Patient characteristics, diagnostic testing, and initial treatment profiles of patients with connective tissue disease-associated pulmonary arterial hypertension (CTD-PAH) using a Japanese claims database.

OBJECTIVES: To investigate the diagnostic and therapeutic landscape for patients with connective tissue disease (CTD) and CTD-associated pulmonary arterial hypertension (CTD-PAH) in acute-care general hospitals in Japan. METHODS: We conducted a retrospective cohort study by analysing the Medical Data Vision (MDV) database from April 2008 and September 2020. CTD patients who prescribed immunosuppressants were included in cohort 1, and CTD-PAH patients extracted from cohort 1 were included in cohort 2. Patient characteristics, diagnostic screening frequencies for PAH, and initial PAH-specific treatment patterns were assessed. RESULTS: Overall, 16648 patients with CTD and 81 patients with CTD-PAH were included in cohorts 1 and 2, respectively. The frequencies of screening tests for PAH, including brain natriuretic peptide (BNP), transthoracic echocardiogram (TTE), and diffusing capacity of the lungs for carbon monoxide (DLCO), among CTD patients were 0.7, 0.3, and 0.1 tests/person-year, respectively. The most common initial PAH-specific treatment therapy was monotherapy (87.7%), followed by dual therapy (7.4%), and triple therapy (2.5%). CONCLUSION: This is the first study to describe the patient flow from PAH diagnosis to initial PAH-specific treatment for real-world patients who were followed regularly due to CTD in Japanese clinical practice.

Real-world diagnostic landscape and incidence of pulmonary hypertension in adult congenital heart disease patients using administrative claims data in Japan.

BACKGROUND: Although pulmonary hypertension (PH) and Eisenmenger's syndrome (ES) are common complications in adult congenital heart disease (ACHD), the frequency of diagnostic tests and the incidence of PH/ES in patients with ACHD in Japanese clinical practice are unclear. Therefore, we sought to clarify the frequency of diagnostic tests and incidence of PH/ES in patients with ACHD using the Medical Data Vision (MDV) database, the largest anonymized database of diagnosis procedure combination hospitals in Japan. METHODS: We conducted a retrospective cohort study using the MDV database (April 2008 to December 2021) of patients with ACHD (International Classificaiton of Diseases, 10th revision codes: Q203-204, Q210-213, Q250) aged ≥15 years. The frequency of laboratory/clinical tests and the incidence of PH/ES were calculated. Subgroup analyses were performed for the periods 2008-2015 and 2016-2021. RESULTS: Overall, 28219 ACHD patients were extracted from the MDV database (females 56.3%, males 43.7%; mean ± standard deviation age 44.7 ± 23.5 years). The mean ± standard deviation follow-up period was 2.5 ± 2.7 years. The frequencies of electrocardiography, ultrasonography, brain natriuretic peptide (BNP), N-terminal pro-BNP (NT-proBNP), right heart catheterization, and pulmonary function tests (DLCO) were 2149.8, 1054, 1233, 340, 40.0, and 6.0 per 1000 person-years, respectively. The incidence rate of PH/ES was 32.8 per 1000 person-years. The incidence rate of PH/ES increased from 24.6 to 46.7 per 1000 person-years from 2008-2015 to 2016-2021. CONCLUSION: We have clarified the frequency of diagnostic tests related to PH/ES and the incidence of PH/ES in patients with ACHD in clinical practice in Japan, including non-specialist institutions for PH.

Patient and Therapeutic Profiles of Pulmonary Hypertension in Chronic Lung Diseases in Japan: A Cohort Study Using a Claims Database.

