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), underlies most heritable forms of PAH. To identify the missing heritability we perform whole-genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses reveal significant overrepresentation of rare variants in ATP13A3, AQP1 and SOX17, and provide independent validation of a critical role for GDF2 in PAH. We demonstrate familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, lead to reduced secretion from transfected cells. In addition, we identify pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these findings contribute new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention.
provides new genetic evidence for the association of GDF2 mutations with PAH and confirms that these mutations cause impaired BMP9 function
Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.
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.
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.
Background: Bone morphogenetic protein 9 (BMP9) circulates at low concentrations maintaining endothelial integrity. BMP9 has potent bone-forming activity at high concentrations.Results: The intermolecular disulfide bond in BMP9 is regulated by redox potential; the loss of which renders BMP9 susceptible to degradation by proteases present in serum.Conclusion: BMP9 is regulated by redox-dependent proteolysis.Significance: Optimum circulating BMP9 levels are essential for endothelium-specific activity.
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.
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 PAHnegative 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.
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