AIMS Upregulated p38MAPK signaling is implicated in the accelerated proliferation of pulmonary artery smooth muscle cells (PA-SMCs) and the pathogenesis of pulmonary artery remodeling observed in pulmonary arterial hypertension (PAH). Previously, we reported that after endothelin-1 (ET-1) pretreatment, bone morphogenetic protein 2 (BMP2) activates p38MAPK signaling and accelerates PA-SMC proliferation. The activity of p38MAPK signaling is tightly regulated by the inactivation of dual-specificity phosphatase 1 (DUSP1). Activated p38MAPK induces DUSP1 expression, forming a negative feedback loop. Prostacyclin IP receptor agonists (prostacyclin and selexipag) are used to treat PAH. In this study, we aimed to verify whether IP receptor agonists affect DUSP1 expression and accelerate the proliferation of PA-SMCs. MAIN METHODS PA-SMCs were treated with BMP2, ET-1, prostacyclin, and MRE-269, an active metabolite of selexipag, either alone or in combination. We quantified mRNA expressions using real-time quantitative polymerase chain reaction. Pulmonary artery specimens and PA-SMCs were obtained during lung transplantation in patients with PAH. KEY FINDINGS Both prostacyclin and MRE-269 increased DUSP1 expression. Combined treatment with BMP2 and ET-1 induced cyclin D1 and DUSP1 expression and increased PA-SMC proliferation. MRE-269 attenuated BMP2/ET-1-induced cell proliferation. ET-1 increased DUSP1 expression in PA-SMCs from control patients but not in PA-SMCs from patients with PAH. SIGNIFICANCE This study showed that the p38MAPK/DUSP1 negative feedback loop is impaired in PAH, contributing to unregulated p38MAPK activation and PA-SMC hyperplasia. IP receptor agonist MRE-269 increases DUSP1 expression and inhibit p38MAPK-mediated PA-SMC proliferation. Future elucidation of the detailed mechanism underlying reduced DUSP1 expression would be informative for PAH treatment.

Background The use of pulmonary vasodilators for pulmonary arterial hypertension (PAH) has led to a favourable prognosis. In contrast, pulmonary veno-occlusive disease (PVOD) is characterized by the possibility of severe pulmonary oedema after specific PAH therapy. Pulmonary oedema presumably develops in association with pulmonary arterial vasodilation without concomitant pulmonary venodilation. The venous circulation maximally dilates with small amounts of nitroglycerine. Case summary A 59-year-old woman with advanced PVOD was referred to our hospital. We performed a right heart catheterization after administering combination therapy with selexipag and macitentan, and intravenous nitroglycerine at 0.2 and 0.4 & mu;g/kg/min decreased pulmonary arterial wedge pressure (PAWP) and mean pulmonary arterial pressure (PAP) to minimal levels. The final dose of 1 & mu;g/kg/min yielded an & SIM;20% decrease in mean PAP and pulmonary vascular resistance (PVR). Discussion Here, we described the acute effect of intravenous nitroglycerine on PAWP and PVR in a patient with PVOD. This case highlights the venodilation response even in advanced PVOD, suggesting the importance of further research into selective venous dilators as potent therapy.

BACKGROUND:Cases of giant coronary artery aneurysms (GCAAs) associated with coronary fistula are rarely reported, and they present with various symptoms, including coronary steal syndrome. We report an uncommon case of an asymptomatic giant coronary fistula aneurysm presenting as a progressing left-sided mediastinal mass that has been tracked for years.CASE SUMMARY:A 67-year-old healthy asymptomatic woman was referred to our hospital because of an abnormal shadow on her chest radiography revealing a left-sided mediastinal mass that had progressed in size over the past 4 years. Computed tomography revealed mass progression from 4 to 5 cm in diameter within 2 years. Coronary computed tomography and coronary angiography identified a GCAAs in a coronary fistula originating in the left anterior descending artery and draining into the main pulmonary artery. Transthoracic Doppler echocardiography revealed a unique systolic dominant flow. She underwent coronary artery aneurysmectomy and fistula ligation. The patient has been in good health without any events for 10 months since her discharge.DISCUSSION:A GCAAs in a coronary fistula can present as an asymptomatic left-sided mediastinal mass that has progressed in size for years in older adults. Echocardiography can provide clues of the steal phenomenon in coronary artery fistula. A close investigation of mediastinal abnormalities can facilitate the detection of coronary aneurysms.

