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Objective: Despite the recent progress in upfront combination therapy for pulmonary arterial hypertension (PAH), useful biomarkers for the disorder still remain to be developed. SeP (Selenoprotein P) is a glycoprotein secreted from various kinds of cells including pulmonary artery smooth muscle cells to maintain cellular metabolism. We have recently demonstrated that SeP production from pulmonary artery smooth muscle cells is upregulated and plays crucial roles in the pathogenesis of PAH. However, it remains to be elucidated whether serum SeP levels could be a useful biomarker for PAH. Approach and Results: We measured serum SeP levels and evaluated their prognostic impacts in 65 consecutive patients with PAH and 20 controls during follow-up (mean, 1520 days; interquartile range, 1393-1804 days). Serum SeP levels were measured using a newly developed sol particle homogeneous immunoassay. The patients with PAH showed significantly higher serum SeP levels compared with controls. Higher SeP levels (cutoff point, 3.47 mg/L) were associated with the outcome (composite end point of all-cause death and lung transplantation) in patients with PAH (hazard ratio, 4.85 [1.42-16.6]; P <0.01). Importantly, we found that the absolute change in SeP of patients with PAH (ΔSeP) in response to the initiation of PAH-specific therapy significantly correlated with the absolute change in mean pulmonary artery pressure, pulmonary vascular resistance (ΔPVR), and cardiac index (ΔCI; R =0.78, 0.76, and -0.71 respectively, all P <0.0001). Moreover, increase in ΔSeP during the follow-up predicted poor outcome of PAH., Conclusions: Serum SeP is a novel biomarker for diagnosis and assessment of treatment efficacy and long-term prognosis in patients with PAH.

Pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH) are fatal diseases; however, their pathogenesis still remains to be elucidated. We have recently screened novel pathogenic molecules and have performed drug discovery targeting those molecules. Pulmonary artery smooth muscle cells (PASMCs) in patients with PAH (PAH-PASMCs) have high proliferative properties like cancer cells, which leads to thickening and narrowing of distal pulmonary arteries. Thus, we conducted a comprehensive analysis of PAH-PASMCs and lung tissues to search for novel pathogenic proteins. We validated the pathogenic role of the selected proteins by using tissue-specific knockout mice. To confirm its clinical significance, we used patient-derived blood samples to evaluate the potential as a biomarker for diagnosis and prognosis. Finally, we conducted a high throughput screening and found inhibitors for the pathogenic proteins.

Rationale: Pulmonary hypertension is a fatal disease; however, its pathogenesis still remains to be elucidated. Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and inhibits fibrinolysis. Plasma TAFI levels are significantly increased in chronic thromboembolic pulmonary hypertension (CTEPH) patients., Objective: To determine the role of activated TAFI (TAFIa) in the development of CTEPH., Methods and Results: Immunostaining showed that TAFI and its binding partner thrombomodulin (TM) were highly expressed in the pulmonary arteries (PAs) and thrombus in patients with CTEPH. Moreover, plasma levels of TAFIa were increased 10-fold in CTEPH patients compared with controls. In mice, chronic hypoxia caused a 25-fold increase in plasma levels of TAFIa with increased plasma levels of thrombin and TM, which led to thrombus formation in PA, vascular remodeling, and pulmonary hypertension. Consistently, plasma clot lysis time was positively correlated with plasma TAFIa levels in mice. Additionally, overexpression of TAFIa caused organized thrombus with multiple obstruction of PA flow and reduced survival rate under hypoxia in mice. Bone marrow transplantation showed that circulating plasma TAFI from the liver, not in the bone marrow, was activated locally in PA endothelial cells through interactions with thrombin and TM. Mechanistic experiments demonstrated that TAFIa increased PA endothelial permeability, smooth muscle cell proliferation, and monocyte/macrophage activation. Importantly, TAFIa inhibitor and peroxisome proliferator-activated receptor-α agonists significantly reduced TAFIa and ameliorated animal models of pulmonary hypertension in mice and rats., Conclusions: These results indicate that TAFIa could be a novel biomarker and realistic therapeutic target of CTEPH., (© 2017 American Heart Association, Inc.)

