Utility of factor D and other alternative complement factors as biomarkers in systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH).

BACKGROUND: Activation of the complement cascade is thought to play a role in scleroderma vasculopathy. We previously showed that complement factor D was elevated in patients with limited cutaneous SSc and pulmonary arterial hypertension (PAH). In this study, we sought to assess multiple relevant components of the complement cascade to determine if they are altered in SSc-PAH, as well as their potential utility as biomarkers of disease severity and progression. METHODS: Complement components (n = 14) were measured using multiplex assays in 156 patients with SSc-PAH from a multi-site repository and were compared to 33 patients with SSc without PAH, and 40 healthy controls. Data were evaluated for correlations between complement levels, right heart catheterization measures, and clinical endpoints including 6-minute walk distance. To assess complement longitudinally, serum complement levels were assayed at 0, 4, 12, 24, 36 and 48 weeks in 52 SSc-PAH patients who participated in a prior clinical trial. RESULTS: We found that factor D was significantly elevated in SSc-PAH compared to SSc without PAH (p < 0.0001) and was highly sensitive and specific for SSc-PAH (AUC=0.82, p < 0.001). In SSc-PAH patients, alterations in factor H, C4, and factor D were associated with measures of PAH disease severity including right heart catheterization measurements (cardiac output, right atrial pressure, and VO2 max), survival, and 6-minute walk distance. No significant changes in complement levels or clinical associations were seen over time or associated with treatment in the longitudinal clinical trial study. CONCLUSION: Our work confirms prior studies demonstrating a role for complement activation in SSc vascular disease and elevations of factor D in a large SSc-PAH population. Further, factor H and other complement factors are associated with severity of PAH including mortality. Taken together, these findings suggest that the alternative complement pathway plays a role in SSc-PAH pathogenesis and may serve as a biomarker to inform diagnosis and prognosis.

Multiple Biomarkers Are Equivalent to Clinical Pulmonary Arterial Hypertension Survival Risk Models.

BACKGROUND: Risk assessment in pulmonary arterial hypertension (PAH) is fundamental to guiding treatment and improved outcomes. Clinical models are excellent at identifying high-risk patients, but leave uncertainty amongst moderate-risk patients. RESEARCH QUESTION: Can a multiple blood biomarker model of PAH, using previously described biomarkers, improve risk discrimination over current models? STUDY DESIGN AND METHODS: Using a multiplex enzyme-linked immunosorbent assay, we measured N-terminal fragment of the prohormone brain natriuretic peptide (NT-proBNP), soluble suppressor of tumorigenicity, IL-6, endostatin, galectin 3, HDGF, and insulin-like growth factor binding proteins (IGFBP1-7) in training (n = 1,623), test (n = 696), and validation (n = 237) cohorts. Clinical variables and biomarkers were evaluated by principal component analysis. NT-proBNP was not included to develop a model independent of NT-proBNP. Unsupervised k-means clustering classified participants into clusters. Transplant-free survival by cluster was examined using Kaplan-Meier and Cox proportional hazard regressions. Hazard by cluster was compared with NT-proBNP, Registry to Evaluate Early and Long-Term PAH Disease Management (REVEAL), and European Society of Cardiology (ESC) and European Respiratory Society (ERS) risk models alone and combined clinical and biomarker models. RESULTS: The algorithm generated 5 clusters with good risk discrimination using 6 biomarkers, weight, height, and age at PAH diagnosis. In the test and validation cohorts, the biomarker model alone performed equivalent to REVEAL (area under the receiver operating characteristic curve, 0.74). Adding the biomarker model to the ESC and ERS score and REVEAL score improved the ESC and ERS score and REVEAL score. The best overall model was the biomarker model adjusted for NT-proBNP with the best C statistic, Akaike information criterion, and calibration for the adjusted model compared with either the biomarker or NT-proBNP model alone. INTERPRETATION: A multibiomarker model alone was equivalent to current PAH clinical mortality risk prediction models and improved performance when combined and added to NT-proBNP. Clinical risk scores offer excellent predictive models, but require multiple tests; adding blood biomarkers to models can improve prediction or can enable more frequent, noninvasive monitoring of risk in PAH to support therapeutic decision-making.

