Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

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.

Bone Morphogenetic Protein 9 Is a Mechanistic Biomarker of Portopulmonary Hypertension.

RATIONALE: BMP9 (bone morphogenetic protein 9) is a circulating endothelial quiescence factor with protective effects in pulmonary arterial hypertension (PAH). Loss-of-function mutations in BMP9, its receptors, and downstream effectors have been reported in heritable PAH. OBJECTIVES: To determine how an acquired deficiency of BMP9 signaling might contribute to PAH. METHODS: Plasma levels of BMP9 and antagonist soluble endoglin were measured in group 1 PAH, group 2 and 3 pulmonary hypertension (PH), and in patients with severe liver disease without PAH. MEASUREMENTS AND MAIN RESULTS: BMP9 levels were markedly lower in portopulmonary hypertension (PoPH) versus healthy control subjects, or other etiologies of PAH or PH; distinguished PoPH from patients with liver disease without PAH; and were an independent predictor of transplant-free survival. BMP9 levels were decreased in mice with PH associated with CCl4-induced portal hypertension and liver cirrhosis, but were normal in other rodent models of PH. Administration of ALK1-Fc, a BMP9 ligand trap consisting of the activin receptor-like kinase-1 extracellular domain, exacerbated PH and pulmonary vascular remodeling in mice treated with hypoxia versus hypoxia alone. CONCLUSIONS: BMP9 is a sensitive and specific biomarker of PoPH, predicting transplant-free survival and the presence of PAH in liver disease. In rodent models, acquired deficiency of BMP9 signaling can predispose to or exacerbate PH, providing a possible mechanistic link between PoPH and heritable PAH. These findings describe a novel experimental model of severe PH that provides insight into the synergy between pulmonary vascular injury and diminished BMP9 signaling in the pathogenesis of PAH.

A self-amplifying loop of YAP and SHH drives formation and expansion of heterotopic ossification.

Heterotopic ossification (HO) occurs as a common complication after injury or in genetic disorders. The mechanisms underlying HO remain incompletely understood, and there are no approved prophylactic or secondary treatments available. Here, we identify a self-amplifying, self-propagating loop of Yes-associated protein (YAP)-Sonic hedgehog (SHH) as a core molecular mechanism underlying diverse forms of HO. In mouse models of progressive osseous heteroplasia (POH), a disease caused by null mutations in GNAS, we found that Gnas-/- mesenchymal cells secreted SHH, which induced osteoblast differentiation of the surrounding wild-type cells. We further showed that loss of Gnas led to activation of YAP transcription activity, which directly drove Shh expression. Secreted SHH further induced YAP activation, Shh expression, and osteoblast differentiation in surrounding wild-type cells. This self-propagating positive feedback loop was both necessary and sufficient for HO expansion and could act independently of Gnas in fibrodysplasia ossificans progressiva (FOP), another genetic HO, and nonhereditary HO mouse models. Genetic or pharmacological inhibition of YAP or SHH abolished HO in POH and FOP and acquired HO mouse models without affecting normal bone homeostasis, providing a previously unrecognized therapeutic rationale to prevent, reduce, and shrink HO.

Saracatinib is an efficacious clinical candidate for fibrodysplasia ossificans progressiva.

Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues owing to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2 (also known as ACVR1). From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2, compared with other receptors of the BMP/TGF-ß signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D-transgenic mouse line, which provides a model of heterotopic ossification (HO), as well as an inducible ACVR1R206H-knockin mouse, which serves as a genetically and physiologically faithful FOP model. In both models, saracatinib was well tolerated and potently inhibited the development of HO, even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in FOP treatment, offering an accelerated path to clinical proof-of-efficacy studies and potentially significant benefits to individuals with this devastating condition.

A novel tissue separation method for determining upper trunk center of gravity in patients with thoracolumbar kyphosis using MIMICS.

