RESUMEN
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
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Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.
Asunto(s)
Hipertensión Arterial Pulmonar , Transducción de Señal , Remodelación Vascular , Animales , Remodelación Vascular/efectos de los fármacos , Remodelación Vascular/fisiología , Ratas , Humanos , Hipertensión Arterial Pulmonar/fisiopatología , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Hipertensión Arterial Pulmonar/metabolismo , Masculino , Modelos Animales de Enfermedad , Arteria Pulmonar/fisiopatología , Arteria Pulmonar/efectos de los fármacos , Ratones , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Factor 5A Eucariótico de Iniciación de Traducción , Proliferación Celular/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Hipertensión Pulmonar/fisiopatología , Hipertensión Pulmonar/metabolismo , Lisina/análogos & derivadosRESUMEN
BACKGROUND: The ability of the right ventricle (RV) to adapt to an increased pressure afterload determines survival in patients with pulmonary arterial hypertension. At present, there are no specific treatments available to prevent RV failure, except for heart/lung transplantation. The wingless/int-1 (Wnt) signaling pathway plays an important role in the development of the RV and may also be implicated in adult cardiac remodeling. METHODS: Molecular, biochemical, and pharmacological approaches were used both in vitro and in vivo to investigate the role of Wnt signaling in RV remodeling. RESULTS: Wnt/ß-catenin signaling molecules are upregulated in RV of patients with pulmonary arterial hypertension and animal models of RV overload (pulmonary artery banding-induced and monocrotaline rat models). Activation of Wnt/ß-catenin signaling leads to RV remodeling via transcriptional activation of FOSL1 and FOSL2 (FOS proto-oncogene [FOS] like 1/2, AP-1 [activator protein 1] transcription factor subunit). Immunohistochemical analysis of pulmonary artery banding -exposed BAT-Gal (ß-catenin-activated transgene driving expression of nuclear ß-galactosidase) reporter mice RVs exhibited an increase in ß-catenin expression compared with their respective controls. Genetic inhibition of ß-catenin, FOSL1/2, or WNT3A stimulation of RV fibroblasts significantly reduced collagen synthesis and other remodeling genes. Importantly, pharmacological inhibition of Wnt signaling using inhibitor of PORCN (porcupine O-acyltransferase), LGKK-974 attenuated fibrosis and cardiac hypertrophy leading to improvement in RV function in both, pulmonary artery banding - and monocrotaline-induced RV overload. CONCLUSIONS: Wnt- ß-Catenin-FOSL signaling is centrally involved in the hypertrophic RV response to increased afterload, offering novel targets for therapeutic interference with RV failure in pulmonary hypertension.
Asunto(s)
Insuficiencia Cardíaca , Hipertensión Arterial Pulmonar , Ratas , Ratones , Animales , Remodelación Ventricular , beta Catenina , Cateninas , Monocrotalina/toxicidad , Transducción de Señal , Modelos Animales de Enfermedad , Función Ventricular DerechaRESUMEN
Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling of small pulmonary arteries (PAs) causing sustained elevation of PA pressure, right ventricular failure, and death. Similar to cancer cells, PA smooth muscle cells (PASMCs), which play a key role in pulmonary vascular remodeling, have adopted multiple mechanisms to sustain their survival and proliferation in the presence of stress. The histone methyltransferase G9a and its partner protein GLP (G9a-like protein) have been shown to exert oncogenic effects and to serve as a buffer against an exaggerated transcriptional response. Therefore, we hypothesized that upregulation of G9a and GLP in PAH plays a pivotal role in pulmonary vascular remodeling by maintaining the abnormal phenotype of PAH-PASMCs. We found that G9a is increased in PASMCs from patients with PAH as well as in remodeled PAs from animal models. Pharmacological inhibition of G9a/GLP activity using BIX01294 and UNC0642 significantly reduced the prosurvival and proproliferative potentials of cultured PAH-PASMCs. Using RNA sequencing, further exploration revealed that G9a/GLP promotes extracellular matrix production and affords protection against the negative effects of an overactive stress response. Finally, we found that therapeutic treatment with BIX01294 reduced pulmonary vascular remodeling and lowered mean PA pressure in fawn-hooded rats. Treatment of Sugen/hypoxia-challenged mice with BIX01294 also improved pulmonary hemodynamics and right ventricular function. In conclusion, these findings indicate that G9a/GLP inhibition may represent a new therapeutic approach in PAH.