Integrative Multiomics in the Lung Reveals a Protective Role of Asporin in Pulmonary Arterial Hypertension.

BACKGROUND: Integrative multiomics can elucidate pulmonary arterial hypertension (PAH) pathobiology, but procuring human PAH lung samples is rare. METHODS: We leveraged transcriptomic profiling and deep phenotyping of the largest multicenter PAH lung biobank to date (96 disease and 52 control) by integration with clinicopathologic data, genome-wide association studies, Bayesian regulatory networks, single-cell transcriptomics, and pharmacotranscriptomics. RESULTS: We identified 2 potentially protective gene network modules associated with vascular cells, and we validated ASPN, coding for asporin, as a key hub gene that is upregulated as a compensatory response to counteract PAH. We found that asporin is upregulated in lungs and plasma of multiple independent PAH cohorts and correlates with reduced PAH severity. We show that asporin inhibits proliferation and transforming growth factor-ß/phosphorylated SMAD2/3 signaling in pulmonary artery smooth muscle cells from PAH lungs. We demonstrate in Sugen-hypoxia rats that ASPN knockdown exacerbated PAH and recombinant asporin attenuated PAH. CONCLUSIONS: Our integrative systems biology approach to dissect the PAH lung transcriptome uncovered asporin as a novel protective target with therapeutic potential in PAH.

Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

RASA3 is a candidate gene in sickle cell disease-associated pulmonary hypertension and pulmonary arterial hypertension.

Pulmonary hypertension (PH) is associated with significant morbidity and mortality. RASA3 is a GTPase activating protein integral to angiogenesis and endothelial barrier function. In this study, we explore the association of RASA3 genetic variation with PH risk in patients with sickle cell disease (SCD)-associated PH and pulmonary arterial hypertension (PAH). Cis-expression quantitative trait loci (eQTL) were queried for RASA3 using whole genome genotype arrays and gene expression profiles derived from peripheral blood mononuclear cells (PBMC) of three SCD cohorts. Genome-wide single nucleotide polymorphisms (SNPs) near or in the RASA3 gene that may associate with lung RASA3 expression were identified, reduced to 9 tagging SNPs for RASA3 and associated with markers of PH. Associations between the top RASA3 SNP and PAH severity were corroborated using data from the PAH Biobank and analyzed based on European or African ancestry (EA, AA). We found that PBMC RASA3 expression was lower in patients with SCD-associated PH as defined by echocardiography and right heart catheterization and was associated with higher mortality. One eQTL for RASA3 (rs9525228) was identified, with the risk allele correlating with PH risk, higher tricuspid regurgitant jet velocity and higher pulmonary vascular resistance in patients with SCD-associated PH. rs9525228 associated with markers of precapillary PH and decreased survival in individuals of EA but not AA. In conclusion, RASA3 is a novel candidate gene in SCD-associated PH and PAH, with RASA3 expression appearing to be protective. Further studies are ongoing to delineate the role of RASA3 in PH.

SOX17 Deficiency Mediates Pulmonary Hypertension: At the Crossroads of Sex, Metabolism, and Genetics.

