Pathology Of Pulmonary Arterial Hypertension Research Articles

Proline and glucose metabolic reprogramming supports vascular endothelial and medial biomass in pulmonary arterial hypertension.

In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-Seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all aa and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest that elevated glucose and proline avidity underlie increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.

Transpulmonary generation of cell-free hemoglobin contributes to vascular dysfunction in pulmonary arterial hypertension via dysregulated clearance mechanisms.

Circulating cell-free hemoglobin (CFH) is elevated in pulmonary arterial hypertension (PAH) and associated with poor outcomes but the mechanisms are unknown. We hypothesized that CFH is generated from the pulmonary circulation and inadequately cleared in PAH. Transpulmonary CFH (difference between wedge and pulmonary artery positions) and lung hemoglobin α were analyzed in patients with PAH and healthy controls. Haptoglobin genotype and plasma hemoglobin processing proteins were analyzed in patients with PAH, unaffected bone morphogenetic protein receptor type II mutation carriers (UMCs), and control subjects. Transpulmonary CFH was increased in patients with PAH (p = 0.04) and correlated with pulmonary vascular resistanc(PVR) (r s = 0.75, p = 0.02) and mean pulmonary arterial pressure(mPAP) (r s = 0.78, p = 0.02). Pulmonary vascular hemoglobin α protein was increased in patients with PAH (p = 0.006), especially in occluded vessels (p = 0.04). Haptoglobin genotype did not differ between groups. Plasma haptoglobin was higher in UMCs compared with both control subjects (p = 0.03) and patients with HPAH (p < 0.0001); patients with IPAH had higher circulating haptoglobin levels than patients with HPAH (p = 0.006). Notably, circulating CFH to haptoglobin ratio was elevated in patients with HPAH compared to control subjects (p = 0.02) and UMCs (p = 0.006). Moreover, in patients with PAH, CFH: haptoglobin correlated with PVR (r s = 0.37, p = 0.0004) and mPAP (r s = 0.25, p = 0.02). Broad alterations in other plasma hemoglobin processing proteins (hemopexin, heme oxygenase-1, and sCD163) were observed. In conclusion, pulmonary vascular CFH is associated with increased PVR and mPAP in PAH and dysregulated CFH clearance may contribute to PAH pathology. Further study is needed to determine whether targeting CFH is a viable therapeutic for pulmonary vascular dysfunction in PAH.

Cross-talk between TSC2 and the extracellular matrix controls pulmonary vascular proliferation and pulmonary hypertension.

Increased proliferation and survival of cells in small pulmonary arteries (PAs) drive pulmonary arterial hypertension (PAH). Because cell growth mediated by the mTOR-containing mTORC1 complex is inhibited by tuberous sclerosis complex 2 (TSC2), we investigated the role of this GTPase-activating protein in PAH pathology. TSC2 abundance was decreased in remodeled small PAs and PA vascular smooth muscle cells (PAVSMCs) from patients with PAH or from rodent pulmonary hypertension (PH) models, as well as PAVSMCs maintained on substrates that reproduced pathology-induced stiffness. Accordingly, mice with smooth muscle-specific reduction in TSC2 developed PH. At the molecular level, decreased TSC2 abundance led to stiffness-induced PAVSMC proliferation, increased abundance of the mechanosensitive transcriptional coactivators YAP/TAZ, and enhanced mTOR kinase activity. Moreover, extracellular matrix (ECM) produced by TSC2-deficient PAVSMCs stimulated the proliferation of nondiseased PA adventitial fibroblasts and PAVSMCs through fibronectin and its receptor, the α5β1 integrin. Reconstituting TSC2 in PAVSMCs from patients with PAH through overexpression or treatment with the SIRT1 activator SRT2104 decreased YAP/TAZ abundance, mTOR activity, and ECM production, as well as inhibited proliferation and induced apoptosis. In two rodent models of PH, SRT2104 treatment restored TSC2 abundance, attenuated pulmonary vascular remodeling, and ameliorated PH. Thus, TSC2 in PAVSMCs integrates ECM composition and stiffness with pro-proliferative and survival signaling, and restoring TSC2 abundance could be an attractive therapeutic option to treat PH.

