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.

Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1 -/-mice were absent in Cav1 -/-Nos3 -/-mice. Further, treatment of Cav1 -/-mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1 -/-lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1 -/-lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration-dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.

Background Vascular occlusion and complex plexiform lesions are hallmarks of the pathology of severe pulmonary arterial hypertension (PAH) in patients. However, mechanisms of obliterative vascular remodeling remain elusive and hence current therapies have not targeted the fundamental disease modifying mechanisms and result in only modest improvement in morbidity and mortality. Methods and Results Mice with Tie2Cre-mediated disruption of Egln1 (encoding prolyl-4 hydroxylase 2, PHD2) (Egln1Tie2) in endothelial cells (ECs) and hematopoietic cells exhibited spontaneous severe PAH with extensive pulmonary vascular remodeling including vascular occlusion and plexiform-like lesions resembling the hallmarks of the pathology of clinical PAH. As seen in idiopathic PAH patients, Egln1Tie2 mice exhibited unprecedented right ventricular hypertrophy and failure and progressive mortality. Consistently, PHD2 expression was diminished in lung ECs of obliterated pulmonary vessels in idiopathic PAH patients. Genetic deletions of both Egln1 and Hif1a or Egln1 and Hif2a identified hypoxia-inducible factor-2α (HIF-2α) as the critical mediator of severe PAH seen in Egln1Tie2 mice. We also observed altered expression of many PH-causing genes in Egln1Tie2 lungs which was also normalized in Egln1Tie2/Hif2aTie2 lungs. PHD2-deficient ECs promoted smooth muscle cell proliferation in part through HIF-2α-activated CXCL12 expression. Genetic deletion of Cxcl12 attenuated PAH in Egln1Tie2 mice. Conclusions These studies defined an unexpected role of PHD2 deficiency in the mechanisms of severe PAH and identified the first genetically modified mouse model with obliterative vascular remodeling and pathophysiology recapitulating clinical PAH. Thus, targeting PHD2/HIF-2α signaling is a promising strategy to reverse vascular remodeling for treatment of severe PAH.

Iron deficiency is common in IPAH patients and associated with disease severity and poor clinical outcome. Inappropriately raised hepcidin levels, which impair iron absorption from the gut, may be a factor.

Phosphodiesterase Type 5 is the main factor regulating cyclic guanosine monophosphate hydrolysis and downstream signaling in human PASMCs. The antiproliferative effects of this signaling pathway may be significant in the chronic treatment of pulmonary hypertension with PDE5 inhibitors such as sildenafil.

We determined the distribution of ET(A) and ET(B) receptors in pulmonary arteries from pulmonary hypertensive patients and control subjects, using in vitro autoradiography, and investigated their role in mediating the proliferative effects of endothelin-1 (ET-1) on distal human pulmonary artery smooth muscle cells (PASMCs). Distal arteries possessed more medial [(125)I]-ET-1 binding sites (105 +/- 10 versus 45 +/- 6 amol/mm(2); p < 0.001) and a greater proportion of ET(B) receptors than proximal arteries (36 +/- 3% versus 3 +/- 1%; p < 0.001). Receptor density in distal arteries and lung parenchyma was twofold greater (p < 0.05) in pulmonary hypertensive patients than in control subjects. ET-1 (10(-9)-10(-7) mol/L) stimulated DNA synthesis (147 +/- 10% of control subjects; p < 0.05) and attenuated the antiproliferative action of cicaprost and forskolin on PASMCs, these effects being mediated via ET(A) and ET(B) receptors. Serum-stimulated proliferation was attenuated by inhibiting either endogenous ET-1 release with phosphoramidon (10(-5) mol/L) or its action with PD145065 (10(-5) mol/L). Cicaprost (10(-10)-10(-7) mol/L) inhibited ET-1 release from PASMCs (49 +/- 16% of control after 24 h; p < 0.001) and increased intracellular cAMP levels, whereas ET(B) receptor stimulation selectively reduced cAMP levels. In conclusion, ET(A) and ET(B) receptors are differentially distributed in human pulmonary arteries. Both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in pulmonary hypertension.

Background-Phosphodiesterase type 5 (PDE5) is a novel therapeutic target for the treatment of pulmonary hypertension.This study examined the distribution of PDE5 in normal and hypoxic lung and the effect of chronic PDE5 inhibition with sildenafil, initiated before and during exposure to hypoxia, on pulmonary artery pressure (PAP) and structure. Methods and Results-Sprague-Dawley rats were exposed to hypoxia (10% O 2 ) for up to 42 days. PAP, measured continuously by telemetry, increased gradually by 20 to 40 mm Hg, reaching a plateau between 10 and 14 days, and declined to normal levels on return to normoxia. PDE5 immunoreactivity was localized to smooth muscle cells in the medial layer of pulmonary arteries and veins in the normal lung and in distal muscularized arteries (Ͻ25 m diameter) after hypoxia-induced pulmonary hypertension. Sildenafil (25 or 75 mg · kg Ϫ1 · d

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