Post-transcriptional regulation of IFI16 promotes inflammatory endothelial pathophenotypes observed in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease driven by endothelial cell inflammation and dysfunction, resulting in the pathological remodeling of the pulmonary vasculature. Innate immune activation has been linked to PAH development; however, the regulation, propagation, and reversibility of the induction of inflammation in PAH is poorly understood. Here, we demonstrate a role for interferon inducible protein 16 (IFI16), an innate immune sensor, as a modulator of endothelial inflammation in pulmonary hypertension, utilizing human pulmonary artery endothelial cells (PAECs). Inflammatory stimulus of PAECs with IL-1ß up-regulates IFI16 expression, inducing proinflammatory cytokine up-regulation and cellular apoptosis. IFI16 mRNA stability is regulated by post-transcriptional m6A modification, mediated by Wilms' tumor 1-associated protein (WTAP), a structural stabilizer of the methyltransferase complex, via regulation of m6A methylation of IFI16. Additionally, m6A levels are increased in the peripheral blood mononuclear cells of PAH patients compared to control, indicating that quantifying this epigenetic change in patients may hold potential as a biomarker for disease identification. In summary, our study demonstrates IFI16 mediates inflammatory endothelial pathophenotypes seen in pulmonary arterial hypertension.

Studying the Pulmonary Endothelium in Health and Disease: An Official American Thoracic Society Workshop Report.

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.

Dietary intake and glutamine-serine metabolism control pathologic vascular stiffness.

Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular fibroblasts rewire their metabolism to sustain collagen biosynthesis remains unknown. Here, we found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAFs). Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions for treating cardiopulmonary vascular disease.

Mitigation of Stress-induced Structural Remodeling and Functional Deficiency in iPSC-CMs with PLN R9C Mutation by Promoting Autophagy.

Background: Phospholamban (PLN) is a key regulator of cardiac function connecting adrenergic signaling and calcium homeostasis. The R9C mutation of PLN is known to cause early onset dilated cardiomyopathy (DCM) and premature death, yet the detailed mechanisms underlie the pathologic remodeling process are not well defined in human cardiomyocytes. The aim of this study is to unravel the role of PLN R9C in DCM and identify potential therapeutic targets. Methods: PLN R9C knock-in (KI) and patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated and comprehensively examined for their expression profile, contractile function, and cellular signaling under both baseline conditions and following functional challenges. Results: PLN R9C KI iPSC-CMs exhibited near-normal morphology and calcium handling, slightly increased contractility, and an attenuated response to ß-adrenergic activation compared to wild-type (WT) cells. However, treatment with a maturation medium (MM) has induced fundamentally different remodeling in the two groups: while it improved the structural integrity and functional performance of WT cells, the same treatment result in sarcomere disarrangement, calcium handling deficiency, and further disrupted adrenergic signaling in PLN R9C KI cells. To understand the mechanism, transcriptomic analysis showed the enrichment of protein homeostasis signaling pathways specifically in PLN R9C KI cells in response to the MM treatment and increased contractile demands. Further studies also indicated elevated ROS levels, interrupted autophagic flux, and increased pentamer PLN aggregation in functionally challenged KI cells. These results were further confirmed in patient-specific iPSC-CM models, suggesting that functional stresses exacerbate the deficiencies in PLN R9C cells through disrupting protein homeostasis. Indeed, treating stressed patient cells with autophagy-accelerating reagents, such as metformin and rapamycin, has restored autophagic flux, mitigated sarcomere disarrangement, and partially rescued ß-adrenergic signaling and cardiac function. Conclusions: PLN R9C leads to a mild increase of calcium recycling and contractility. Functional challenges further enhanced contractile and proteostasis stress, leading to autophagic overload, structural remodeling, and functional deficiencies in PLN R9C cardiomyocytes. Activation of autophagy signaling partially rescues these effects, revealing a potential therapeutic target for DCM patients with the PLN R9C mutation. Graphic abstracts: A graphic abstract is available for this article.

A roadmap for therapeutic discovery in pulmonary hypertension associated with left heart failure. A scientific statement of the Heart Failure Association (HFA) of the ESC and the ESC Working Group on Pulmonary Circulation & Right Ventricular Function.