Pulmonary arterial hypertension (PAH) is characterized by abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin-1 (ET-1) and activated p38 mitogen-activated protein kinase (p38MAPK) has been observed in PAH patients. p38MAPK has been implicated in cell proliferation. An unspecified disturbance in bone morphogenetic protein (BMP) signaling may be involved in the development of PAH. Type I receptors (BMPR1A and BMPR1B) and type II receptor (BMPR2) transduce signals via two distinct pathways, i.e., canonical and non-canonical pathways, activating Smad1/5/8 and p38MAPK, respectively. BMPR1B expression was previously reported to be enhanced in the PASMCs of patients with idiopathic PAH. BMP15 binds specifically to BMPR1B. We assessed the effects of ET-1 on BMP receptor expression and cell proliferation. BMP2 increased BMPR1B expression in human PASMCs after pretreatment with ET-1 in vitro. Although BMP2 alone did not affect PASMC proliferation, BMP2 treatment after ET-1 pretreatment significantly accelerated PASMC proliferation. PH-797804, a selective p38MAPK inhibitor, abrogated this proliferation. Similarly, after ET-1 pretreatment, BMP15 significantly accelerated the proliferation of PASMCs, whereas stimulation with BMP15 alone did not. In conclusion, in PASMCs, ET-1 exposure under pathological conditions alters BMP signaling to activate p38MAPK, resulting in cell proliferation.

The increase in thickening of the arterial wall of pulmonary arterial hypertension (PAH) includes cellular proliferation as well as matrix deposition and interrupted internal elastic lamina (IEL) consisting of a thick homogeneous sheet of elastin. Little is, although, known about the detail of IEL formation in PAH. Endothelin-1 is overexpressed in pulmonary arterioles of PAH. We aimed to examine the expression of genes contributing to IEL formation in pulmonary artery smooth muscle cells (PASMCs) especially focused on lysyl oxidase (LOx), an exreacellular matrix enzyme that catalyzes the cross-linking of collagens or elastin. We quantified mRNA expressions of genes contributing to IEL formation including LOx in PASMCs using real-time quantitative polymerase chain reaction. We stimulated human PASMCs with endothelin-1 with prostacyclin or trapidil. Endothelin-1 significantly increased LOx expression. Prostacyclin and trapidil restored endothelin-1-induced LOx expression to the basal level. Endothelin-1 increased LOx expression strongly in PASMCs from PAH patients compared to those from controls. Trapidil reduced LOx expression only in PASMCs from PAH patients. Overexpressed endothelin-1 in PAH patients can increase expression of LOx and agitate cross-linking of elastin and collagen, resulting in ectopic deposition of these in the vascular media.

Introduction: The Glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide is an incretin hormone mimetics, a drug for diabetes mellitus. Recently, it is reported that the GLP-1R agonist inhibits endothelial dysfunction, inflammation, and cell proliferation, which are the major molecular findings for pathophysiology of pulmonary arterial hypertension (PAH). However, it is unclear whether the GLP-1R agonist ameliorates PAH. Objective: To investigate the effect of a GLP-1R agonist on the development of PAH. Methods: 8 week-old C57BL6/J mice were injected liraglutide (0.3 mg/kg/day) or saline for total 5 weeks. One week after starting injection, mice were placed in room air or exposed to chronic hypoxia (10% O2) for further 4 weeks. Results: Right ventricular systolic pressure of hypoxia + liraglutide group were significantly reduced compared with that of hypoxia + saline group (30.0 ± 1.5 mmHg vs 34.3 ± 2.2 mmHg, P < .01, n = 7). Right ventricular/body weight ratio of hypoxia + liraglutide group were significantly smaller than that of hypoxia + saline group (P < .01). The expression of eNOS mRNA and the protein expression of phospho-eNOS in the lung was significantly augmented by liraglutide administration under hypoxia (P < .05). Conclusion: The GLP-1R agonist liraglutide reduces right ventricular pressure in hypoxia-induced PAH mice model. This effect is suggested to be through the eNOS activation in pulmonary artery.