Asunto(s)
Hipertensión Arterial Pulmonar , Ratas , Ratones , Animales , Hipertensión Arterial Pulmonar/tratamiento farmacológico , Remodelación Vascular , Proliferación Celular , Hipertensión Pulmonar Primaria Familiar , Modelos Animales de Enfermedad , Miocitos del Músculo Liso , Arteria PulmonarRESUMEN
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.
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RATIONALE: The MSTs (mammalian Ste20-like kinases) 1/2 are members of the HIPPO pathway that act as growth suppressors in adult proliferative diseases. Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small PAs, pulmonary vascular remodeling, and the rise of pulmonary arterial pressure. The role of MST1/2 in PAH is currently unknown. OBJECTIVE: To investigate the roles and mechanisms of the action of MST1 and MST2 in PAH. METHODS AND RESULTS: Using early-passage pulmonary vascular cells from PAH and nondiseased lungs and mice with smooth muscle-specific tamoxifen-inducible Mst1/2 knockdown, we found that, in contrast to canonical antiproliferative/proapoptotic roles, MST1/2 act as proproliferative/prosurvival molecules in human PAH pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts and support established pulmonary vascular remodeling and pulmonary hypertension in mice with SU5416/hypoxia-induced pulmonary hypertension. By using unbiased proteomic analysis, gain- and loss-of function approaches, and pharmacological inhibition of MST1/2 kinase activity by XMU-MP-1, we next evaluated mechanisms of regulation and function of MST1/2 in PAH pulmonary vascular cells. We found that, in PAH pulmonary arterial adventitial fibroblasts, the proproliferative function of MST1/2 is caused by IL-6-dependent MST1/2 overexpression, which induces PSMC6-dependent downregulation of forkhead homeobox type O 3 and hyperproliferation. In PAH pulmonary arterial vascular smooth muscle cells, MST1/2 acted via forming a disease-specific interaction with BUB3 and supported ECM (extracellular matrix)- and USP10-dependent BUB3 accumulation, upregulation of Akt-mTORC1, cell proliferation, and survival. Supporting our in vitro observations, smooth muscle-specific Mst1/2 knockdown halted upregulation of Akt-mTORC1 in small muscular PAs of mice with SU5416/hypoxia-induced pulmonary hypertension. CONCLUSIONS: Together, this study describes a novel proproliferative/prosurvival role of MST1/2 in PAH pulmonary vasculature, provides a novel mechanistic link from MST1/2 via BUB3 and forkhead homeobox type O to the abnormal proliferation and survival of pulmonary arterial vascular smooth muscle cells and pulmonary arterial adventitial fibroblasts, remodeling and pulmonary hypertension, and suggests new target pathways for therapeutic intervention.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Factores de Transcripción Forkhead/metabolismo , Hipertensión Pulmonar , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Hipertensión Arterial Pulmonar , Animales , Proliferación Celular , Células Cultivadas , Hipertensión Pulmonar/metabolismo , Hipoxia/metabolismo , Mamíferos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Miocitos del Músculo Liso/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Hipertensión Arterial Pulmonar/genética , Arteria Pulmonar/metabolismo , Remodelación Vascular/fisiologíaRESUMEN
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.
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Hipertensión Pulmonar , Fibrosis Pulmonar Idiopática , Animales , Fibroblastos/metabolismo , Humanos , Hipertensión Pulmonar/tratamiento farmacológico , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/metabolismo , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
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.