Pulmonary hypertension (PH) associated with left heart failure (LHF) (PH-LHF) is one of the most common causes of PH. It directly contributes to symptoms and reduced functional capacity and negatively affects right heart function, ultimately leading to a poor prognosis. There are no specific treatments for PH-LHF, despite the high number of drugs tested so far. This scientific document addresses the main knowledge gaps in PH-LHF with emphasis on pathophysiology and clinical trials. Key identified issues include better understanding of the role of pulmonary venous versus arteriolar remodelling, multidimensional phenotyping to recognize patient subgroups positioned to respond to different therapies, and conduct of rigorous pre-clinical studies combining small and large animal models. Advancements in these areas are expected to better inform the design of clinical trials and extend treatment options beyond those effective in pulmonary arterial hypertension. Enrichment strategies, endpoint assessments, and thorough haemodynamic studies, both at rest and during exercise, are proposed to play primary roles to optimize early-stage development of candidate therapies for PH-LHF.

Comparison of Contemporary Risk Scores in All Groups of Pulmonary Hypertension: A Pulmonary Vascular Research Institute GoDeep Meta-Registry Analysis.

BACKGROUND: Pulmonary hypertension (PH) is a heterogeneous disease with a poor prognosis. Accurate risk stratification is essential for guiding treatment decisions in pulmonary arterial hypertension (PAH). Although various risk models have been developed for PAH, their comparative prognostic potential requires further exploration. Additionally, the applicability of risk scores in PH groups beyond group 1 remains to be investigated. RESEARCH QUESTION: Are risk scores originally developed for PAH predictive in PH groups 1 through 4? STUDY DESIGN AND METHODS: We conducted a comprehensive analysis of outcomes among patients with incident PH enrolled in the multicenter worldwide Pulmonary Vascular Research Institute GoDeep meta-registry. Analyses were performed across PH groups 1 through 4 and further subgroups to evaluate the predictive value of PAH risk scores, including the Registry to Evaluate Early and Long-Term PAH Disease Mangement (REVEAL) Lite 2, REVEAL 2.0, European Society of Cardiology/European Respiratory Society 2022, Comparative, Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension (COMPERA) 3-strata, and COMPERA 4-strata. RESULTS: Eight thousand five hundred sixty-five patients were included in the study, of whom 3,537 patients were assigned to group 1 PH, whereas 1,807 patients, 1,635 patients, and 1,586 patients were assigned to group 2 PH, group 3 PH, and group 4 PH, respectively. Pulmonary hemodynamics were impaired with median mean pulmonary arterial pressure of 42 mm Hg (interquartile range, 33-52 mm Hg) and pulmonary vascular resistance of 7 Wood units (WU) (interquartile range, 4-11 WU). All risk scores were prognostic in the entire PH population and in each of the PH groups 1 through 4. The REVEAL scores, when used as continuous prediction models, demonstrated the highest statistical prognostic power and granularity; the COMPERA 4-strata risk score provided subdifferentiation of the intermediate-risk group. Similar results were obtained when separately analyzing various subgroups (PH subgroups 1.1, 1.4.1, and 1.4.4; PH subgroups 3.1 and 3.2; group 2 with isolated postcapillary PH vs combined precapillary and postcapillary PH; patients of all groups with concomitant cardiac comorbidities; and severe [> 5 WU] vs nonsevere PH). INTERPRETATION: This comprehensive study with real-world data from 15 PH centers showed that PAH-designed risk scores possess predictive power in a large PH cohort, whether considered as common to the group or calculated separately for each PH group (1-4) and various subgroups. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT05329714; URL: www. CLINICALTRIALS: gov.

Genetic regulation and targeted reversal of lysosomal dysfunction and inflammatory sterol metabolism in pulmonary arterial hypertension.