Rationale: Genetic studies suggest that SOX17 (SRY-related HMG-box 17) deficiency increases pulmonary arterial hypertension (PAH) risk. Objectives: On the basis of pathological roles of estrogen and HIF2α (hypoxia-inducible factor 2α) signaling in pulmonary artery endothelial cells (PAECs), we hypothesized that SOX17 is a target of estrogen signaling that promotes mitochondrial function and attenuates PAH development via HIF2α inhibition. Methods: We used metabolic (Seahorse) and promoter luciferase assays in PAECs together with the chronic hypoxia murine model to test the hypothesis. Measurements and Main Results: Sox17 expression was reduced in PAH tissues (rodent models and from patients). Chronic hypoxic pulmonary hypertension was exacerbated by mice with conditional Tie2-Sox17 (Sox17EC-/-) deletion and attenuated by transgenic Tie2-Sox17 overexpression (Sox17Tg). On the basis of untargeted proteomics, metabolism was the top pathway altered by SOX17 deficiency in PAECs. Mechanistically, we found that HIF2α concentrations were increased in the lungs of Sox17EC-/- and reduced in those from Sox17Tg mice. Increased SOX17 promoted oxidative phosphorylation and mitochondrial function in PAECs, which were partly attenuated by HIF2α overexpression. Rat lungs in males displayed higher Sox17 expression versus females, suggesting repression by estrogen signaling. Supporting 16α-hydroxyestrone (16αOHE; a pathologic estrogen metabolite)-mediated repression of SOX17 promoter activity, Sox17Tg mice attenuated 16αOHE-mediated exacerbations of chronic hypoxic pulmonary hypertension. Finally, in adjusted analyses in patients with PAH, we report novel associations between a SOX17 risk variant, rs10103692, and reduced plasma citrate concentrations (n = 1,326). Conclusions: Cumulatively, SOX17 promotes mitochondrial bioenergetics and attenuates PAH, in part, via inhibition of HIF2α. 16αOHE mediates PAH development via downregulation of SOX17, linking sexual dimorphism and SOX17 genetics in PAH.

Integrative Multiomics to Dissect the Lung Transcriptional Landscape of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) remains an incurable and often fatal disease despite currently available therapies. Multiomics systems biology analysis can shed new light on PAH pathobiology and inform translational research efforts. Using RNA sequencing on the largest PAH lung biobank to date (96 disease and 52 control), we aim to identify gene co-expression network modules associated with PAH and potential therapeutic targets. Co-expression network analysis was performed to identify modules of co-expressed genes which were then assessed for and prioritized by importance in PAH, regulatory role, and therapeutic potential via integration with clinicopathologic data, human genome-wide association studies (GWAS) of PAH, lung Bayesian regulatory networks, single-cell RNA-sequencing data, and pharmacotranscriptomic profiles. We identified a co-expression module of 266 genes, called the pink module, which may be a response to the underlying disease process to counteract disease progression in PAH. This module was associated not only with PAH severity such as increased PVR and intimal thickness, but also with compensated PAH such as lower number of hospitalizations, WHO functional class and NT-proBNP. GWAS integration demonstrated the pink module is enriched for PAH-associated genetic variation in multiple cohorts. Regulatory network analysis revealed that BMPR2 regulates the main target of FDA-approved riociguat, GUCY1A2, in the pink module. Analysis of pathway enrichment and pink hub genes (i.e. ANTXR1 and SFRP4) suggests the pink module inhibits Wnt signaling and epithelial-mesenchymal transition. Cell type deconvolution showed the pink module correlates with higher vascular cell fractions (i.e. myofibroblasts). A pharmacotranscriptomic screen discovered ubiquitin-specific peptidases (USPs) as potential therapeutic targets to mimic the pink module signature. Our multiomics integrative study uncovered a novel gene subnetwork associated with clinicopathologic severity, genetic risk, specific vascular cell types, and new therapeutic targets in PAH. Future studies are warranted to investigate the role and therapeutic potential of the pink module and targeting USPs in PAH.

COL18A1 genotypic associations with endostatin levels and clinical features in pulmonary arterial hypertension: a quantitative trait association study.

Variation around the COL18A1 gene, which encodes the angiostatic peptide endostatin, may influence disease heterogeneity in pulmonary arterial hypertension https://bit.ly/3shXrNR.

Hepatoma-derived growth factor is associated with pulmonary vascular remodeling and PAH disease severity and survival.

