Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.

Reduced bone morphogenetic protein receptor 2 (BMPR2) expression in patients with pulmonary arterial hypertension (PAH) can impair pulmonary arterial EC (PAEC) function. This can adversely affect EC survival and promote SMC proliferation. We hypothesized that interventions to normalize expression of genes that are targets of BMPR2 signaling could restore PAEC function and prevent or reverse PAH. Here we have characterized, in human PAECs, a BMPR2-mediated transcriptional complex between PPARγ and β-catenin and shown that disruption of this complex impaired BMP-mediated PAEC survival. Using whole genome-wide ChIP-Chip promoter analysis and gene expression microarrays, we delineated PPARγ/β-catenin–dependent transcription of target genes including APLN, which encodes apelin. We documented reduced PAEC expression of apelin in PAH patients versus controls. In cell culture experiments, we showed that apelin-deficient PAECs were prone to apoptosis and promoted pulmonary arterial SMC (PASMC) proliferation. Conversely, we established that apelin, like BMPR2 ligands, suppressed proliferation and induced apoptosis of PASMCs. Consistent with these functions, administration of apelin reversed PAH in mice with reduced production of apelin resulting from deletion of PPARγ in ECs. Taken together, our findings suggest that apelin could be effective in treating PAH by rescuing BMPR2 and PAEC dysfunction.

Rationale: Pulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown. Objectives: To determine the mechanisms by which autophagic proteins regulate tissue responses during PH. Methods: Lungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B−/−), or early growth response-1 (Egr-1−/−), were evaluated for vascular morphology and hemodynamics. Measurements and Main Results: Human PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B−/− or Egr-1−/−, but not Beclin 1+/−, mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia. Conclusions: The results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B−/− and Egr-1−/− mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.

Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34–PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)–positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

Background— Coronary sequelae that persist after Kawasaki disease (KD) have been associated with obstructive changes of the lesions and coronary vascular events in adolescents and young adults. However, little is known about the association between sequelae late after KD and inflammatory markers, which are potential mediators and markers for atherogenesis. Methods and Results— Cross-sectional study was performed to test the hypothesis that coronary sequelae are associated with elevated levels of inflammatory markers in patients late after KD (mean time interval after the onset, 10 years, 10 months). Levels of high-sensitivity C-reactive protein (CRP), serum amyloid-A (SAA), interleukin-6, and soluble intercellular adhesion molecule-1 were measured in the 4 groups (n=80): the referent group (n=15) and KD subgroups with normal coronary arteries from the onset (n=27); with regressed aneurysms (n=18); and with coronary artery lesions, such as persistent aneurysms, stenosis, and occlusion (n=20). CRP levels were significantly elevated in a KD subgroup with coronary artery lesions compared with the referent or other KD subgroups, as analyzed by ANOVA and ANCOVA after adjustment for a confounding factor body mass index. Levels of CRP, SAA, and interleukin-6 were positively correlated. Stepwise regression and logistic regression analyses support the association between the persistence of coronary artery lesions and the levels of CRP and SAA. Conclusions— Results demonstrate that the persistence of coronary lesions late after KD was independently associated with levels of CRP and SAA, suggesting that inflammation may be a novel functional aspect of coronary artery diseases late after KD.