Vascular inflammation critically regulates endothelial cell (EC) pathophenotypes, particularly in pulmonary arterial hypertension (PAH). Dysregulation of lysosomal activity and cholesterol metabolism have known inflammatory roles in disease, but their relevance to PAH is unclear. In human pulmonary arterial ECs and in PAH, we found that inflammatory cytokine induction of the nuclear receptor coactivator 7 (NCOA7) both preserved lysosomal acidification and served as a homeostatic brake to constrain EC immunoactivation. Conversely, NCOA7 deficiency promoted lysosomal dysfunction and proinflammatory oxysterol/bile acid generation that, in turn, contributed to EC pathophenotypes. In vivo, mice deficient for Ncoa7 or exposed to the inflammatory bile acid 7α-hydroxy-3-oxo-4-cholestenoic acid (7HOCA) displayed worsened PAH. Emphasizing this mechanism in human PAH, an unbiased, metabolome-wide association study (N=2,756) identified a plasma signature of the same NCOA7-dependent oxysterols/bile acids associated with PAH mortality (P<1.1x10-6). Supporting a genetic predisposition to NCOA7 deficiency, in genome-edited, stem cell-derived ECs, the common variant intronic SNP rs11154337 in NCOA7 regulated NCOA7 expression, lysosomal activity, oxysterol/bile acid production, and EC immunoactivation. Correspondingly, SNP rs11154337 was associated with PAH severity via six-minute walk distance and mortality in discovery (N=93, P=0.0250; HR=0.44, 95% CI [0.21-0.90]) and validation (N=630, P=2x10-4; HR=0.49, 95% CI [0.34-0.71]) cohorts. Finally, utilizing computational modeling of small molecule binding to NCOA7, we predicted and synthesized a novel activator of NCOA7 that prevented EC immunoactivation and reversed indices of rodent PAH. In summary, we have established a genetic and metabolic paradigm and a novel therapeutic agent that links lysosomal biology as well as oxysterol and bile acid processes to EC inflammation and PAH pathobiology. This paradigm carries broad implications for diagnostic and therapeutic development in PAH and in other conditions dependent upon acquired and innate immune regulation of vascular disease.

Pulmonary primary oxysterol and bile acid synthesis as a predictor of outcomes in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare and fatal vascular disease with heterogeneous clinical manifestations. To date, molecular determinants underlying the development of PAH and related outcomes remain poorly understood. Herein, we identify pulmonary primary oxysterol and bile acid synthesis (PPOBAS) as a previously unrecognized pathway central to PAH pathophysiology. Mass spectrometry analysis of 2,756 individuals across five independent studies revealed 51 distinct circulating metabolites that predicted PAH-related mortality and were enriched within the PPOBAS pathway. Across independent single-center PAH studies, PPOBAS pathway metabolites were also associated with multiple cardiopulmonary measures of PAH-specific pathophysiology. Furthermore, PPOBAS metabolites were found to be increased in human and rodent PAH lung tissue and specifically produced by pulmonary endothelial cells, consistent with pulmonary origin. Finally, a poly-metabolite risk score comprising 13 PPOBAS molecules was found to not only predict PAH-related mortality but also outperform current clinical risk scores. This work identifies PPOBAS as specifically altered within PAH and establishes needed prognostic biomarkers for guiding therapy in PAH.

Omics and Extreme Phenotyping Reveal Longitudinal Association Between Left Atrial Size and Pulmonary Vascular Resistance in Group 2 Pulmonary Hypertension.

BACKGROUND: Left heart disease is the most common cause of pulmonary hypertension (PH) and is frequently accompanied by increases in pulmonary vascular resistance. However, the distinction between phenotypes of PH due to left heart disease with a normal or elevated pulmonary vascular resistance-isolated postcapillary PH (IpcPH) and combined pre- and postcapillary PH (CpcPH), respectively-has been incompletely defined using unbiased methods. METHODS AND RESULTS: Patients with extremes of IpcPH versus CpcPH were identified from a single-center record of those who underwent right heart catheterization. Individuals with left ventricular ejection fraction <40% or with potential causes of PH beyond left heart disease were excluded. Medication usage in IpcPH and CpcPH was compared across Anatomical Therapeutic Chemical classes and identified vitamin K antagonists as the only medication with pharmacome-wide significance, being more commonly used in CpcPH and for an indication of atrial fibrillation in ≈90% of instances. Accordingly, atrial fibrillation prevalence was significantly higher in CpcPH in a phenome-wide analysis. Review of echocardiographic data most proximal to right heart catheterization revealed that left atrial diameter indexed to body surface area-known to be associated with atrial fibrillation-was increased in CpcPH regardless of the presence of atrial fibrillation. An independent cohort with serial right heart catheterizations and PH-left heart disease showed a significant positive correlation between change in left atrial diameter indexed to body surface area and change in pulmonary vascular resistance. CONCLUSIONS: Guided by pharmacomic and phenomic screens in a rigorously phenotyped cohort, we identify a longitudinal association between left atrial diameter indexed to body surface area and pulmonary vascular resistance with implications for the future development of diagnostic, prognostic, and therapeutic tools.