Diagnosis and management of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary vasculature that is characterised by elevated pressures within the pulmonary vascular tree. Recent decades have witnessed a dramatic expansion in our understanding of the pathobiology and the epidemiology of PAH, and improvements in treatment options and outcomes. The prevalence of PAH is estimated to be between 48 and 55 cases per million adults. The definition was recently amended and a diagnosis of PAH now requires evidence of a mean pulmonary artery pressure >20 mmHg, a pulmonary vascular resistance >2 Wood units and a pulmonary artery wedge pressure ≤15 mmHg at right heart catheterisation. Detailed clinical assessment and a number of additional diagnostic tests are required to assign a clinical group. Biochemistry, echocardiography, lung imaging and pulmonary function tests provide valuable information to assist in the assignment of a clinical group. Risk assessment tools have been refined, and these greatly facilitate risk stratification and enhance treatment decisions and prognostication. Current therapies target three therapeutic pathways: the nitric oxide, prostacyclin and endothelin pathways. While lung transplantation remains the only curative intervention for PAH, there are a number of promising therapies under investigation which may further reduce morbidity and improve outcomes. This review describes the epidemiology, pathology and pathobiology of PAH and introduces important concepts regarding the diagnosis and risk stratification of PAH. The management of PAH is also discussed, with a special focus on PAH specific therapy and key supportive measures.

Pipersentan: A De Novo Synthetic Endothelin Receptor Antagonist that Inhibits Monocrotaline- and Hypoxia-Induced Pulmonary Hypertension.

Background: Although major advances have been made in the pathogenesis and management of pulmonary arterial hypertension (PAH), the endothelin system is still considered to play a vital role in the pathology of PAH due to its vasoconstrictive action. Endothelin receptor antagonists (ERAs), either as monotherapy or in combination with other drugs, have attracted much attention in the treatment of this lethal disease, and research is continuing. Methods: A novel ERA, pipersentan 5-(1,3-Benzodioxol-5-yl)-6-[2-(5-bromopyrimidin-2-yl)oxyethoxy]-N-(2-methoxyethylsulfamoyl)pyrimidin-4-amine, was recently synthesized and the physicochemical characterizations and the pharmacology both in vitro and in vivo were studied. Results: This orally administered ERA can both competitively and selectively inhibit the binding of endothelin-1 (ET-1) to its receptors with good physicochemical characteristics. Pipersentan efficaciously antagonized the effects of ET-1 on pulmonary artery smooth muscle cell proliferation, migration and calcium mobilization and effectively improved right ventricular hypertrophy and pulmonary arterial pressure in both monocrotaline- and hypoxia-induced pulmonary hypertension (PH) rat models. Conclusions: This profile identifies pipersentan as a new agent for treating ET-1 system activation-related PH.

A Literature Review of Pulmonary Arterial Hypertension (PAH)

Abstract: In 1891, German doctor E. Romberg was the first to report PAH. It is widespread throughout the world, but it is particularly problematic in India and other developing countries. Pulmonary arterial hypertension (PAH) is characterised by an increase in pulmonary arterial pres-sure as well as the emergence of progressive symptoms, such as a loss of functional ability, short-ness of breath, and fatigue. Blood flows from the right side of the heart to the lungs through the pulmonary arteries. Pulmonary arterial pressure refers to the pressure in the arteries of the lungs (PAH). It necessitates immediate treatment because high blood pressure in the lungs causes the right side of the heart to work much harder, increasing the risk of heart failure. This article aimed to provide brief information about the prevalence, pathology, classification, and different therapies of PAH.