INTRODUCTION: Pulmonary hypertension (PH) is often complicated by chronic lung diseases (CLDs) such as chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). Differentiating between PH associated with CLD (group 3 PH) and pulmonary arterial hypertension (PAH) in CLD is often difficult and reporting on the efficacy of PAH-specific therapies is inconsistent as a result of the lack of understanding of the heterogeneity of patients with PH. METHODS: A retrospective observational cohort study was conducted to understand the baseline characteristics, comorbidities, and treatment profiles of patients with PH in CLD in a real-world setting using a large-scale claims database (Medical Data Vision). Administrative and clinical data for patients admitted to acute-care hospitals in Japan between April 2008 and January 2021 were analyzed. RESULTS: A total of 115,921 patients with CLD (109,578 with COPD and 6343 with ILD, of whom 569 and 176 had PH, respectively) were analyzed. This study found lower PH diagnosis rates among patients with COPD and patients with ILD than in previous studies. The majority of PH with CLD patients were elderly (mean age 75.7 years) and male (80.81%). Among patients with CLD prescribed PAH-specific therapies (105 patients with COPD; 64 patients with ILD), most received these as monotherapy (COPD, 84.76%; ILD, 75.56%); the most common were phosphodiesterase 5 inhibitors (COPD, 42.70%; ILD, 18.37%), prostacyclins (oral; COPD, 48.31%; ILD, 24.49%), and endothelin receptor antagonists (ERA) (COPD, 8.99%; ILD, 18.37%). Comorbidities (e.g., pulmonary, cardiac, kidney), home oxygen therapy (HOT), and echocardiography (ECHO) were factors associated with the diagnosis of PH. CONCLUSION: This is the first study using an administrative database that provides real-world data on patients with PH in CLD in Japan. Our results indicate that PH may be misdiagnosed or underdiagnosed in Japan which may lead to suboptimal treatment for patients, and supports the need for further evidence to guide appropriate treatment.

Pulmonary hypertension is a disorder affecting the arteries in the lungs and the right heart. It can be associated with a variety of heart and lung conditions, including many chronic lung diseases such as chronic obstructive pulmonary disease and interstitial lung disease. Patients with pulmonary hypertension with chronic lung disease and/or hypoxia can be hard to tell apart from patients with pulmonary arterial hypertension coinciding with chronic lung disease. In Japan, there is not enough data on patient demographics and their disease characteristics for patients with pulmonary hypertension and chronic lung disease, including treatment profiles, and disease management. We identified these patients from a large medical claims database in Japan and analyzed their data. Our study focused on the use of therapies for pulmonary arterial hypertension on patients with pulmonary hypertension and chronic lung disease. The diagnosis rates of pulmonary hypertension for patients with chronic obstructive pulmonary disease and interstitial lung disease were low compared to previous reports, meaning patients with pulmonary hypertension may be misdiagnosed or underdiagnosed which may be resulting in suboptimal treatments. Furthermore, the majority of patients with pulmonary hypertension treated with pulmonary arterial hypertension medication received a single drug as treatment, even though the guidelines recommend the use of combination therapies in certain situations. This study emphasizes the need for further evidence generation for improvements in diagnoses and treatment of patients with pulmonary hypertension in Japan.

Comparative Treatment Persistence and Adherence to Endothelin Receptor Antagonists Among Patients with Pulmonary Arterial Hypertension in Japan: A Real-World Administrative Claims Database Study.

INTRODUCTION: Real-world data on the comparative effectiveness of endothelin receptor antagonists (ERAs; macitentan, bosentan, ambrisentan) for pulmonary arterial hypertension (PAH), particularly in Asian countries, are scarce. We evaluated the persistence of these ERAs before and after macitentan approval in Japan (2015). METHODS: We used real-world data from the Japanese Medical Data Vision administrative claims database between April 2008 and November 2020. Patients with PAH were identified from the dataset. Persistence to ERA treatment before and after approval of macitentan in Japan was defined as the time between start of the index ERA and treatment discontinuation or death. Propensity score adjustment was applied to minimize confounding effects among treatment groups. RESULTS: In the pre-macitentan approval cohort, 153 and 51 patients received bosentan and ambrisentan, respectively. In the post-macitentan approval cohort, 331, 284, and 91 patients received macitentan, bosentan, and ambrisentan, respectively. Unadjusted median persistence for ambrisentan- and bosentan-treated patients was 19 and 10 months, respectively (adjusted HR 0.87 [95% CI 0.61-1.24]; P = 0.434 [bosentan as reference]). In the post-macitentan approval cohort, unadjusted median persistence was 18 months for macitentan-treated patients versus 6 and 8 months for ambrisentan- and bosentan-treated patients, respectively. Adjusted HRs for ambrisentan and bosentan were 1.48 (95% CI 1.12-1.95; P = 0.006) and 1.63 (95% CI 1.30-2.04; P < 0.001 [macitentan as reference]), respectively. CONCLUSIONS: Real-world data for Japanese patients with PAH showed that persistence was significantly higher for macitentan, versus ambrisentan and bosentan, since its approval.