Aims: Vascular remodeling results from aberrations in the balance between cell proliferation and death, which is seen in the obstructive vasculature of pulmonary arterial hypertension (PAH). Endothelin (ET)-1 has a potent proliferative activity on vascular smooth muscle cells, and ET receptor inhibitors are used to treat PAH; however, it remains unclear whether ET receptor inhibition contributes to the apoptosis of pulmonary arterial smooth muscle cells (PASMCs), another cause of pulmonary vascular remodeling.Main methods: Cultured human PASMCs were treated with the ETA receptor antagonist BQ-123 (100 mu M), or the ETB antagonist A-192621 (1 - 100 mu M) or BQ-788 (1 - 100 mu M) for 48 h. The cells were then incubated for another 24 h with or without doxorubicin (DOX, 1 mu M), an anthracyclin antitumor antibiotic that promotes p53-mediated apoptosis. Cell viability and apoptosis were evaluated by MTT assays, caspase-3/7 activity assays, and Western blots for cleaved caspase-3 expression.Key findings: The viability of PASMCs was significantly decreased by A-192621 and BQ-788, in a dose-dependent manner. A-192621 and BQ-788 significantly increased the caspase-3/7 activity and cleaved caspase-3 expression in PASMCs. The PASMCs' susceptibility to DOX-induced apoptosis was significantly higher in the presence of A-192621 and BQ-788 than with vehicle. However, BQ-123 did not affect these parameters.Significance: Blockade of the ETB receptor increases the extent of apoptosis and susceptibility to DOX-induced apoptosis in PASMCs. Apoptosis caused by ETB receptor blockade in PASMCs may be one of the mechanisms by which vascular remodeling is reduced in ET receptor inhibitor-based PAH treatments. (C) 2016 Elsevier Inc. All rights reserved.

Background: Congenital diaphragmatic hernia (CDH) has a high mortality rate mainly due to lung hypoplasia and persistent pulmonary hypertension of the newborn (PPHN). We recently reported that antenatal treatment with sildenafil or simvastatin prevented pulmonary hypoplasia and PPHN in experimental CDH. Objectives. We sought to evaluate the efficacy of antenatal sildenafil-simvastatin combination on pulmonary hypoplasia and PPHN associated to nitrofen-induced CDH. Methods: On embryonic day (E)9.5, nitrofen or vehicle was administered to pregnant rats. Nitrofen-treated rats were randomly assigned to antenatal sildenafil (100 mg/kg/day) and simvastatin (20 mg/kg/day) combination or placebo administration from E11 to E21. On E21, foetuses were delivered by caesarean section, killed and checked for left-sided CDH. Results: In nitrofen-induced CDH, simvastatin-sildenafil totally prevented left-sided CDH in the offspring, but failed to increase body weight and lung-to-body weight ratio. Antenatal simvastatin-sildenafil improved lung parenchyma structure, with decreased alveolar septal thickness and increased radial alveolar count. Antenatal simvastatin-sildenafil restored pulmonary vascular density and external diameter, and reduced pulmonary arteriolar remodeling. This was associated with restored epithelial and vascular activation of apoptotic processes in the lungs. Antenatal combination therapy was more efficacious than antenatal therapy with simvastatin or sildenafil alone. Conclusions: Antenatal simvastatin-sildenafil combination therapy improves pathological features of lung hypoplasia and PPHN in experimental nitrofen-induced CDH.