Mining the Plasma Proteome for Insights into the Molecular Pathology of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. Objectives: To quantify and analyze the plasma proteome of patients with PAH using inherited genetic variation to inform on underlying molecular drivers. Methods: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers, and 23 relatives of patients with PAH. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared, and the relationship to transplantation-free survival in PAH was determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomization (MR) analysis. Measurements and Main Results: From 4,152 annotated plasma proteins, levels of 208 differed between patients with PAH and healthy subjects, and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR, 0.83; 95% CI, 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. Conclusions: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

Hsa_circWDR37_016 Regulates Hypoxia-Induced Proliferation of Pulmonary Arterial Smooth Muscle Cells.

Pulmonary arterial hypertension (PAH) is characterized by abnormal remodeling of pulmonary vessel walls caused by excessive pulmonary arterial smooth muscle cell (PASMC) proliferation. Our previous clinical studies have demonstrated the importance of the downregulated circRNA in PAH. However, the role of upregulated circRNAs is still elusive. Here, we identified the upregulated circRNA in PAH patients, hsa_circWDR37_016 (circWDR37), as a key regulator of hypoxic proliferative disorder of pulmonary arterial smooth muscle cells (PASMCs). Quantitative real-time PCR (qRT-PCR) analysis validated that exposure to hypoxia markedly increased the circWDR37 level in cultured human PASMCs. As evidenced by flow cytometry, 5-ethynyl-2′-deoxyuridine (EdU) incorporation, wound healing, and Tunel assay, silencing of endogenous circWDR37 attenuated proliferation and cell-cycle progression in hypoxia-exposed human PASMCs in vitro. Furthermore, bioinformatics and Luciferase assay showed that circWDR37 directly sponged hsa-miR-138-5p (miR-138) and was involved in the immunoregulatory and inflammatory processes of PAH. Together, these studies suggested new insights into circRNA regulated the pathology of PAH, providing a new potential therapeutic target for PAH treatment.

Epigenetic Mechanisms as Emerging Therapeutic Targets and Microfluidic Chips Application in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a disease that progress over time and is defined as an increase in pulmonary arterial pressure and pulmonary vascular resistance that frequently leads to right-ventricular (RV) failure and death. Epigenetic modifications comprising DNA methylation, histone remodeling, and noncoding RNAs (ncRNAs) have been established to govern chromatin structure and transcriptional responses in various cell types during disease development. However, dysregulation of these epigenetic mechanisms has not yet been explored in detail in the pathology of pulmonary arterial hypertension and its progression with vascular remodeling and right-heart failure (RHF). Targeting epigenetic regulators including histone methylation, acetylation, or miRNAs offers many possible candidates for drug discovery and will no doubt be a tempting area to explore for PAH therapies. This review focuses on studies in epigenetic mechanisms including the writers, the readers, and the erasers of epigenetic marks and targeting epigenetic regulators or modifiers for treatment of PAH and its complications described as RHF. Data analyses from experimental cell models and animal induced PAH models have demonstrated that significant changes in the expression levels of multiple epigenetics modifiers such as HDMs, HDACs, sirtuins (Sirt1 and Sirt3), and BRD4 correlate strongly with proliferation, apoptosis, inflammation, and fibrosis linked to the pathological vascular remodeling during PAH development. The reversible characteristics of protein methylation and acetylation can be applied for exploring small-molecule modulators such as valproic acid (HDAC inhibitor) or resveratrol (Sirt1 activator) in different preclinical models for treatment of diseases including PAH and RHF. This review also presents to the readers the application of microfluidic devices to study sex differences in PAH pathophysiology, as well as for epigenetic analysis.

PH-ILD: Identification, Evaluation, and Monitoring: A Diagnostic View From Both Sides

PH-ILD: Identification, Evaluation, and Monitoring: A Diagnostic View From Both Sides

PULMONARY ARTERIAL HYPERTENSION AND INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS: A NOVEL ASSOCIATION?

PULMONARY ARTERIAL HYPERTENSION AND INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS: A NOVEL ASSOCIATION?