Hepatoma-derived growth factor (HDGF) was previously shown to be associated with increased mortality in a small study of idiopathic and connective tissue disease-associated pulmonary arterial hypertension (PAH). In this study, we measured serum HDGF levels in a large multicenter cohort (total 2017 adult PAH-Biobank enrollees), we analyzed the associations between HDGF levels and various clinical measures using linear or logistic regression models. Higher HDGF levels were found to be significantly associated with worse pulmonary hemodynamics, prostacyclin treatment; among PAH subtypes, higher HDGF levels were most associated with portopulmonary hypertension (beta = 0.469, p < 0.0001). Both Kaplan-Meier curve and Cox proportional hazard regression demonstrated that higher HDGF levels are associated with a higher risk of mortality (COX hazard ratio 1.31, p < 0.0001). Further, in the Sugen hypoxia (SuHx) rat model, the highest HDGF levels were post-pulmonary circulation, and HDGF levels significantly increased with the development of PAH. In pulmonary arteries, immunohistochemistry staining showed that HDGF was highly expressed in pulmonary smooth muscle cells in both PAH patients and SuHx rats. In conclusion, we found that higher serum HDGF was linked with increased mortality, and associated with disease severity in a large multi-center adult PAH cohort (n = 2017). In the SuHX PAH models, circulating HDGF levels are pulmonary in origin and increase with PAH progression. HDGF may be actively involved in vascular remodeling in PAH.

Mendelian randomisation and experimental medicine approaches to interleukin-6 as a drug target in pulmonary arterial hypertension.

BACKGROUND: Inflammation and dysregulated immunity are important in the development of pulmonary arterial hypertension (PAH). Compelling preclinical data supports the therapeutic blockade of interleukin-6 (IL-6) signalling. METHODS: We conducted a phase 2 open-label study of intravenous tocilizumab (8 mg·kg-1) over 6 months in patients with group 1 PAH. Co-primary end-points were safety, defined by incidence and severity of adverse events, and change in pulmonary vascular resistance. Separately, a mendelian randomisation study was undertaken on 11 744 individuals with European ancestry including 2085 patients with idiopathic/heritable disease for the IL-6 receptor (IL6R) variant (rs7529229), known to associate with circulating IL-6R levels. RESULTS: We recruited 29 patients (male/female 10/19; mean±sd age 54.9±11.4 years). Of these, 19 had heritable/idiopathic PAH and 10 had connective tissue disease-associated PAH. Six were withdrawn prior to drug administration; 23 patients received at least one dose of tocilizumab. Tocilizumab was discontinued in four patients owing to serious adverse events. There were no deaths. Despite evidence of target engagement in plasma IL-6 and C-reactive protein levels, both intention-to-treat and modified intention-to-treat analyses demonstrated no change in pulmonary vascular resistance. Inflammatory markers did not predict treatment response. Mendelian randomisation did not support an effect of the lead IL6R variant on risk of PAH (OR 0.99, p=0.88). CONCLUSION: Adverse events were consistent with the known safety profile of tocilizumab. Tocilizumab did not show any consistent treatment effect.

Biomarkers of Pulmonary Hypertension Are Altered in Children with Down Syndrome and Pulmonary Hypertension.

OBJECTIVE: To evaluate the performance of pulmonary hypertension (PH) biomarkers in children with Down syndrome, an independent risk factor for PH, in whom biomarker performance may differ compared with other populations. STUDY DESIGN: Serum endostatin, interleukin (IL)-1 receptor 1 (ST2), galectin-3, N-terminal pro hormone B-natriuretic peptide (NT-proBNP), IL-6, and hepatoma-derived growth factor (HDGF) were measured in subjects with Down syndrome and PH (n = 29), subjects with Down syndrome and resolved PH (n = 13), subjects with Down syndrome without PH (n = 49), and subjects without Down syndrome with World Symposium on Pulmonary Hypertension group I pulmonary arterial hypertension (no Down syndrome PH group; n = 173). Each biomarker was assessed to discriminate PH in Down syndrome. A classification tree was created to distinguish PH from resolved PH and no PH in children with Down syndrome. RESULTS: Endostatin, galectin-3, HDGF, and ST2 were elevated in subjects with Down syndrome regardless of PH status. Not all markers differed between subjects with Down syndrome and PH and subjects with Down syndrome and resolved PH. NT-proBNP and IL-6 levels were similar in the Down syndrome with PH group and the no Down syndrome PH group. A classification tree identified NT-proBNP and galectin-3 as the best markers for sequentially distinguishing PH, resolved PH, and no PH in subjects with Down syndrome. CONCLUSIONS: Proteomic markers are used to improve the diagnosis and prognosis of PH but, as demonstrated here, can be altered in genetically unique populations such as individuals with Down syndrome. This further suggests that clinical biomarkers should be evaluated in unique groups with the development of population-specific nomograms.