AIMS:Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases. Bone morphogenetic protein (BMP) and endothelin-1 signaling pathways have been shown to be altered in hypoxic PH and to play crucial roles in the associated pulmonary artery remodeling. We, therefore, aimed to study the potential link between hypoxia and the alteration of BMP and endothelin-1 signaling observed in pulmonary artery smooth muscle cells (PA-SMCs) in hypoxic PH.MATERIALS AND METHODS:Human PA-SMCs were treated with hypoxia-mimetic agent cobalt chloride (CoCl2; 100μM), with or without pretreatment with a dual endothelin receptor antagonist bosentan (10μM). Expressions of preproendothelin-1 (PPET1), BMP type 2 receptor (BMPR-2), and one BMP signaling target gene, the inhibitor of DNA binding 1 (ID1) were evaluated by real time quantitative polymerase chain reaction. BMP2-treated PA-SMCs were assessed for Smad1/5/8 signaling activation by Western Blotting.KEY FINDINGS:Treatment of PA-SMCs with CoCl2 increased PPET1 gene expression, while it did not alter expressions of endothelin converting enzyme, endothelin receptor type A or type B. Hypoxia-mimetic agent CoCl2 decreased the expressions of BMPR-2 and ID1 maximally after 3- and 6-hour treatment respectively, while CoCl2 treatment progressively increased noggin expression. Bosentan pretreatment restored expressions of BMPR-2 and ID1, as well as the activation (by phosphorylation) of Smad1/5/8 signaling induced by BMP2.SIGNIFICANCE:Hypoxia induces the downregulation of the BMP signaling in PA-SMCs, at least, partly through the endothelin system. In hypoxic PH, increased endothelin-1 production might therefore contribute to the altered BMP signaling and subsequent PA-SMC hyperplasia.

Congenital diaphragmatic hernia (CDH) has a high mortality rate mainly due to lung hypoplasia and persistent pulmonary hypertension of the newborn (PPHN). Simvastatin has been shown to prevent the development of pulmonary hypertension (PH) in experimental models of PH. We, therefore, hypothesized that antenatal simvastatin would attenuate PPHN in nitrofen-induced CDH in rats. The efficacy of antenatal simvastatin was compared with antenatal sildenafil, which has already been shown to improve pathological features of PPHN in nitrofen-induced CDH. On embryonic day (E) 9.5, nitrofen or vehicle was administered to pregnant Sprague-Dawley rats. On E11, nitrofen-treated rats were randomly assigned to antenatal simvastatin (20 mg·kg(-1)·day(-1) orally), antenatal sildenafil (100 mg·kg(-1)·day(-1) orally), or placebo administration from E11 to E21. On E21, fetuses were delivered by cesarean section, killed, and checked for left-sided CDH. Lung tissue was then harvested for further pathobiological evaluation. In nitrofen-induced CDH, simvastatin failed to reduce the incidence of nitrofen-induced CDH in the offspring and to increase the body weight, but improved the lung-to-body weight ratio and lung parenchyma structure. Antenatal simvastatin restored the pulmonary vessel density and external diameter, and reduced the pulmonary arteriolar remodeling compared with nitrofen-induced CDH. This was associated with decreased lung expression of endothelin precursor, endothelin type A and B receptors, endothelial and inducible nitric oxide synthase, together with restored lung activation of apoptotic processes mainly in the epithelium. Antenatal simvastatin presented similar effects as antenatal therapy with sildenafil on nitrofen-induced CDH. Antenatal simvastatin improves pathological features of lung hypoplasia and PPHN in experimental nitrofen-induced CDH.

Background: Pulmonary hypertension (PH) is a common complication of chronic hypoxic lung diseases. Alterations in bone morphogenetic protein (BMP) and endothelin signaling have been described in hypoxic PH. We recently reported on endothelin-induced downregulation of BMP signaling in pulmonary artery smooth muscle cells (PA-SMCs), partly through increased expression of BMP antagonist gremlin1. Here, we aimed to study the alterations in BMP signaling in hypoxia. Methods: Lung expressions of BMP antagonists noggin and gremlin1 were evaluated in 4 patients with group3 PH, 6 with idiopathic pulmonary arterial hypertension (iPAH) and 6 controls. Control PA-SMCs were treated with the hypoxia-mimetic agent cobalt chloride (CoCl2; 100 µM), with or without pre-treatment with bosentan (1 µM). Gene expressions of preproendothelin-1 (PPET1), endothelin converting enzyme 1 (ECE1), BMP type 2 receptor (BMPR2), noggin, gremlin1 and inhibitor of DNA binding 1 (Id1) were evaluated by RTQ-PCR. Results: Lung tissue from group3 PH patients presented with increased expression of noggin, gremlin1 and 2, while a less important increase was observed in iPAH lungs. Treatment of PA-SMCs with CoCl2 increased PPET1 expression, while it did not alter ECE1 expression. CoCl2 decreased BMPR2 and Id1 expressions, while it increased noggin and gremlin1 expression. In PA-SMCs, bosentan pretreatment prevented the alteration of Id1 and gremlin1 expressions induced by CoCl2, while it did not alter noggin and BMPR2. Conclusion: Hypoxia induces the downregulation of the BMP signaling in control PA-SMCs, partly through the endothelin system. This may account for the increased expression of gremlin1 observed in hypoxic PH patients.

