJ, Bidart M, Feige JJ, Bailly S . BMP9 and BMP10 are critical for postnatal retinal vascular remodeling.Blood. 2012; 119:6162–6171. doi: 10.1182/blood-2012-01-407593CrossrefMedlineGoogle Scholar - 18.
Levet S, Ciais D, Merdzhanova G, Mallet C, Zimmers TA, Lee SJ, Navarro FP, Texier I, Feige JJ, Bailly S, Vittet D . Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation.Blood. 2013; 122:598–607. doi: 10.1182/blood-2012-12-472142CrossrefMedlineGoogle Scholar - 19.
Gupta S, Gill D, Pal SK, Agarwal N . Activin receptor inhibitors–dalantercept.Curr Oncol Rep. 2015; 17:14. doi: 10.1007/s11912-015-0441-5CrossrefMedlineGoogle Scholar - 20.
Long L, Ormiston ML, Yang X, . Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension.Nat Med. 2015; 21:777–785. doi: 10.1038/nm.3877CrossrefMedlineGoogle Scholar - 21.
Gräf S, Haimel M, Bleda M, . Identification of rare sequence variation underlying heritable pulmonary arterial hypertension.Nat Commun. 2018; 9:1416. doi: 10.1038/s41467-018-03672-4CrossrefMedlineGoogle Scholar - 22.
Wang G, Fan R, Ji R, Zou W, Penny DJ, Varghese NP, Fan Y . Novel homozygous BMP9 nonsense mutation causes pulmonary arterial hypertension: a case report.BMC Pulm Med. 2016; 16:17. doi: 10.1186/s12890-016-0183-7CrossrefMedlineGoogle Scholar - 23.
Eyries M, Montani D, Nadaud S, . Widening the landscape of heritable pulmonary hypertension mutations in pediatric and adult cases. [published online December 21, 2018].Eur Respir J. 2018;1801371. doi: 10.1183/13993003.01371-2018CrossrefMedlineGoogle Scholar - 24.
Wang XJ, Lian TY, Jiang X, . Germline BMP9 mutation causes idiopathic pulmonary arterial hypertension. [published online December 21, 2018].Eur Respir J. 2018;1801609. doi: 10.1183/13993003.01609-2018CrossrefMedlineGoogle Scholar - 25.
Perron JC, Dodd J . ActRIIA and BMPRII Type II BMP receptor subunits selectively required for Smad4-independent BMP7-evoked chemotaxis.PLoS One. 2009; 4:e8198. doi: 10.1371/journal.pone.0008198CrossrefMedlineGoogle Scholar - 26.
Yu PB, Beppu H, Kawai N, Li E, Bloch KD . Bone morphogenetic protein (BMP) type II receptor deletion reveals BMP ligand-specific gain of signaling in pulmonary artery smooth muscle cells.J Biol Chem. 2005; 280:24443–24450. doi: 10.1074/jbc.M502825200CrossrefMedlineGoogle Scholar - 27.
Olsen OE, Sankar M, Elsaadi S, Hella H, Buene G, Darvekar SR, Misund K, Katagiri T, Knaus P, Holien T . BMPR2 inhibits activin and BMP signaling via wild-type ALK2.J Cell Sci. 2018; 131:jcs213512. doi: 10.1242/jcs.213512CrossrefMedlineGoogle Scholar - 28.
Nikolic I, Yung LM, Yang P, . Bone morphogenetic protein 9 is a mechanistic biomarker of portopulmonary hypertension. [published online October 12, 2018].Am J Respir Crit Care Med. 2018. doi: 10.1164/rccm.201807-1236OCCrossrefMedlineGoogle Scholar - 29.
Srinivasan S, Hanes MA, Dickens T, Porteous ME, Oh SP, Hale LP, Marchuk DA . A mouse model for hereditary hemorrhagic telangiectasia (HHT) type 2.Hum Mol Genet. 2003; 12:473–482.CrossrefMedlineGoogle Scholar - 30.
Torsney E, Charlton R, Diamond AG, Burn J, Soames JV, Arthur HM . Mouse model for hereditary hemorrhagic telangiectasia has a generalized vascular abnormality.Circulation. 2003; 107:1653–1657. doi: 10.1161/01.CIR.0000058170.92267.00LinkGoogle Scholar - 31.
Roman BL, Pham VN, Lawson ND, Kulik M, Childs S, Lekven AC, Garrity DM, Moon RT, Fishman MC, Lechleider RJ, Weinstein BM . Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels.Development. 2002; 129:3009–3019.CrossrefMedlineGoogle Scholar - 32.
Levet S, Ouarné M, Ciais D, Coutton C, Subileau M, Mallet C, Ricard N, Bidart M, Debillon T, Faravelli F, Rooryck C, Feige JJ, Tillet E, Bailly S . BMP9 and BMP10 are necessary for proper closure of the ductus arteriosus.Proc Natl Acad Sci USA. 2015; 112:E3207–E3215. doi: 10.1073/pnas.1508386112CrossrefMedlineGoogle Scholar - 33.
Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, Gomez Sanchez MA, Krishna Kumar R, Landzberg M, Machado RF, Olschewski H, Robbins IM, Souza R . Updated clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2013; 62:D34–D41. doi: 10.1016/j.jacc.2013.10.029CrossrefMedlineGoogle Scholar - 34.
Telli G, Tel BC, Yersal N, Korkusuz P, Gumusel B . Effect of intermedin/adrenomedullin2 on the pulmonary vascular bed in hypoxia-induced pulmonary hypertensive rats.Life Sci. 2018; 192:62–67. doi: 10.1016/j.lfs.2017.11.031CrossrefMedlineGoogle Scholar - 35.
Alastalo TP, Li M, Perez Vde J, Pham D, Sawada H, Wang JK, Koskenvuo M, Wang L, Freeman BA, Chang HY, Rabinovitch M . Disruption of PPARγ/β-catenin-mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival.J Clin Invest. 2011; 121:3735–3746. doi: 10.1172/JCI43382CrossrefMedlineGoogle Scholar - 36.
Star GP, Giovinazzo M, Langleben D . ALK2 and BMPR2 knockdown and endothelin-1 production by pulmonary microvascular endothelial cells.Microvasc Res. 2013; 85:46–53. doi: 10.1016/j.mvr.2012.10.012CrossrefMedlineGoogle Scholar - 37.
Chen X, Orriols M, Walther FJ, Laghmani EH, Hoogeboom AM, Hogen-Esch ACB, Hiemstra PS, Folkerts G, Goumans MTH, Ten Dijke P, Morrell NW, Wagenaar GTM . Bone morphogenetic protein 9 protects against neonatal hyperoxia-induced impairment of alveolarization and pulmonary inflammation.Front Physiol. 2017; 8:486. doi: 10.3389/fphys.2017.00486CrossrefMedlineGoogle Scholar - 38.
Mitrofan CG, Appleby SL, Nash GB, Mallat Z, Chilvers ER, Upton PD, Morrell NW . Bone morphogenetic protein 9 (BMP9) and BMP10 enhance tumor necrosis factor-α-induced monocyte recruitment to the vascular endothelium mainly via activin receptor-like kinase 2.J Biol Chem. 2017; 292:13714–13726. doi: 10.1074/jbc.M117.778506CrossrefMedlineGoogle Scholar - 39.
Young K, Tweedie E, Conley B, Ames J, FitzSimons M, Brooks P, Liaw L, Vary CP . BMP9 Crosstalk with the hippo pathway regulates endothelial cell matricellular and chemokine responses.PLoS One. 2015; 10:e0122892. doi: 10.1371/journal.pone.0122892CrossrefMedlineGoogle Scholar - 40.
Le Hiress M, Tu L, Ricard N, Phan C, Thuillet R, Fadel E, Dorfmüller P, Montani D, de Man F, Humbert M, Huertas A, Guignabert C . Proinflammatory signature of the dysfunctional endothelium in pulmonary hypertension. role of the macrophage migration inhibitory factor/CD74 complex.Am J Respir Crit Care Med. 2015; 192:983–997. doi: 10.1164/rccm.201402-0322OCCrossrefMedlineGoogle Scholar
Novelty and Significance
What Is Known?
Autosomal dominant mutations in the BMPR2 (type 2 BMP receptor), ACVLR1 (ALK1), GDF2 (BMP9), and in the BMP10 genes predispose to heritable pulmonary arterial hypertension.
BMP9 and BMP10 are 2 high affinity ligands for ALK1 (activin receptor-like kinase 1) and BMPRII present in a heterotetrameric complex on pulmonary endothelial cells.
Perturbation in the BMP9/BMP10 signaling pathway have emerged as essential in endothelial (dys)function and vascular remodeling.
What New Information Does This Article Contribute?
Using 3 different approaches of suppressing BMP9 action in rodents, we provide evidence that the selective loss or inhibition of BMP9 does not predispose, but partially prevent or protect against experimental pulmonary hypertension.
We show that BMP9 regulates the endothelial synthesis/release of potent vasoreactive factors in vitro and in vivo, namely endothelin-1, apelin, and adrenomedullin.
These findings offer new insight into the complexity of endothelial BMP9 signaling and may have important implications for human pulmonary arterial hypertension. Our data support the notion that blockade of BMP9 signaling can protect the pulmonary endothelium and attenuate the structural and functional remodeling of the lung vasculature. Further studies are needed to better understand the differences between experimental models and the human predisposition to pulmonary arterial hypertension identified in subjects carrying BMP9 germline mutations. The apparent ambivalent role of the BMP9/BMP10 signaling pathway should stimulate work to better understand this feature and the underlying mechanisms.
eLetters(0)
eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.
Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.