Real-world clinical practice of pulmonary arterial hypertension in Japan: Insights from a large administrative database.

Pulmonary arterial hypertension (PAH) is a fatal disease that often occurs at an early age. In recent years, aggressive treatment with multiple drugs from the early-stage diagnosis is expected to improve the prognosis. Indeed, a high rate of initial combination therapy and excellent treatment outcomes have been reported from specialized centers for PAH in Japan. However, information on PAH epidemiology, including non-PAH specialized centers in Japan, is unclear. To address the above, we conducted a retrospective observational cohort study from April 2008 to September 2020 using real-world evidence from a large-scale administrative database (Medical Data Vision) to examine baseline characteristics, comorbidities, and treatment profiles of Japanese patients with PAH. Five hundred and eighteen patients with PAH (treatment-naive PAH, age 67.2 ± 15.9) were identified through our comprehensive approach which combined PAH disease codes, medications, and diagnostic procedures. Moreover, we showed that a larger proportion of patients received monotherapy in their initial treatment (66%) compared to those receiving combination therapy (34%). During the 1-year follow-up after PAH diagnosis, 13% of patients increased their PAH medications while other patients either decreased their PAH medications (6%) or discontinued PAH treatment (27%). The 3- and 5-year event-free survival rates of all-cause death were 72% and 64%, respectively. This is the first large-scale administrative database study that provides insights into real-world PAH management in Japan. This study highlighted a different PAH clinical landscape which included a larger portion of the elderly population, higher initial monotherapy treatment, and lower survival rates than previous studies.

Treatment pattern and clinical outcomes in portopulmonary hypertension: A database study in Japan.

Background and Aim: Portopulmonary hypertension (PoPH) is a complication associated with portal hypertension. Since the epidemiological study in Japan was limited, this study aimed to investigate the characteristics, treatment patterns, and prognosis of PoPH patients in real-world data. Methods: The characteristics and treatment patterns of PoPH (n = 386) and portal hypertension without pulmonary arterial hypertension (portal hypertension w/o PAH) (n = 96 463) were analyzed based on the Medical Data Vision (MDV) dataset from April 2008 to September 2020. Survival-time analyses of emergency hospitalization and mortality were also conducted between matched pair cohorts of PoPH (n = 210) and portal hypertension w/o PAH (n = 840). Results: Among 386 PoPH patients, the Child-Pugh classification of PoPH group comprised patients with Class A (59 [15.3%]), B (109 [28.2%]), and C (42 [10.9%]), and missing (176 [45.6%]). Regarding the feature of PoPH group, the proportion of primary biliary cholangitis (PBC) (13.7%) and splenomegaly (9.8%) was higher compared with portal hypertension w/o PAH group. The survival time of all-cause hospitalization in PoPH group was shorter than portal hypertension w/o PAH group in matched pair cohort (P < 0.001 by log-rank test). Of PoPH patients, the frequency of PAH-specific medicine usage within 90 days was monotherapy of 79 patients (20.5%), combination therapy of 64 patients (16.6%), and PAH-specific medicine usage of 243 patients (63.0%). Conclusion: This was the first study demonstrating that high proportion of PBC and splenomegaly and a greater risk of hospitalization were observed in PoPH patients based on the analysis using administrative claim database.

PARP1-PKM2 Axis Mediates Right Ventricular Failure Associated With Pulmonary Arterial Hypertension.