BACKGROUND: In the past, a variety of methods have been explored to determine the center of gravity or gravity line, such as the hanging method or force plate analysis. However, these methods possess limitations related to precise location of center of gravity. The aim of this study was to describe a tissue separation method to precisely calculate the center of gravity in upper trunk. METHODS: Twenty post-operative patients with thoracolumbar kyphosis were retrospectively studied. Center of gravity models were computed: T1-T5 segment, T6-T10 segment and T1-T10 segment. The tissue separation method was used to calculate the center of gravity in each segment. A new center of gravity was composited from T1-T5 segment and T6-T10 segment by composition formula. Similarity and collinearity between center of gravity models was analyzed to verify the reliability of tissue separation method. Correlation between gravity line and theoretic hip axis was compared on pre- and post-operative radiographs to explore their potential application for surgical plan. FINDINGS: Composited center of gravity had significant correlation and high similarity with center of gravity in T1-T10 segment. There was high collinearity between center of gravity points. The post-operative included angle between the gravity line and theoretic hip axis significantly decreased to nearly 0°. INTERPRETATION: These findings demonstrate the MIMICS can reliably calculate the center of gravity in the upper trunk by a tissue separation method. The pre-operative included angle between the gravity line and theoretic hip axis was suggested as a parameter to improve surgical design for thoracolumbar kyphosis correction.

ACTRIIA-Fc rebalances activin/GDF versus BMP signaling in pulmonary hypertension.

Human genetics, biomarker, and animal studies implicate loss of function in bone morphogenetic protein (BMP) signaling and maladaptive transforming growth factor-ß (TGFß) signaling as drivers of pulmonary arterial hypertension (PAH). Although sharing common receptors and effectors with BMP/TGFß, the function of activin and growth and differentiation factor (GDF) ligands in PAH are less well defined. Increased expression of GDF8, GDF11, and activin A was detected in lung lesions from humans with PAH and experimental rodent models of pulmonary hypertension (PH). ACTRIIA-Fc, a potent GDF8/11 and activin ligand trap, was used to test the roles of these ligands in animal and cellular models of PH. By blocking GDF8/11- and activin-mediated SMAD2/3 activation in vascular cells, ACTRIIA-Fc attenuated proliferation of pulmonary arterial smooth muscle cells and pulmonary microvascular endothelial cells. In several experimental models of PH, prophylactic administration of ACTRIIA-Fc markedly improved hemodynamics, right ventricular (RV) hypertrophy, RV function, and arteriolar remodeling. When administered after the establishment of hemodynamically severe PH in a vasculoproliferative model, ACTRIIA-Fc was more effective than vasodilator in attenuating PH and arteriolar remodeling. Potent antiremodeling effects of ACTRIIA-Fc were associated with inhibition of SMAD2/3 activation and downstream transcriptional activity, inhibition of proliferation, and enhancement of apoptosis in the vascular wall. ACTRIIA-Fc reveals an unexpectedly prominent role of GDF8, GDF11, and activin as drivers of pulmonary vascular disease and represents a therapeutic strategy for restoring the balance between SMAD1/5/9 and SMAD2/3 signaling in PAH.

Pharmacologic Strategies for Assaying BMP Signaling Function.

The bone morphogenetic protein (BMP) signaling pathway, a subset of the transforming growth factor ß (TGF-ß) signaling family, consists of structurally diverse receptors and ligands whose combinatorial specificity encodes autocrine, paracrine, and endocrine signals essential for regulating tissue growth, differentiation, and survival during embryonic patterning and postnatal tissue remodeling. Aberrant signaling of these receptors and ligands is implicated in a variety of inborn and acquired diseases. The roles of various receptors and their ligands can be explored using small molecule inhibitors of the BMP receptor kinases. Several BMP type I receptor kinase inhibitor tool compounds have been described that exhibit sufficient selectivity to discriminate BMP receptor signaling in vitro or in vivo, with various trade-offs in selectivity, potency, cell permeability, and pharmacokinetics. Several methods for assaying BMP function via pharmacologic inhibition are presented. Two in vitro methods, an In-Cell Western assay of BMP-mediated SMAD1/5/8 phosphorylation and an alkaline phosphatase osteogenic differentiation assay, represent efficient high-throughput methodologies for assaying pharmacologic inhibitors. Two in vivo methods are described for assaying the effects of BMP signaling inhibition in embryonic zebrafish and mouse development. Small molecule inhibitors of BMP receptor kinases represent an important complementary strategy to genetic gain- and loss-of-function and ligand-trap approaches for targeting this signaling system in biology and disease.