Background: Right ventricular (RV) function is the major determinant for both functional capacity and survival in patients with pulmonary arterial hypertension (PAH). Despite the recognized clinical importance of preserving RV function, the subcellular mechanisms that govern the transition from a compensated to a decompensated state remain poorly understood and as a consequence there are no clinically established treatments for RV failure and a paucity of clinically useful biomarkers. Accumulating evidence indicates that long noncoding RNAs are powerful regulators of cardiac development and disease. Nonetheless, their implication in adverse RV remodeling in PAH is unknown.Methods: Expression of the long noncoding RNA H19 was assessed by quantitative PCR in plasma and RV from patients categorized as control RV, compensated RV or decompensated RV based on clinical history and cardiac index. The impact of H19 suppression using GapmeR was explored in 2 rat models mimicking RV failure, namely the monocrotaline and pulmonary artery banding. Echocardiographic, hemodynamic, histological, and biochemical analyses were conducted. In vitro gain- and loss-of-function experiments were performed in rat cardiomyocytes.Results: We demonstrated that H19 is upregulated in decompensated RV from PAH patients and correlates with RV hypertrophy and fibrosis. Similar findings were observed in monocrotaline and pulmonary artery banding rats. We found that silencing H19 limits pathological RV hypertrophy, fibrosis and capillary rarefaction, thus preserving RV function in monocrotaline and pulmonary artery banding rats without affecting pulmonary vascular remodeling. This cardioprotective effect was accompanied by E2F transcription factor 1-mediated upregulation of enhancer of zeste homolog 2. In vitro, knockdown of H19 suppressed cardiomyocyte hypertrophy induced by phenylephrine, while its overexpression has the opposite effect. Finally, we demonstrated that circulating H19 levels in plasma discriminate PAH patients from controls, correlate with RV function and predict long-term survival in 2 independent idiopathic PAH cohorts. Moreover, H19 levels delineate subgroups of patients with differentiated prognosis when combined with the NT-proBNP (N-terminal pro-B-type natriuretic peptide) levels or the risk score proposed by both REVEAL (Registry to Evaluate Early and Long-Term PAH Disease Management) and the 2015 European Pulmonary Hypertension Guidelines.Conclusions: Our findings identify H19 as a new therapeutic target to impede the development of maladaptive RV remodeling and a promising biomarker of PAH severity and prognosis. [ABSTRACT FROM AUTHOR]

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by a sustained elevation of pulmonary artery (PA) pressure, right ventricular failure, and premature death. Enhanced proliferation and resistance to apoptosis (as seen in cancer cells) of PA smooth muscle cells (PASMCs) is a major pathological hallmark contributing to pulmonary vascular remodeling in PAH, for which current therapies have only limited effects. Emerging evidence points toward a critical role for Enhancer of Zeste Homolog 2 (EZH2) in cancer cell proliferation and survival. However, its role in PAH remains largely unknown. The aim of this study was to determine whether EZH2 represents a new factor critically involved in the abnormal phenotype of PAH-PASMCs. We found that EZH2 is overexpressed in human lung tissues and isolated PASMCs from PAH patients compared to controls as well as in two animal models mimicking the disease. Through loss- and gain-of-function approaches, we showed that EZH2 promotes PAH-PASMC proliferation and survival. By combining quantitative transcriptomic and proteomic approaches in PAH-PASMCs subjected or not to EZH2 knockdown, we found that inhibition of EZH2 downregulates many factors involved in cell-cycle progression, including E2F targets, and contributes to maintain energy production. Notably, we found that EZH2 promotes expression of several nuclear-encoded components of the mitochondrial translation machinery and tricarboxylic acid cycle genes. Overall, this study provides evidence that, by overexpressing EZH2, PAH-PASMCs remove the physiological breaks that normally restrain their proliferation and susceptibility to apoptosis and suggests that EZH2 or downstream factors may serve as therapeutic targets to combat pulmonary vascular remodeling. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs). Hypothesis: To discover a novel compound for PAH patients from the original 5,562 compounds of Tohoku University. Methods: We performed high-throughput screening using PASMCs from PAH patients (PAH-PASMCs). Next, we performed functional tests for emetine in vivo. Results: We found that emetine inhibits PAH-PASMC proliferation. Interestingly, emetine significantly reduced activities of HIF-1α and -2α in PAH-PASMCs (n=3 each, P <0.05). Consistently, emetine reduced secretion of inflammatory cytokines/chemokines and growth factors, and oxidative stress-augmenting proteins such as cyclophilin A and its receptor basigin in PAH-PASMCs (n=3 each, P <0.05). Moreover, emetine significantly suppressed Rho-kinase activity in PAH-PASMCs (n=3 each, P <0.05). Additionally, emetine increased apoptosis, detected by TUNEL assay and reduced the expression of the anti-apoptotic protein Bcl2 in PAH-PASMCs (n=3 each, P <0.05). Next, we performed functional tests for emetine in vivo. Six-week-old male SD rats were used for the two experimental PH models; monocrotaline-induced PH and Sugen/hypoxia-induced PH. In the prevention protocol, after subcutaneous injection of monocrotaline (60 mg/kg), the animals were assigned to the simultaneous oral administration with emetine (0.05 mg/kg/day) or vehicle for 3 weeks. Emetine significantly reduced right ventricular (RV) systolic pressure (57±3 vs. 77±4 mmHg), RV hypertrophy (0.40±0.02 vs. 0.50±0.02), and PA remodeling compared with controls (all P <0.01, n=10~15). In the treatment protocol, after establishment of PH with Sugen (20 mg/kg) and hypoxia (10% O2) for 3 weeks followed by normoxia for 6 weeks, the animals were assigned to the oral administration of emetine (0.05 mg/kg/day) or vehicle for additional 4 weeks. Emetine significantly reduced RV systolic pressure (93±5 vs. 59±5 mmHg), RV hypertrophy (0.52±0.04 vs. 0.35±0.02), and PA remodeling compared with controls (all P <0.01, n=10~12). Importantly, emetine significantly reduced TNF-α and MCP-1 in serum compared with vehicle controls, suggesting its potent anti-inflammatory effects in vivo. Finally, emetine significantly reduced protein levels of cyclophilin A, basigin, and Rho-kinase activities in both models in vivo. Conclusions: We identified emetine as a novel promising drug for the treatment of PAH. [ABSTRACT FROM AUTHOR]