BACKGROUND: Endothelin receptor antagonists improve pulmonary arterial hypertension (PAR). Mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) predispose to PAR. Here, we sought to determine whether there might exist interactions between these 2 signaling pathways and their effect on the acquisition of the altered phenotype of pulmonary artery smooth muscle cells (PA-SMCs) observed in PAR.METHODS: Expression of BMPR2, of the BMP agonist BMP4, and of the BMP antagonists gremlin1 and gremlin2 was evaluated in lungs and in PA-SMCs from 6 PAR patients and 14 controls treated with endothelin-1 Endothelin-1 pre-treated PA-SMCs were assessed for proliferation, apoptosis, and downstream signaling activation of Smad1/5/8 and p38 mitogen-activated protein kinase (p38(MAPK)) after BMP2 treatment.RESULTS: In PA-SMCs from PAR patients, expression of BMPR2 and BMP4 decreased, whereas expression of gremlin1 and gremlin2 increased compared with controls. Treatment of control PA-SMCs with endothelin-1 induced a dose-dependent increase in gremlin1 and gremlin2, whereas BMPR2 and BMP4 expression decreased, reaching similar levels as those observed in PAR cells. In control PA-SMCs, endothelin-1 pre-treatment reduced inhibitor of DNA binding 1 (Id1) expression and Smad1/5/8 activation induced by BMP2, whereas it enhanced p38(MAPK) activation. Moreover, BMP2 decreased serum-induced proliferation and increased the pro-apoptotic Bax/Bcl-2 ratio. These effects were attenuated by endothelin-1 pre-treatment. Endothelin-1 did not alter BMPR2 signaling in PA-SMCs from PAR patients.CONCLUSIONS: Endothelin-1 downregulates canonical BMPR2 signaling. This is related to decreased BMPR2 and increased anti-BMP gremlin expression associated with increased activation of p38(MAPK) and results in PA-SMC proliferation. (C) 2015 International Society for Heart and Lung Transplantation. All rights reserved.

In cardiac hypertrophy, the activation of RNA polymerase II (RNAPII) by phosphorylation of C-terminal domain (CTD) is a crucial response for cardiomyocytes; the phosphorylation and dephosphorylation of CTD is regulated by p-TEFb and the phosphatase FCP1, respectively. Although the role of p-TEFb in heart pathogenesis is well studied, the precise of FCP1 involvement is unclear. We created cardiomyocyte-specific FCP1 transgenic mice (FCP1-TG) using αMHC promoter. FCP1-TG showed the decreased level of myocardial CTD phosphorylation. FCP1-TG developed severe cardiac dilatation and dysfunction at 22-week old but not at 5-week old revealed by histopathology and echocardiography; however, a marker for failing heart, ANP expression, was markedly augmented even from 5-week old. The apoptosis marker cleaved caspase-3 and pro-apoptosis signal Bax was augmented in a time-dependent manner in FCP1-TG. These changes were not observed in dominant-negative FCP1 (dnFCP1)-TG. In transient transfection study in neonatal rat cardiomyocytes, FCP1 inhibited CTD phosphorylation and a hypertrophic agent (endothelin-1)-induced increase of RNA and protein synthesis; however, dnFCP1 did not affect on this phenomenon. In conclusion, myocardial FCP1-overexpression induces cardiac dysfunction accordant with apoptosis signaling probably via inhibition of RNA synthesis secondary to the suppression of CTD phosphorylation. FCP1 may be an important molecule to properly coordinate the RNAPII function and the inhibition of RNA synthesis induces heart failure probably due not able to respond to environment-associated stimuli.

AIMS:Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions.MAIN METHODS:We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [(3)H]-leucine incorporation.KEY FINDINGS:Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [(3)H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin.SIGNIFICANCE:This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.