The authors show that increased poly(adenosine diphosphate-ribose) polymerase 1 (PARP1) and pyruvate kinase muscle isozyme 2 (PKM2) expression is a common feature of a decompensated right ventricle in patients with pulmonary arterial hypertension and animal models. The authors find in vitro that overactivated PARP1 promotes cardiomyocyte dysfunction by favoring PKM2 expression and nuclear function, glycolytic gene expression, activation of nuclear factor κB-dependent proinflammatory factors. Pharmacologic and genetic inhibition of PARP1 or enforced tetramerization of PKM2 attenuates maladaptive remodeling improving right ventricular (RV) function in multiple rodent models. Taken together, these data implicate the PARP1/PKM2 axis as a critical driver of maladaptive RV remodeling and a new promising target to directly sustain RV function in patients with pulmonary arterial hypertension.

Potential for inhibition of checkpoint kinases 1/2 in pulmonary fibrosis and secondary pulmonary hypertension.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterised by exuberant tissue remodelling and associated with high unmet medical needs. Outcomes are even worse when IPF results in secondary pulmonary hypertension (PH). Importantly, exaggerated resistance to cell death, excessive proliferation and enhanced synthetic capacity are key endophenotypes of both fibroblasts and pulmonary artery smooth muscle cells, suggesting shared molecular pathways. Under persistent injury, sustained activation of the DNA damage response (DDR) is integral to the preservation of cells survival and their capacity to proliferate. Checkpoint kinases 1 and 2 (CHK1/2) are key components of the DDR. The objective of this study was to assess the role of CHK1/2 in the development and progression of IPF and IPF+PH. METHODS AND RESULTS: Increased expression of DNA damage markers and CHK1/2 were observed in lungs, remodelled pulmonary arteries and isolated fibroblasts from IPF patients and animal models. Blockade of CHK1/2 expression or activity-induced DNA damage overload and reverted the apoptosis-resistant and fibroproliferative phenotype of disease cells. Moreover, inhibition of CHK1/2 was sufficient to interfere with transforming growth factor beta 1-mediated fibroblast activation. Importantly, pharmacological inhibition of CHK1/2 using LY2606368 attenuated fibrosis and pulmonary vascular remodelling leading to improvement in respiratory mechanics and haemodynamic parameters in two animal models mimicking IPF and IPF+PH. CONCLUSION: This study identifies CHK1/2 as key regulators of lung fibrosis and provides a proof of principle for CHK1/2 inhibition as a potential novel therapeutic option for IPF and IPF+PH.

Implication of EZH2 in the Pro-Proliferative and Apoptosis-Resistant Phenotype of Pulmonary Artery Smooth Muscle Cells in PAH: A Transcriptomic and Proteomic Approach.

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.

17ß-Estradiol and estrogen receptor α protect right ventricular function in pulmonary hypertension via BMPR2 and apelin.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17ß-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α-null or ER-ß-null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

Identification of Celastrol as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension and Right Ventricular Failure Through Suppression of Bsg (Basigin)/CyPA (Cyclophilin A).

OBJECTIVE: Pulmonary arterial hypertension is characterized by abnormal proliferation of pulmonary artery smooth muscle cells and vascular remodeling, which leads to right ventricular (RV) failure. Bsg (Basigin) is a transmembrane glycoprotein that promotes myofibroblast differentiation, cell proliferation, and matrix metalloproteinase activation. CyPA (cyclophilin A) binds to its receptor Bsg and promotes pulmonary artery smooth muscle cell proliferation and inflammatory cell recruitment. We previously reported that Bsg promotes cardiac fibrosis and failure in the left ventricle in response to pressure-overload in mice. However, the roles of Bsg and CyPA in RV failure remain to be elucidated. Approach and Results: First, we found that protein levels of Bsg and CyPA were upregulated in the heart of hypoxia-induced pulmonary hypertension (PH) in mice and monocrotaline-induced PH in rats. Furthermore, cardiomyocyte-specific Bsg-overexpressing mice showed exacerbated RV hypertrophy, fibrosis, and dysfunction compared with their littermates under chronic hypoxia and pulmonary artery banding. Treatment with celastrol, which we identified as a suppressor of Bsg and CyPA by drug screening, decreased proliferation, reactive oxygen species, and inflammatory cytokines in pulmonary artery smooth muscle cells. Furthermore, celastrol treatment ameliorated RV systolic pressure, hypertrophy, fibrosis, and dysfunction in hypoxia-induced PH in mice and SU5416/hypoxia-induced PH in rats with reduced Bsg, CyPA, and inflammatory cytokines in the hearts and lungs. CONCLUSIONS: These results indicate that elevated Bsg in pressure-overloaded RV exacerbates RV dysfunction and that celastrol ameliorates RV dysfunction in PH model animals by suppressing Bsg and its ligand CyPA. Thus, celastrol can be a novel drug for PH and RV failure that targets Bsg and CyPA. Graphic Abstract: A graphic abstract is available for this article.

