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Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

RationalePulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS), which carries a high mortality. Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.ObjectiveTo determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS)-induced lung inflammation and permeability.MethodsA BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human primary lung endothelial cells. Impact of BMP9 was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and endotoxemic mice.Main ResultsSubacute neutralization of endogenous BMP9 in mice resulted in increased lung vascular permeability, interstitial edema and neutrophil extravasation. In lung endothelial cells, BMP9 regulated a programme of gene expression and pathways controlling vascular permeability and cell membrane integrity. Augmentation of BMP9 signalling in mice with exogenous BMP9 prevented inhaled LPS-caused lung injury and edema. In endotoxemic mice, endogenous BMP9 levels were markedly reduced, due to a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human sepsis patients, circulating levels of BMP9 were also markedly reduced.ConclusionsEndogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Supplementation with exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in the setting of lung injury.Short summaryScientific Knowledge on the SubjectIncreased pulmonary endothelial permeability is a major factor in the development of acute respiratory distress syndrome (ARDS). Evidence is emerging that circulating BMP9, secreted from the liver, might protect the pulmonary endothelium from injury. For example, loss of BMP9 levels or signalling receptor contributes to the development of pulmonary arterial hypertension. The role of endogenous BMP9 in endothelial permeability remains unclear.What This Study Adds to the FieldHere we show that subacute neutralization of endogenous BMP9 leads to lung vascular injury, including enhanced permeability and neutrophil extravasation. BMP9 levels were markedly reduced in the setting of inflammation in mice and humans. Conversely, exogenous supplementation of BMP9 protected the lung from LPS-induced injury. This study suggests that exogenous BMP9 could offer a novel approach to prevent increased pulmonary endothelial permeability in the setting of lung injury and ARDS.

International audience; Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor.Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH.Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260).Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations.Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor. Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH. Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260). Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations. Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

Bone morphogenetic protein (BMP)9 and BMP10 are high affinity ligands for activin receptor-like kinase 1 (ALK1), a type I BMP receptor mainly expressed on vascular endothelial cells (ECs). ALK1-mediated BMP9/BMP10 signalling pathways have emerged as essential in EC biology and in angiogenesis. Several genetic mutations in the genes encoding the ligands and receptors of this pathway have been reported in two cardiovascular diseases, pulmonary arterial hypertension (PAH) and hereditary haemorrhagic telangiectasia (HHT). Administration of recombinant BMP9 reverses experimental PAH in preclinical rodent models. Dalantercept, an Fc-fusion protein of the extracellular domain of ALK1 and a ligand trap for BMP9 and BMP10, is in phase II clinical trials for anti-tumour angiogenesis. Understanding the regulation of BMP9 and BMP10, at both gene and protein levels, under physiological and pathological conditions, will reveal essential information and potential novel prognostic markers for the BMP9/BMP10-targeted therapies.

Introduction and objective Heightened endothelial cell permeability is a feature of life-threatening conditions such as sepsis and acute respiratory distress syndrome. However, there is a lack of pharmacological therapies targeting this pathological hyper-permeability. Bone morphogenetic protein 9 (BMP9) is a circulating vascular quiescence factor, signalling on vascular endothelial cells (ECs) through activin receptor-like kinase 1 (ALK1) and BMP receptor type II (BMPRII). Since recombinant BMP9 affords protection from endothelial permeability induced by inflammatory mediators, our objective was to investigate whether endogenous BMP9 plays a constitutive role in maintaining endothelial barrier function. Methods Wild-type mice were treated with BMP9 neutralising antibody at 5 mg/kg to assess: 1) rapid vascular leak using live imaging in mouse cremaster muscle and 2) more sustained vascular leak using Evans Blue extravasation, neutrophil accumulation and lung histology. Changes in endogenous BMP9 during LPS-induced endotoxemia were assessed by mRNA expression in the liver and protein levels in circulation. Anti-BMP9 effects on endothelial VE-cadherin were assessed by immunostaining. BMP9 signalling on EC receptors was profiled using microarray. Effects of BMP9 administration were investigated using an intranasal LPS-induced lung injury model. Results Selective inhibition of circulating BMP9 alone resulted in a rapid ( Conclusions Endogenous circulating BMP9 plays a constitutive role in maintaining lung endothelial barrier function. Remarkably, inhibition of BMP9 alone was sufficient to increase lung vascular permeability and promote neutrophil extravasation. The reduction in circulating BMP9 associated with LPS-induced inflammation, and the rescue of lung permeability with exogenous BMP9 suggests that supplementation of this factor may be a useful therapeutic approach in conditions associated with lung endothelial injury.