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Proteína Potenciadora del Homólogo Zeste 2/genética , Proteoma/genética , Hipertensión Arterial Pulmonar/genética , Transcriptoma/genética , Animales , Apoptosis/genética , Proliferación Celular/genética , Ciclo del Ácido Cítrico/genética , Epigénesis Genética/genética , Femenino , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Humanos , Pulmón/metabolismo , Pulmón/patología , Masculino , Persona de Mediana Edad , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Hipertensión Arterial Pulmonar/patología , Arteria Pulmonar/crecimiento & desarrollo , Arteria Pulmonar/patología , RatasRESUMEN
Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.
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Fármacos Cardiovasculares/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hipertensión Pulmonar/tratamiento farmacológico , Hipoxia/prevención & control , Miocitos del Músculo Liso/efectos de los fármacos , Trifluoperazina/farmacología , Animales , Antipsicóticos/farmacología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Reposicionamiento de Medicamentos , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Hemodinámica/efectos de los fármacos , Humanos , Hipertensión Pulmonar/inducido químicamente , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/fisiopatología , Hipoxia/inducido químicamente , Hipoxia/genética , Hipoxia/fisiopatología , Indoles/administración & dosificación , Monocrotalina/administración & dosificación , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Pirroles/administración & dosificación , Ratas , Ratas Sprague-Dawley , Survivin/genética , Survivin/metabolismoRESUMEN
Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness.Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets.Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH.Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro, molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function.Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.
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Enzimas Reparadoras del ADN/genética , ADN/metabolismo , Estrés Oxidativo/genética , Monoéster Fosfórico Hidrolasas/genética , Hipertensión Arterial Pulmonar/genética , Arteria Pulmonar/metabolismo , Remodelación Vascular/genética , 8-Hidroxi-2'-Desoxicoguanosina/metabolismo , Adulto , Anciano , Animales , Apoptosis/genética , Western Blotting , Estudios de Casos y Controles , Proliferación Celular/genética , Cromatografía Liquida , Ensayo Cometa , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteína Forkhead Box M1/metabolismo , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Oxidación-Reducción , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Hipertensión Arterial Pulmonar/metabolismo , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Espectrometría de Masas en Tándem , Regulación hacia ArribaRESUMEN
OBJECTIVE: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by the narrowing of pulmonary arteries (PAs). It is now established that this phenotype is associated with enhanced PA smooth muscle cells (PASMCs) proliferation and suppressed apoptosis. This phenotype is sustained in part by the activation of several DNA repair pathways allowing PASMCs to survive despite the unfavorable environmental conditions. PIM1 (Moloney murine leukemia provirus integration site) is an oncoprotein upregulated in PAH and involved in many prosurvival pathways, including DNA repair. The objective of this study was to demonstrate the implication of PIM1 in the DNA damage response and the beneficial effect of its inhibition by pharmacological inhibitors in human PAH-PASMCs and in rat PAH models. Approach and Results: We found in vitro that PIM1 inhibition by either SGI-1776, TP-3654, siRNA (silencer RNA) decreased the phosphorylation of its newly identified direct target KU70 (lupus Ku autoantigen protein p70) resulting in the inhibition of double-strand break repair (Comet Assay) by the nonhomologous end-joining as well as reduction of PAH-PASMCs proliferation (Ki67-positive cells) and resistance to apoptosis (Annexin V positive cells) of PAH-PASMCs. In vivo, SGI-1776 and TP-3654 given 3× a week, improved significantly pulmonary hemodynamics (right heart catheterization) and vascular remodeling (Elastica van Gieson) in monocrotaline and Fawn-Hooded rat models of PAH. CONCLUSIONS: We demonstrated that PIM1 phosphorylates KU70 and initiates DNA repair signaling in PAH-PASMCs and that PIM1 inhibitors represent a therapeutic option for patients with PAH.