Identification of Long Noncoding RNA H19 as a New Biomarker and Therapeutic Target in Right Ventricular Failure in Pulmonary Arterial Hypertension.

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.

Identification of Adipsin as a Novel Prognostic Biomarker in Patients With Coronary Artery Disease.

Background Circulating proteins are exposed to vascular endothelial layer and influence their functions. Among them, adipsin is a member of the trypsin family of peptidases and is mainly secreted from adipocytes, monocytes, and macrophages, catalyzing the rate-limiting step of the alternative complement pathway. However, its pathophysiological role in cardiovascular disease remains to be elucidated. Here, we examined whether serum adipsin levels have a prognostic impact in patients with coronary artery disease. Methods and Results In 370 consecutive patients undergoing diagnostic coronary angiography, we performed a cytokine array analysis for screening serum levels of 50 cytokines/chemokines and growth factors. Among them, classification and regression analysis identified adipsin as the best biomarker for prediction of their long-term prognosis (median 71 months; interquartile range, 55-81 months). Kaplan-Meier curve showed that higher adipsin levels (≥400 ng/mL) were significantly associated with all-cause death (hazard ratio [HR], 4.2; 95% CI, 1.7-10.6 [P<0.001]) and rehospitalization (HR, 2.4; 95% CI, 1.7-3.5 [P<0.001]). Interestingly, higher high-sensitivity C-reactive protein levels (≥1 mg/L) were significantly correlated with all-cause death (HR, 3.2; 95% CI, 1.7-5.9 [P<0.001]) and rehospitalization (HR, 1.5, 95% CI, 1.1-1.9 [P<0.01]). Importantly, the combination of adipsin (≥400 ng/mL) and high-sensitivity C-reactive protein (≥1 mg/L) was more significantly associated with all-cause death (HR, 21.0; 95% CI, 2.9-154.1 [P<0.001]). Finally, the receiver operating characteristic curve demonstrated that serum adipsin levels predict the death caused by acute myocardial infarction in patients with coronary artery disease (C-statistic, 0.847). Conclusions These results indicate that adipsin is a novel biomarker that predicts all-cause death and rehospitalization in patients with coronary artery disease, demonstrating the novel aspects of the alternative complementary system in the pathogenesis of coronary artery disease.

Diagnostic and Prognostic Significance of Serum Levels of SeP (Selenoprotein P) in Patients With Pulmonary Hypertension.

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.