The angiostatic peptide endostatin enhances mortality risk prediction in pulmonary arterial hypertension.

Currently available noninvasive markers for assessing disease severity and mortality risk in pulmonary arterial hypertension (PAH) are unrelated to fundamental disease biology. Endostatin, an angiostatic peptide known to inhibit pulmonary artery endothelial cell migration, proliferation and survival in vitro, has been linked to adverse haemodynamics and shortened survival in small PAH cohorts. This observational cohort study sought to assess: 1) the prognostic performance of circulating endostatin levels in a large, multicentre PAH cohort; and 2) the added value gained by incorporating endostatin into existing PAH risk prediction models. Endostatin ELISAs were performed on enrolment samples collected from 2017 PAH subjects with detailed clinical data, including survival times. Endostatin associations with clinical variables, including survival, were examined using multivariable regression and Cox proportional hazards models. Extended survival models including endostatin were compared to null models based on the REVEAL risk prediction tool and European Society of Cardiology/European Respiratory Society (ESC/ERS) low-risk criteria using likelihood ratio tests, Akaike and Bayesian information criteria and C-statistics. Higher endostatin was associated with higher right atrial pressure, mean pulmonary arterial pressure and pulmonary vascular resistance, and with shorter 6-min walk distance (p<0.01). Mortality risk doubled for each log higher endostatin (hazard ratio 2.3, 95% CI 1.6-3.4, p<0.001). Endostatin remained an independent predictor of survival when incorporated into existing risk prediction models. Adding endostatin to REVEAL-based and ESC/ERS criteria-based risk assessment strategies improved mortality risk prediction. Endostatin is a robust, independent predictor of mortality in PAH. Adding endostatin to existing PAH risk prediction strategies improves PAH risk assessment.

Angiostatic Peptide, Endostatin, Predicts Severity in Pediatric Congenital Heart Disease-Associated Pulmonary Hypertension.

Background Endostatin, an angiogenic inhibitor, is associated with worse pulmonary arterial hypertension (PAH) outcomes in adults and poor lung growth in children. This study sought to assess whether endostatin is associated with disease severity and outcomes in pediatric PAH. Methods and Results Serum endostatin was measured in cross-sectional (N=160) and longitudinal cohorts (N=64) of pediatric subjects with PAH, healthy pediatric controls and pediatric controls with congenital heart disease (CHD) (N=54, N=15), and adults with CHD associated PAH (APAH-CHD, N=185). Outcomes, assessed by regression and Kaplan-Meier analysis, included hemodynamics, change in endostatin over time, and transplant-free survival. Endostatin secretion was evaluated in pulmonary artery endothelial and smooth muscle cells. Endostatin was higher in those with PAH compared with healthy controls and controls with CHD and was highest in those with APAH-CHD. In APAH-CHD, endostatin was associated with a shorter 6-minute walk distance and increased mean right atrial pressure. Over time, endostatin was associated with higher pulmonary artery pressure and pulmonary vascular resistance index, right ventricular dilation, and dysfunction. Endostatin decreased with improved hemodynamics over time. Endostatin was associated with worse transplant-free survival. Addition of endostatin to an NT-proBNP (N-terminal pro-B-type natriuretic peptide) based survival analysis improved risk stratification, reclassifying subjects with adverse outcomes. Endostatin was secreted primarily by pulmonary artery endothelial cells. Conclusions Endostatin is associated with disease severity, disease improvement, and worse survival in APAH-CHD. Endostatin with NT-proBNP improves risk stratification, better predicting adverse outcomes. The association of elevated endostatin with shunt lesions suggests that endostatin could be driven by both pulmonary artery flow and pressure. Endostatin could be studied as a noninvasive prognostic marker, particularly in APAH-CHD.