Ion channels as convergence points in the pathology of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a fatal disease of the cardiopulmonary system that lacks curative treatments. The main pathological event in PAH is elevated vascular resistance in the pulmonary circulation, caused by abnormal vasoconstriction and vascular remodelling. Ion channels are key determinants of vascular smooth muscle tone and homeostasis, and four PAH channelopathies (KCNK3, ABCC8, KCNA5, TRPC6) have been identified so far. However, the contribution of ion channels in other forms of PAH, which account for the majority of PAH patients, has been less well characterised. Here we reason that a variety of triggers of PAH (e.g. BMPR2 mutations, hypoxia, anorectic drugs) that impact channel function may contribute to the onset of the disease. We review the molecular mechanisms by which these ‘extrinsic’ factors converge on ion channels and provoke their dysregulation to promote the development of PAH. Ion channels of the pulmonary vasculature are therefore promising therapeutic targets because of the modulation they provide to both vasomotor tone and proliferation of arterial smooth muscle cells.

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by a progressive increase in pulmonary artery pressure due to an increase in vessel tone and occlusion of vessels. The endogenous vasodilator prostacyclin and its analogs are used as therapeutic agents for PAH. However, their pharmacological effects on occlusive vascular remodeling have not been elucidated yet. Selexipag is a recently approved, orally available and selective prostacyclin receptor agonist with a non-prostanoid structure. In this study, we investigated the pharmacological effects of selexipag on the pathology of chronic severe PAH in Sprague-Dawley and Fischer rat models in which PAH was induced by a combination of injection with the vascular endothelial growth factor receptor antagonist Sugen 5416 and exposure to hypoxia (SuHx). Oral administration of selexipag for three weeks significantly improved right ventricular systolic pressure and right ventricular (RV) hypertrophy in Sprague-Dawley SuHx rats. Selexipag attenuated the proportion of lung vessels with occlusive lesions and the medial wall thickness of lung arteries, corresponding to decreased numbers of Ki-67-positive cells and a reduced expression of collagen type 1 in remodeled vessels. Administration of selexipag to Fischer rats with SuHx-induced PAH reduced RV hypertrophy and mortality caused by RV failure. These effects were probably based on the potent prostacyclin receptor agonistic effect of selexipag on pulmonary vessels. Selexipag has been approved and is used in the clinical treatment of PAH worldwide. It is thought that these beneficial effects of prostacyclin receptor agonists on multiple aspects of PAH pathology contribute to the clinical outcomes in patients with PAH.

Potential damage in pulmonary arterial hypertension: An experimental study of pressure-induced damage of pulmonary artery.

Pulmonary arterial hypertension (PAH) is associated with elevated pulmonary arterial pressure. PAH prognosis remains poor with a 15% mortality rate within 1 year, even with modern clinical management. Previous clinical studies proposed wall shear stress (WSS) to be an important hemodynamic factor affecting cell mechanotransduction, growth and remodeling, and disease progress in PAH. However, WSS in vivo is typically at most 2.5 Pa and a doubt has been cast whether WSS alone can drive disease progress. Furthermore, our current understanding of PAH pathology largely comes from small animals' studies in which caliber enlargement, a hallmark of PAH in humans, is rarely reported. Therefore, a large-animal experiment on pulmonary arteries (PAs) is needed to validate whether increased pressure can induce enlargement of PAs caliber. In this study, we use an inflation testing device to characterize the mechanical behavior, both nonlinear elastic behavior and irreversible damage of porcine arteries. The parameters of elastic behavior are estimated from the inflation test at a low-pressure range before and after over-pressurization. Then, histological images are qualitatively examined for medial and adventitial layers. This study sheds light on the relevance of pressure-induced damage mechanism in human PAH.

Multimodal Imaging Mass Spectrometry to Identify Markers of Pulmonary Arterial Hypertension in Human Lung Tissue Using MALDI-ToF, ToF-SIMS, and Hybrid SIMS.