ADAMTS8 Promotes the Development of Pulmonary Arterial Hypertension and Right Ventricular Failure: A Possible Novel Therapeutic Target.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling with aberrant pulmonary artery smooth muscle cells (PASMCs) proliferation, endothelial dysfunction, and extracellular matrix remodeling. OBJECTIVE: Right ventricular (RV) failure is an important prognostic factor in PAH. Thus, we need to elucidate a novel therapeutic target in both PAH and RV failure. METHODS AND RESULTS: We performed microarray analysis in PASMCs from patients with PAH (PAH-PASMCs) and controls. We found a ADAMTS8 (disintegrin and metalloproteinase with thrombospondin motifs 8), a secreted protein specifically expressed in the lung and the heart, was upregulated in PAH-PASMCs and the lung in hypoxia-induced pulmonary hypertension (PH) in mice. To elucidate the role of ADAMTS8 in PH, we used vascular smooth muscle cell-specific ADAMTS8-knockout mice (ADAMTSΔSM22). Hypoxia-induced PH was attenuated in ADAMTSΔSM22 mice compared with controls. ADAMTS8 overexpression increased PASMC proliferation with downregulation of AMPK (AMP-activated protein kinase). In contrast, deletion of ADAMTS8 reduced PASMC proliferation with AMPK upregulation. Moreover, deletion of ADAMTS8 reduced mitochondrial fragmentation under hypoxia in vivo and in vitro. Indeed, PASMCs harvested from ADAMTSΔSM22 mice demonstrated that phosphorylated DRP-1 (dynamin-related protein 1) at Ser637 was significantly upregulated with higher expression of profusion genes (Mfn1 and Mfn2) and improved mitochondrial function. Moreover, recombinant ADAMTS8 induced endothelial dysfunction and matrix metalloproteinase activation in an autocrine/paracrine manner. Next, to elucidate the role of ADAMTS8 in RV function, we developed a cardiomyocyte-specific ADAMTS8 knockout mice (ADAMTS8ΔαMHC). ADAMTS8ΔαMHC mice showed ameliorated RV failure in response to chronic hypoxia. In addition, ADAMTS8ΔαMHC mice showed enhanced angiogenesis and reduced RV ischemia and fibrosis. Finally, high-throughput screening revealed that mebendazole, which is used for treatment of parasite infections, reduced ADAMTS8 expression and cell proliferation in PAH-PASMCs and ameliorated PH and RV failure in PH rodent models. CONCLUSIONS: These results indicate that ADAMTS8 is a novel therapeutic target in PAH.

Identification of Emetine as a Therapeutic Agent for Pulmonary Arterial Hypertension: Novel Effects of an Old Drug.

OBJECTIVE: Excessive proliferation and apoptosis resistance are special characteristics of pulmonary artery smooth muscle cells (PASMCs) in pulmonary arterial hypertension (PAH). However, the drugs in clinical use for PAH target vascular dilatation, which do not exert adequate effects in patients with advanced PAH. Here, we report a novel therapeutic effect of emetine, a principal alkaloid extracted from the root of ipecac clinically used as an emetic and antiprotozoal drug. Approach and Results: We performed stepwise screenings for 5562 compounds from original library. First, we performed high-throughput screening with PASMCs from patients with PAH (PAH-PASMCs) and found 80 compounds that effectively inhibited proliferation. Second, we performed the repeatability and counter assay. Finally, we performed a concentration-dependent assay and found that emetine inhibits PAH-PASMC proliferation. Interestingly, emetine significantly reduced protein levels of HIFs (hypoxia-inducible factors; HIF-1α and HIF-2α) and downstream PDK1 (pyruvate dehydrogenase kinase 1). Moreover, emetine significantly reduced the protein levels of RhoA (Ras homolog gene family, member A), Rho-kinases (ROCK1 and ROCK2 [rho-associated coiled-coil containing protein kinases 1 and 2]), and their downstream CyPA (cyclophilin A), and Bsg (basigin) in PAH-PASMCs. Consistently, emetine treatment significantly reduced the secretion of cytokines/chemokines and growth factors from PAH-PASMCs. Interestingly, emetine reduced protein levels of BRD4 (bromodomain-containing protein 4) and downstream survivin, both of which are involved in many cellular functions, such as cell cycle, apoptosis, and inflammation. Finally, emetine treatment ameliorated pulmonary hypertension in 2 experimental rat models, accompanied by reduced inflammatory changes in the lungs and recovered right ventricular functions. CONCLUSIONS: Emetine is an old but novel drug for PAH that reduces excessive proliferation of PAH-PASMCs and improves right ventricular functions.

Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) accompanying increased production of inflammatory factors and adaptation of the mitochondrial metabolism to a hyperproliferative state. However, all the drugs in clinical use target pulmonary vascular dilatation, which may not be effective for patients with advanced PAH. OBJECTIVE: We aimed to discover a novel drug for PAH that inhibits PASMC proliferation. METHODS AND RESULTS: We screened 5562 compounds from original library using high-throughput screening system to discover compounds which inhibit proliferation of PASMCs from patients with PAH (PAH-PASMCs). We found that celastramycin, a benzoyl pyrrole-type compound originally found in a bacteria extract, inhibited the proliferation of PAH-PASMCs in a dose-dependent manner with relatively small effects on PASMCs from healthy donors. Then, we made 25 analogs of celastramycin and selected the lead compound, which significantly inhibited cell proliferation of PAH-PASMCs and reduced cytosolic reactive oxygen species levels. Mechanistic analysis demonstrated that celastramycin reduced the protein levels of HIF-1α (hypoxia-inducible factor 1α), which impairs aerobic metabolism, and κB (nuclear factor-κB), which induces proinflammatory signals, in PAH-PASMCs, leading to reduced secretion of inflammatory cytokine. Importantly, celastramycin treatment reduced reactive oxygen species levels in PAH-PASMCs with increased protein levels of Nrf2 (nuclear factor erythroid 2-related factor 2), a master regulator of cellular response against oxidative stress. Furthermore, celastramycin treatment improved mitochondrial energy metabolism with recovered mitochondrial network formation in PAH-PASMCs. Moreover, these celastramycin-mediated effects were regulated by ZFC3H1 (zinc finger C3H1 domain-containing protein), a binding partner of celastramycin. Finally, celastramycin treatment ameliorated pulmonary hypertension in 3 experimental animal models, accompanied by reduced inflammatory changes in the lungs. CONCLUSIONS: These results indicate that celastramycin ameliorates pulmonary hypertension, reducing excessive proliferation of PAH-PASMCs with less inflammation and reactive oxygen species levels, and recovered mitochondrial energy metabolism. Thus, celastramycin is a novel drug for PAH that targets antiproliferative effects on PAH-PASMCs.

Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease.

RATIONALE: Pulmonary hypertension (PH) due to left heart disease (LHD), or group 2 PH, is the most prevalent form of PH worldwide. PH due to LHD is often associated with metabolic syndrome (MetS). In 12% to 13% of cases, patients with PH due to LHD display vascular remodeling of pulmonary arteries (PAs) associated with poor prognosis. Unfortunately, the underlying mechanisms remain unknown; PH-targeted therapies for this group are nonexistent, and the development of a new preclinical model is crucial. Among the numerous pathways dysregulated in MetS, inflammation plays also a critical role in both PH and vascular remodeling. OBJECTIVE: We hypothesized that MetS and inflammation may trigger the development of vascular remodeling in group 2 PH. METHODS AND RESULTS: Using supracoronary aortic banding, we induced diastolic dysfunction in rats. Then we induced MetS by a combination of high-fat diet and olanzapine treatment. We used metformin treatment and anti-IL-6 (interleukin-6) antibodies to inhibit the IL-6 pathway. Compared with sham conditions, only supracoronary aortic banding+MetS rats developed precapillary PH, as measured by both echocardiography and right/left heart catheterization. PH in supracoronary aortic banding+MetS was associated with macrophage accumulation and increased IL-6 production in lung. PH was also associated with STAT3 (signal transducer and activator of transcription 3) activation and increased proliferation of PA smooth muscle cells, which contributes to remodeling of distal PA. We reported macrophage accumulation, increased IL-6 levels, and STAT3 activation in the lung of group 2 PH patients. In vitro, IL-6 activates STAT3 and induces human PA smooth muscle cell proliferation. Metformin treatment decreased inflammation, IL-6 levels, STAT3 activation, and human PA smooth muscle cell proliferation. In vivo, in the supracoronary aortic banding+MetS animals, reducing IL-6, either by anti-IL-6 antibody or metformin treatment, reversed pulmonary vascular remodeling and improve PH due to LHD. CONCLUSIONS: We developed a new preclinical model of group 2 PH by combining MetS with LHD. We showed that MetS exacerbates group 2 PH. We provided evidence for the importance of the IL-6-STAT3 pathway in our experimental model of group 2 PH and human patients.