Profiling of repetitive RNA sequences in the blood plasma of patients with cancer.

Liquid biopsies provide a means for the profiling of cell-free RNAs secreted by cells throughout the body. Although well-annotated coding and non-coding transcripts in blood are readily detectable and can serve as biomarkers of disease, the overall diagnostic utility of the cell-free transcriptome remains unclear. Here we show that RNAs derived from transposable elements and other repeat elements are enriched in the cell-free transcriptome of patients with cancer, and that they serve as signatures for the accurate classification of the disease. We used repeat-element-aware liquid-biopsy technology and single-molecule nanopore sequencing to profile the cell-free transcriptome in plasma from patients with cancer and to examine millions of genomic features comprising all annotated genes and repeat elements throughout the genome. By aggregating individual repeat elements to the subfamily level, we found that samples with pancreatic cancer are enriched with specific Alu subfamilies, whereas other cancers have their own characteristic cell-free RNA profile. Our findings show that repetitive RNA sequences are abundant in blood and can be used as disease-specific diagnostic biomarkers.

Innovations in RNA therapy for hemophilia.

Given the shortcomings of current factor-, nonfactor-, and adeno-associated virus gene-based therapies, the recent advent of RNA-based therapeutics for hemophilia is changing the fundamental approach to hemophilia management. From small interfering RNA therapeutics that knockdown clot regulators antithrombin, protein S, and heparin cofactor II, to CRISPR/Cas9 gene editing that may personalize treatment, improved technologies have the potential to reduce bleeds and factor use and avoid inhibitor formation. These novel agents, some in preclinical studies and others in early phase trials, have the potential to simplify treatment and improve hemostasis and quality of life. Furthermore, because these therapies arise from manipulation of the coagulation cascade and thrombin generation and its regulation, they will enhance our understanding of hemostasis and thrombosis and ultimately lead to better therapies for children and adults with inherited bleeding disorders. What does the future hold? With the development of novel preclinical technologies at the bench, there will be fewer joint bleeds, debilitating joint disease, orthopedic surgery, and improved physical and mental health, which were not previously possible. In this review, we identify current limitations of treatment and progress in the development of novel RNA therapeutics, including messenger RNA nanoparticle delivery and gene editing for the treatment of hemophilia.

Effects of ranolazine on right ventricular function, fluid dynamics, and metabolism in patients with precapillary pulmonary hypertension: insights from a longitudinal, randomized, double-blinded, placebo controlled, multicenter study.

Introduction: Right ventricular (RV) function is a major determinant of outcome in patients with precapillary pulmonary hypertension (PH). We studied the effect of ranolazine on RV function over 6 months using multi-modality imaging and biochemical markers in patients with precapillary PH (groups I, III, and IV) and RV dysfunction [CMR imaging ejection fraction (EF) < 45%] in a longitudinal, randomized, double-blinded, placebo-controlled, multicenter study of ranolazine treatment. Methods: Enrolled patients were assessed using cardiac magnetic resonance (CMR) imaging, 11C-acetate and 18-F-FDG positron emission tomography (PET), and plasma metabolomic profiling, at baseline and at the end of treatment. Results: Twenty-two patients were enrolled, and 15 patients completed all follow-up studies with 9 in the ranolazine arm and 6 in the placebo arm. RVEF and RV/Left ventricle (LV) mean glucose uptake were significantly improved after 6 months of treatment in the ranolazine arm. Metabolomic changes in aromatic amino acid metabolism, redox homeostasis, and bile acid metabolism were observed after ranolazine treatment, and several changes significantly correlated with changes in PET and CMR-derived fluid dynamic measurements. Discussion: Ranolazine may improve RV function by altering RV metabolism in patients with precapillary PH. Larger studies are needed to confirm the beneficial effects of ranolazine.

Frataxin deficiency disrupts mitochondrial respiration and pulmonary endothelial cell function.

