Hirofumi Sawada

FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension

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.

Disruption of PPARγ/β-catenin–mediated regulation of apelin impairs BMP-induced mouse and human pulmonary arterial EC survival

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.

Autophagic Protein LC3B Confers Resistance against Hypoxia-induced Pulmonary Hypertension

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.

Tie2-mediated loss of peroxisome proliferator-activated receptor-γ in mice causes PDGF receptor-β-dependent pulmonary arterial muscularization

Peroxisome proliferator-activated receptor (PPAR)-gamma is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPARgamma in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPARgamma in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPARgamma(flox/flox) mice to induce EC loss of PPARgamma (Tie2 PPARgamma(-/-)), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPARgamma(-/-) mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPARgamma(-/-) mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPARgamma(-/-) vs. WT mice in RA had increased platelet-derived growth factor receptor-beta (PDGF-Rbeta) expression and signaling, despite an elevation in the PPARgamma target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rbeta signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPARgamma(-/-) mice. Thus the disruption of PPARgamma signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rbeta signaling is sufficient to reverse PAH in this genetic model.

Elevated Levels of High-Sensitivity C-Reactive Protein and Serum Amyloid-A Late After Kawasaki Disease: Association Between Inflammation and Late Coronary Sequelae in Kawasaki Disease

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.

Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension

Reduced expression of bone morphogenetic protein receptor 2 subverts a stress granule response, heightens GM-CSF mRNA translation, and increases inflammatory cell recruitment to exacerbate pulmonary arterial hypertension.

BMP promotes motility and represses growth of smooth muscle cells by activation of tandem Wnt pathways

Vascular smooth muscle cell motility relies on interdependent activation of canonical and noncanonical Wnt signal transduction pathways; fibronectin, produced in response to BMP-2–mediated activation of β-catenin, promotes motility by activating an integrin-linked kinase via α4-integrin.

In Vivo Plaque Composition and Morphology in Coronary Artery Lesions in Adolescents and Young Adults Long After Kawasaki Disease. A Virtual Histology–Intravascular Ultrasound Study

Background— Coronary artery lesions (CALs) late after Kawasaki disease were characterized by endothelial dysfunction and low-grade inflammation, surrogate markers for atherosclerosis. We tested the hypothesis that CALs in patients long after Kawasaki disease are accompanied by atheroma-like features, as assessed by virtual histology–intravascular ultrasound, a new method to assess coronary plaque composition and morphology in vivo. Methods and Results— Virtual histology–intravascular ultrasound was performed in 13 Japanese Kawasaki disease patients (median age, 18.3 years; interquartile range, 16.9 to 23.3 years) an interval after Kawasaki disease (median, 15.9 years; interquartile range, 14.3 to 21.9 years). We investigated 6 sites with localized stenosis, 15 sites with an aneurysm, 29 sites with a regressed aneurysm, and 50 sites with a normal coronary segment. Plaque components were categorized into 4 parts: fibrous, fibrofatty, necrotic core, and dense calcium areas. Qualitatively, the normal segment had no or trivial intravascular ultrasound–visible plaque area, whereas the CAL exhibited a heterogeneous plaque area with the 4 components in different amounts and proportions. Quantitatively, a combined group of CALs had a higher absolute value of fibrous, dense calcium, and necrotic core areas than the normal segment. In further analyses of 3 subtypes of CALs, localized stenosis, an advanced lesion, exhibited higher absolute and relative values of dense calcium and necrotic core areas and a lower relative value of the fibrous area than regressed and persistent aneurysms. Conclusion— The present limited but initial virtual histology–intravascular ultrasound findings give new insight into the potential role of atherogenesis in the evolution of CALs in adolescents and young adults long after Kawasaki disease and therefore warrant further investigation.

Public access defibrillation improved the outcome after out-of-hospital cardiac arrest in school-age children: a nationwide, population-based, Utstein registry study in Japan.

Aims The purpose of this study was to determine whether implementation of public access defibrillation (PAD) improves the outcome after out-of-hospital cardiac arrest (OHCA) in school-age children at national level. Methods and results We conducted a prospective, nationwide, population-based Japanese Utstein registry study of consecutive OHCA cases in elementary and middle school children (7–15 years of age) who had a bystander-witnessed arrest of presumed cardiac origin during 2005–09 and received pre-hospital resuscitation by emergency responders. The primary endpoint was a favourable neurological outcome 1 month after an arrest. Among 230 eligible patients enrolled, 128 had ventricular fibrillation (VF) as an initial rhythm. Among these 128 patients, 29 (23%) children received a first shock by a bystander. Among these 29 patients, the proportion of the favourable neurological outcome after OHCA was 55%. During the study period, the proportion of patients initially shocked by a bystander among eligible patients increased from 2 to 21% (P = 0.002 for trend). The proportion of patients with a favourable neurological outcome after OHCA increased from 12 to 36% overall (P = 0.006). The collapse to defibrillation time was shorter in bystander-initiated defibrillation when compared with defibrillation by emergency responders (3.3 ± 3.7 vs. 12.9 ± 5.8 min, P < 0.001), and was independently associated with a favourable neurological outcome after OHCA [P = 0.03, odds ratio (OR) per 1 min increase, 0.90 (95% confidence interval 0.82–0.99)]. A non-family members witness was independently associated with VF as the initial rhythm [P < 0.001, OR 4.03 (2.08–7.80)]. Conclusion Implementation of PAD improved the outcome after OHCA in school-age children at national level in Japan.

Neutrophil Elastase Is Produced by Pulmonary Artery Smooth Muscle Cells and Is Linked to Neointimal Lesions

Previously, we reported that murine gammaherpesvirus-68 (M1-MHV-68) induces pulmonary artery (PA) neointimal lesions in S100A4-overexpressing, but not in wild-type (C57), mice. Lesions were associated with heightened lung elastase activity and PA elastin degradation. We now investigate a direct relationship between elastase and PA neointimal lesions, the nature and source of the enzyme, and its presence in clinical disease. We found an association exists between the percentage of PAs with neointimal lesions and elastin fragmentation in S100A4 mice 6 months after viral infection. Confocal microscopy documented the heightened susceptibility of S100A4 versus C57 PA elastin to degradation by elastase. A transient increase in lung elastase activity occurs in S100A4 mice, 7 days after M1-MHV-68, unrelated to inflammation or viral load and before neointimal lesions. Administration of recombinant elafin, an elastase-specific inhibitor, ameliorates early increases in serine elastase and attenuates later development of neointimal lesions. Neutrophils are the source of elevated elastase (NE) in the S100A4 lung, and NE mRNA and protein levels are greater in PA smooth muscle cells (SMC) from S100A4 mice than from C57 mice. Furthermore, elevated NE is observed in cultured PA SMC from idiopathic PA hypertension versus that in control lungs and localizes to neointimal lesions. Thus, PA SMC produce NE, and heightened production and activity of NE is linked to experimental and clinical pulmonary vascular disease.