Introduction: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs). However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be an effective target for patients with advanced PAH. Celastramycin is a benzoyl pyrrole-type compound originally found in a bacteria extract with anti-inflammatory effects. We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. Methods and Results: We screened 5,562 compounds from original library using high-throughput screening (HTS) system. Among the compounds, we identified that celastramycin (CEL), a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PASMCs from patients with PAH (PAH-PASMCs) in a dose-dependent manner with minimal effects on PASMCs from healthy donors (n=8 each, P <0.05). Moreover, cell toxicity assays showed that CEL inhibited cell proliferation without any cell death in PAH-PASMCs (n=8 each, P <0.05). Mechanistic analysis demonstrated that CEL significantly inhibited ERK and NF-κB signaling in PAH-PAMSCs compared with vehicle controls (both P <0.05, n=4 each). Moreover, CEL significantly reduced protein levels of bromodomain-containing protein 4 (BRD4), a member of the BET family with inflammatory property, compared with vehicle controls (both P <0.05, n=6 each). Consistently, CEL treatment reduced reactive oxygen species (ROS) production in PAH-PASMCs with increased protein levels of NF-E2-related factor 2, a master regulator of cellular response against oxidative stress (n=8 each, P <0.05). Furthermore, using a Seahorse apparatus, which provides information on mitochondrial function through real-time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), we found that CEL recovered mitochondrial function in PAH-PASMCs. Indeed, metabolome analysis showed that CEL increased metabolites in TCA cycle in PAH-PASMCs. Finally, CEL treatment significantly ameliorated hypoxia-induced pulmonary hypertension (PH) in mice, monocrotaline-induced PH in rats, and Sugen/hypoxia-induced PH in rats (all P <0.05), in which we found significantly reduced cytokines/chemokines and growth factors in lung homogenates (e.g. IL-2, IL-6, and M-CSF). Conclusions: CEL is a potential novel drug for the treatment of PAH with anti-proliferative, anti-oxidative, and anti-inflammatory effects. [ABSTRACT FROM AUTHOR]

Background: Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by persistent elevation of pulmonary vascular resistance, which results in a striking increase in right ventricular (RV) afterload and subsequent failure. Pulmonary artery (PA) smooth muscle cells (PASMCs) from PAH patients exhibit a cancer-like phenotype with increased glycolysis (Hexokinase 2 activity) and reduced glucose oxidation (inhibition of pyruvate dehydrogenase (PDH)) promoting cell proliferation and apoptosis-resistant contributing to obliteration of the PA lumen. Emerging evidence have demonstrated a critical role for the histone methyltransferase EZH2 in cancer cell survival and several EZH2 inhibitors are now clinically investigated. However, its role in PAH remains unknown. We hypothesized that EZH2 expression is upregulated in PAH-PASMCs leading to PA remodeling. Methods and Results: Using Western blot (WB), we showed that EZH2 is upregulated in lungs, distal PAs and isolated PASMCs from PAH patients (p<0.01, n=10) compared to controls. Consistently, increased EZH2 expression was found in distal PAs from animal models mimicking PAH (monocrotaline and Sugen/Hypoxia). Using proteomic LC-MSMS approach followed by Maxquant/Andromeda analysis in PASMCs isolated from 4 PAH patients, we identified 139 proteins differentially expressed (27 up-regulated ≥ 1.2 or and 112 down-regulated ≤ 0.84; p ≤ 0.05) following EZH2 knockdown. Results of KEGG pathway analysis revealed that the tricarboxylic acid (TCA) cycle, carbon metabolism and metabolic pathways were the top significant pathways (FDR p-value <10-4). This suggests that EZH2 is important for regulating bioenergetic metabolism in PAH-PASMCs. In agreement with this, pharmacological (GSK126 or EPZ-6438) or molecular (siEZH2) inhibition of EZH2 leads to reduced expression (WB) of Hexokinase 2, pPDH and PDH. This was associated with a significant reduction in PAH-PASMC proliferation (Ki67 labeling and EdU assay, p<0.001) and resistance to apoptosis (Annexin V assay p<0.001). Conclusion: We provide evidence that EZH2 is overexpressed in human PAH and implicated in the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs. [ABSTRACT FROM AUTHOR]