Pulmonary arterial hypertension (PAH) is a rare and deadly disease affecting roughly 15-60 people per million in Europe with a poorly understood pathology. There are currently no diagnostic tools for early detection nor does a curative treatment exist. The lipid composition of arteries in lung tissue samples from human PAH and control patients were investigated using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) combined with time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging. Using random forests as an IMS data analysis technique, it was possible to identify the ion at m/z 885.6 as a marker of PAH in human lung tissue. The m/z 885.6 ion intensity was shown to be significantly higher around diseased arteries and was confirmed to be a diacylglycerophosphoinositol PI(C18:0/C20:4) via MS/MS using a novel hybrid SIMS instrument. The discovery of a potential biomarker opens up new research avenues which may finally lead to a better understanding of the PAH pathology and highlights the vital role IMS can play in modern biomedical research.

Plasma insulin-like growth factor binding protein 1 in pulmonary arterial hypertension

Background Beside the pulmonary vasoconstriction observed in pulmonary arterial hypertension (PAH), severe proliferative and antiapoptotic cellular phenotypes result in vascular remodelling. Many recent findings indicate similarities between PAH and tumour pathology. For instance, insulin-like growth factor (IGF)-1 signalling, which is known to promote tumour development, is implicated in PAH. Higher circulating IGF binding protein (IGFBP)-1 levels are associated with worse survival in PAH. The present study aimed to investigate the relationship between plasma levels of various tumour-related biomarkers and PAH. Methods: IGFBP-1, -2 and -7, along with other tumour-related biomarkers, were measured in plasma from 48 treatment-naïve PAH patients and 16 healthy controls, using proximity extension assays. Among the PAH patients, 33 were also studied at an early treatment follow-up. Results: Plasma IGFBP-1 (p < .003), IGFBP-2 (p < .001), IGFBP-7 (p < .008), vimentin (p < .001), carbonic anhydrase 9 (p < .001), S100A11 (p < .001), human epididymis protein 4 (p < .001) and folate receptor-α (p < .004) were elevated in PAH, compared to controls. IGFBP-1 exhibited the most interesting correlations to clinical parameters and was selected for further analyses. IGFBP-1 correlated specifically to N-terminal prohormone of brain natriuretic peptide (NT-proBNP) (r = 0.44, p < .002), mean right atrial pressure (r = 0.41, p < .004), venous oxygen saturation (r = –0.43, p < .003), cardiac index (r = –0.32, p < .03) and 6-minute walking distance (r = –0.29, p < .05). Plasma IGFBP-1 also correlated to risk scores based on the European Society of Cardiology/European Respiratory Society (ESC/ERS) PAH guidelines (r = 0.43, p < .003) and the REVEAL model (r = 0.46, p < .001). PAH patients with supra-median baseline IGFBP-1 levels showed a trend for worse overall survival than those with infra-median levels (p = .087). IGFBP-1 was unaltered between baseline and an early treatment follow-up. However, IGFBP-1 changes, between baseline and follow-up, correlated to changes in NT-proBNP (r = 0.48, p < .006). Conclusion: Plasma IGFBP-1 levels at PAH diagnosis show moderate association to NT-proBNP and hemodynamics as well as with ESC/ERS and REVEAL risk scores.

Endothelin Receptor Antagonists: Status Quo and Future Perspectives for Targeted Therapy.

The endothelin axis, recognized for its vasoconstrictive action, plays a central role in the pathology of pulmonary arterial hypertension (PAH). Treatment with approved endothelin receptor antagonists (ERAs), such as bosentan, ambrisentan, or macitentan, slow down PAH progression and relieves symptoms. Several findings have indicated that endothelin is further involved in the pathogenesis of certain other diseases, making ERAs potentially beneficial in the treatment of various conditions. In addition to PAH, this review summarizes the use and perspectives of ERAs in cancer, renal disease, fibrotic disorders, systemic scleroderma, vasospasm, and pain management. Bosentan has proven to be effective in systemic sclerosis PAH and in decreasing the development of vasospasm-related digital ulcers. The selective ERA clazosentan has been shown to be effective in preventing cerebral vasospasm and delaying ischemic neurological deficits and new infarcts. Furthermore, in the SONAR (Study Of Diabetic Nephropathy With Atrasentan) trial, the selective ERA atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease. These data suggest atrasentan as a new therapy in the treatment of diabetic nephropathy and possibly other renal diseases. Preclinical studies regarding heart failure, cancer, and fibrotic diseases have demonstrated promising effects, but clinical trials have not yet produced measurable results. Nevertheless, the potential benefits of ERAs may not be fully realized.