Most heritable cases of pulmonary arterial hypertension (PAH) are explained by mutations in BMPR2, which encodes the bone morphogenetic protein type 2 receptor (BMPR-II). Additionally, mutations in the endothelial type 1 receptor, ALK1, can cause PAH. Growth and differentiation factor 2 (GDF2; also known as BMP9) is a circulating vascular quiescence factor that possesses high affinity for the BMPR-II/ALK1 complex. Furthermore, exogenous GDF2 administration reverses PAH in animal models. Recently, an unbiased case-control analysis revealed rare missense mutations in GDF2 in PAH cases.1 Here we determined the functional impact and pathogenicity of disease-associated mutations in GDF2. Sixteen GDF2 missense variants were generated in an overexpression system and characterised by ELISAs, western blotting, signalling and apoptosis assays. BMP9 and BMP10 levels were assayed in plasma samples from PAH patients harbouring GDF2 missense variants and deletions at the GDF2 locus, and age/sex matched healthy controls. Missense variants predicted to be pathogenic by in silico analysis exhibited severely reduced secretion and activity of GDF2 in vitro. For most variants, reduced secretion was associated with disruption of the interaction between the prodomain and growth factor domain of GDF2, leading to destabilisation of the molecule. In contrast, missense variants predicted to be benign exhibited normal signalling and functional responses. Similarly, plasma from pathogenic GDF2 mutation carriers exhibited greatly reduced levels of the GDF2 growth factor domain compared to healthy controls and benign missense carriers. Intriguingly, plasma levels of BMP10, the other circulating ligand for ALK1/BMPR-II, were also markedly reduced in patients with pathogenic GDF2 variants, suggesting co-regulation of these circulating BMPs. These studies provide evidence for the pathogenicity of rare missense mutations in GDF2 and demonstrate that the major mechanism involves destabilisation of the GDF2 molecule leading to reduced secretion of the growth factor domain. These data provide support for the central role of the GDF2/BMPR-II/ALK1 axis in PAH and suggest that GDF2 replacement may be an effective therapeutic strategy in these individuals. This study was produced by the Morrell group on behalf of the UK PAH Cohort Study. Reference Nat Commun 12 April 2018;9(1):1416. doi:10.1038/s41467-018-03672-4

Pulmonary arterial hypertension (PAH) is a rare disorder with a poor prognosis. Deleterious variation within components of the transforming growth factor-β pathway, particularly the bone morphogenetic protein type 2 receptor (BMPR2), underlie most heritable forms of PAH. Since the missing heritability likely involves genetic variation confined to small numbers of cases, we performed whole genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses revealed significant overrepresentation of rare variants in novel genes, namely ATP13A3, AQP1 and SOX17, and provided independent validation of a critical role for GDF2 in PAH. We provide evidence for familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, led to reduced secretion from transfected cells. In addition, we identified pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these findings provide new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention.

BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling.

Introduction A critical reduction of bone morphogenetic protein type II receptor (BMPRII) in the pulmonary circulation, either due to the genetic loss-of-function mutations, heightened inflammation or prolonged hypoxia, is one of the major causes behind pulmonary arterial hypertension (PAH), a fatal disease with poor prognosis. BMPRII is highly expressed in the vascular endothelium and undergoes rapid turnover. Bone morphogenetic protein 9 (BMP9), the only active circulating BMP, signals via endothelial BMPRII, inducing BMPRII expression and maintaining endothelial homeostasis. Although BMPRII function has been studied extensively, factors that regulate BMP9 stability and activity remain unclear. Objective To investigate how BMP9 activity and stability are regulated and whether this regulation plays a role in pulmonary arterial hypertension. Results Two forms of BMP9 dimer could be co-purified, with (D-form) or without (M-form) intermolecular disulphide bond. M- and D-forms BMP9 are interchangeable with redox potential, but have different stability. While the M-form is more susceptible to redox-dependent cleavage and proteases present in serum, the D-form is a preferred substrate for neutrophil elastase. Freshly isolated human peripheral blood neutrophils, when activated by hypoxia or inflammatory stimuli, released elastase that cleaved BMP9 effectively. Conclusions and Discussions This study demonstrates a novel proteolytic regulation of BMP9 under physiological and pathological conditions, suggesting neutrophil elastase could be a potential link between inflammation/hypoxia and BMPRII signalling, and the recognised benefits of elastase inhibition in rodent models of PAH may be due in part to reduced degradation of BMP9 and preservation of endothelial BMPR-II signalling.