Genetic evidence implicates the loss of bone morphogenetic protein type II receptor (BMPR-II) signaling in the endothelium as an initiating factor in pulmonary arterial hypertension (PAH). However, selective targeting of this signaling pathway using BMP ligands has not yet been explored as a therapeutic strategy. We identified BMP9 as the preferred ligand for preventing apoptosis and enhancing monolayer integrity in both pulmonary arterial endothelial cells and blood outgrowth endothelial cells from subjects with PAH bearing mutations in BMPR-II. In vivo, we report the spontaneous generation of PAH in a mouse model bearing a heterozygous knock-in of a human BMPR-II mutation, R899X. Administration of BMP9 reversed established PAH in Bmpr2+/R899X mice, as well as in models of disease developed in response to either monocrotaline or VEGF receptor inhibition combined with chronic hypoxia. These results demonstrate the promise of direct enhancement of endothelial BMP signaling as a novel therapeutic strategy for PAH.

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.

Abstract-Mutations in the bone morphogenetic protein type II receptor gene (BMPR2) are the major genetic cause of familial pulmonary arterial hypertension (FPAH). Although smooth muscle cell proliferation contributes to the vascular remodeling observed in PAH, the role of BMPs in this process and the impact of BMPR2 mutation remains unclear. Studies involving normal human pulmonary artery smooth muscle cells (PASMCs) suggest site-specific responses to BMPs. Thus, BMP-4 inhibited proliferation of PASMCs isolated from proximal pulmonary arteries, but stimulated proliferation of PASMCs from peripheral arteries, and conferred protection from apoptosis. These differences were not caused by differential activation of BMP signaling pathways because exogenous BMP-4 led to phosphorylation of Smad1, p38 MAPK , and ERK1/2 in both cell types. However, the proproliferative effect of BMP-4 on peripheral PASMCs was found to be p38 MAPK /ERK-dependent. Conversely, overexpression of dominant-negative Smad1 converted the response to BMP-4 in proximal PASMCs from inhibitory to proliferative. Furthermore, we confirmed that proximal PASMCs harboring kinase domain mutations in BMPR2 are deficient in Smad signaling and are unresponsive to the growth suppressive effect of BMP-4. Moreover, we show that the pulmonary vasculature of patients with familial and idiopathic PAH are deficient in the activated form of Smad1. We conclude that defective Smad signaling and unopposed p38 MAPK /ERK signaling, as a consequence of mutation in BMPR2, underlie the abnormal vascular cell proliferation observed in familial PAH. Key Words: vascular remodeling Ⅲ pulmonary hypertension Ⅲ transforming growth factor-␤ Ⅲ smooth muscle cells Ⅲ cell signaling P rimary, or idiopathic, pulmonary arterial hypertension (IPAH) is a rare disorder that is progressive and often fatal, leading to death within a median of 3 years from right ventricular failure without treatment. 1 The disease is characterized by vascular cell proliferation and obliteration of small pulmonary arteries by smooth muscle cells and myofibroblasts. 2 In addition, plexiform lesions comprising endothelial cells and myofibroblasts are found in Ϸ50% of cases. 3 Genetic studies have revealed heterozygous mutations in the BMPR2 gene encoding the type II bone morphogenetic protein receptor (BMPR-II), a member of the TGF-␤ superfamily of receptors, 4,5 underlying the familial form of the disease (FPAH). Subsequently, BMPR2 mutations were found in Ϸ25% of apparently sporadic cases of IPAH, many of which are examples of familial transmission with low disease gene penetrance. 6,7 Although these genetic studies point toward a critical role for the TGF-␤ superfamily in the regulation of pulmonary vascular cell growth and differentiation, the precise molecular mechanisms leading to disease pathogenesis remain to be elucidated.Signaling by BMP receptors involves heterodimerization of 2 transmembrane serine/threonine kinases: the constitutively active type II receptor, BMPR-II, and a corresponding type I re...

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