Platelet glycolytic metabolism correlates with hemodynamic severity in pulmonary arterial hypertension.

Group 1 pulmonary hypertension (PH), i.e., pulmonary arterial hypertension (PAH), is associated with a metabolic shift favoring glycolysis in cells comprising the lung vasculature as well as skeletal muscle and right heart. We sought to determine whether this metabolic switch is also detectable in circulating platelets from PAH patients. We used Seahorse Extracellular Flux to measure bioenergetics in platelets isolated from group 1 PH (PAH), group 2 PH, patients with dyspnea and normal pulmonary artery pressures, and healthy controls. We show that platelets from group 1 PH patients exhibit enhanced basal glycolysis and lower glycolytic reserve compared with platelets from healthy controls but do not differ from platelets of group 2 PH or dyspnea patients without PH. Although we were unable to identify a glycolytic phenotype unique to platelets from PAH patients, we found that platelet glycolytic metabolism correlated with hemodynamic severity only in group 1 PH patients, supporting the known link between PAH pathology and altered glycolytic metabolism and extending this association to ex vivo platelets. Pulmonary artery pressure and pulmonary vascular resistance in patients with group 1 PH were directly associated with basal platelet glycolysis and inversely associated with maximal and reserve glycolysis, suggesting that PAH progression reduces the capacity for glycolysis even while demanding an increase in glycolytic metabolism. Therefore, platelets may provide an easy-to-harvest, real-time window into the metabolic shift occurring in the lung vasculature and represent a useful surrogate for interrogating the glycolytic shift central to PAH pathology.

P6012Active selexipag metabolite MRE-269 increases endothelin receptors in pulmonary artery smooth muscle cells

Abstract Background The pathology of pulmonary arterial hypertension (PAH) indicates the abnormal outgrowth of pulmonary artery smooth muscle cells (PASMCs) of the media. Abundant expression of endothelin 1 (ET-1) is observed in vessels of PAH, and has been considered to play a pathogenic role. There are several endothelin receptors including ETA, ETB. Compared to ETA, ETB mRNA is less expressed in PASMCs from control individuals, and is reported to be increased in those from PAH patients. However, how ETB is involved in PAH remains unclear. Selexipag, a non-prostanoid IP receptor agonist, was recently authorized for treating PAH. Compared to selexipag, the active metabolite MRE-269 has a higher affinity for the IP receptor. Initial combination therapy come to be accepted as a standard strategy for this disease, although the interaction of each drug has not been discussed enough. Purpose To assess the effect of selexipag on ET-1 receptors in PASMCs. Methods We stimulated purchased human PASMCs and endothelial cells by MRE-269 (300 nM), ET-1 (100 nM) or combination of them in vitro. Quantitative PCR was performed to quantify mRNA expressions. Cell proliferation was assessed by CCK8 cell proliferation assay kit. BQ123, A192621, bosentan was used as blocker against ETA, ETB, or both, respectively. Results In PASMCs, MRE-269 increased ETA and ETB expressions 2- and 7-fold, respectively. On the other hand, it increased ETB 1.2-fold in pulmonary artery endothelial cells; ETA was not detected in those cells. After pretreatment by MRE-269, ET-1 accelerated the proliferation of PASMCs. A192621 and bosentan abrogated this proliferation. In contrast, BQ123 did not abrogate it. Conclusions In PASMCs, active selexipag metabolite MRE-269 increases ETB more strongly than ETA, resulting in accelerated cell proliferation by ET-1 predominantly via ETB. These data call for further study focused on the choice of ET-1 receptor antagonists in the case of combination therapy with selexipag.

Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation.

Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4, are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2-mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes. Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated leukotriene B4 biosynthesis. Bmpr2-mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-β) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation. In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.