Deficiency of iron­sulfur (FeS) clusters promotes metabolic rewiring of the endothelium and the development of pulmonary hypertension (PH) in vivo. Joining a growing number of FeS biogenesis proteins critical to pulmonary endothelial function, recent data highlighted that frataxin (FXN) reduction drives Fe-S-dependent genotoxic stress and senescence across multiple types of pulmonary vascular disease. Trinucleotide repeat mutations in the FXN gene cause Friedreich's ataxia, a disease characterized by cardiomyopathy and neurodegeneration. These tissue-specific phenotypes have historically been attributed to mitochondrial reprogramming and oxidative stress. Whether FXN coordinates both nuclear and mitochondrial processes in the endothelium is unknown. Here, we aim to identify the mitochondria-specific effects of FXN deficiency in the endothelium that predispose to pulmonary hypertension. Our data highlight an Fe-S-driven metabolic shift separate from previously described replication stress whereby FXN knockdown diminished mitochondrial respiration and increased glycolysis and oxidative species production. In turn, FXN-deficient endothelial cells had increased vasoconstrictor production (EDN1) and decreased nitric oxide synthase expression (NOS3). These data were observed in primary pulmonary endothelial cells after pharmacologic inhibition of FXN, mice carrying a genetic endothelial deletion of FXN, and inducible pluripotent stem cell-derived endothelial cells from patients with FXN mutations. Altogether, this study indicates FXN is an upstream driver of pathologic aberrations in metabolism and genomic stability. Moreover, our study highlights FXN-specific vasoconstriction in vivo, prompting future studies to investigate available and novel PH therapies in contexts of FXN deficiency.

Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4-Human Leukocyte Antigen-DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.

A p53-TLR3 axis ameliorates pulmonary hypertension by inducing BMPR2 via IRF3.

Pulmonary arterial hypertension (PAH) features pathogenic and abnormal endothelial cells (ECs), and one potential origin is clonal selection. We studied the role of p53 and toll-like receptor 3 (TLR3) in clonal expansion and pulmonary hypertension (PH) via regulation of bone morphogenetic protein (BMPR2) signaling. ECs of PAH patients had reduced p53 expression. EC-specific p53 knockout exaggerated PH, and clonal expansion reduced p53 and TLR3 expression in rat lung CD117+ ECs. Reduced p53 degradation (Nutlin 3a) abolished clonal EC expansion, induced TLR3 and BMPR2, and ameliorated PH. Polyinosinic/polycytidylic acid [Poly(I:C)] increased BMPR2 signaling in ECs via enhanced binding of interferon regulatory factor-3 (IRF3) to the BMPR2 promoter and reduced PH in p53-/- mice but not in mice with impaired TLR3 downstream signaling. Our data show that a p53/TLR3/IRF3 axis regulates BMPR2 expression and signaling in ECs. This link can be exploited for therapy of PH.

Metabolomic Profiles Differentiate Scleroderma-PAH From Idiopathic PAH and Correspond With Worsened Functional Capacity.

BACKGROUND: The prognosis and therapeutic responses are worse for pulmonary arterial hypertension associated with systemic sclerosis (SSc-PAH) compared with idiopathic pulmonary arterial hypertension (IPAH). This discrepancy could be driven by divergence in underlying metabolic determinants of disease. RESEARCH QUESTION: Are circulating bioactive metabolites differentially altered in SSc-PAH vs IPAH, and can this alteration explain clinical disparity between these PAH subgroups? STUDY DESIGN AND METHODS: Plasma biosamples from 400 patients with SSc-PAH and 1,082 patients with IPAH were included in the study. Another cohort of 100 patients with scleroderma with no PH and 44 patients with scleroderma with PH was included for external validation. More than 700 bioactive lipid metabolites, representing a range of vasoactive and immune-inflammatory pathways, were assayed in plasma samples from independent discovery and validation cohorts using liquid chromatography/high-resolution mass spectrometry-based approaches. Regression analyses were used to identify metabolites that exhibited differential levels between SSc-PAH and IPAH and associated with disease severity. RESULTS: From hundreds of circulating bioactive lipid molecules, five metabolites were found to distinguish between SSc-PAH and IPAH, as well as associate with markers of disease severity. Relative to IPAH, patients with SSc-PAH carried increased levels of fatty acid metabolites, including lignoceric acid and nervonic acid, as well as eicosanoids/oxylipins and sex hormone metabolites. INTERPRETATION: Patients with SSc-PAH are characterized by an unfavorable bioactive metabolic profile that may explain the poor and limited response to therapy. These data provide important metabolic insights into the molecular heterogeneity underlying differences between subgroups of PAH.