Rare variant analysis of 4241 pulmonary arterial hypertension cases from an international consortium implicates FBLN2, PDGFD, and rare de novo variants in PAH.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a lethal vasculopathy characterized by pathogenic remodeling of pulmonary arterioles leading to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. PAH can be associated with other diseases (APAH: connective tissue diseases, congenital heart disease, and others) but often the etiology is idiopathic (IPAH). Mutations in bone morphogenetic protein receptor 2 (BMPR2) are the cause of most heritable cases but the vast majority of other cases are genetically undefined. METHODS: To identify new risk genes, we utilized an international consortium of 4241 PAH cases with exome or genome sequencing data from the National Biological Sample and Data Repository for PAH, Columbia University Irving Medical Center, and the UK NIHR BioResource - Rare Diseases Study. The strength of this combined cohort is a doubling of the number of IPAH cases compared to either national cohort alone. We identified protein-coding variants and performed rare variant association analyses in unrelated participants of European ancestry, including 1647 IPAH cases and 18,819 controls. We also analyzed de novo variants in 124 pediatric trios enriched for IPAH and APAH-CHD. RESULTS: Seven genes with rare deleterious variants were associated with IPAH with false discovery rate smaller than 0.1: three known genes (BMPR2, GDF2, and TBX4), two recently identified candidate genes (SOX17, KDR), and two new candidate genes (fibulin 2, FBLN2; platelet-derived growth factor D, PDGFD). The new genes were identified based solely on rare deleterious missense variants, a variant type that could not be adequately assessed in either cohort alone. The candidate genes exhibit expression patterns in lung and heart similar to that of known PAH risk genes, and most variants occur in conserved protein domains. For pediatric PAH, predicted deleterious de novo variants exhibited a significant burden compared to the background mutation rate (2.45×, p = 2.5e-5). At least eight novel pediatric candidate genes carrying de novo variants have plausible roles in lung/heart development. CONCLUSIONS: Rare variant analysis of a large international consortium identified two new candidate genes-FBLN2 and PDGFD. The new genes have known functions in vasculogenesis and remodeling. Trio analysis predicted that ~ 15% of pediatric IPAH may be explained by de novo variants.

ST2 Is a Biomarker of Pediatric Pulmonary Arterial Hypertension Severity and Clinical Worsening.

BACKGROUND: Pediatric pulmonary hypertension is a severe disease defined by sustained elevation of pulmonary artery pressures and pulmonary vascular resistance (PVR). Noninvasive diagnostic and prognostic markers that are more pulmonary vascular specific have been elusive because of disease heterogeneity and patient growth. RESEARCH QUESTION: Is soluble suppressor of tumorigenicity (ST2) associated with pulmonary hemodynamic and functional changes in pediatric pulmonary hypertension? Does ST2 improve mortality risk models in pediatric pulmonary hypertension? STUDY DESIGN AND METHODS: Two pediatric cohorts (age < 21 years) were assayed for ST2 and N-terminal prohormone B-natriuretic peptide: a cross-sectional cohort from the National Heart Lung and Blood Institute-funded National Biological Sample and Data Repository for PAH (PAHB) (N = 182), and a second longitudinal cohort from Children's Hospital of Colorado (N = 61). Adjusted linear regression was used for association with clinical variables. Clinical mortality models (the Registry to Evaluate Early and Long-Term PAH Disease Management [REVEAL] score) with and without ST2 were used to predict worsening outcomes and compared. Pulmonary artery endothelial and smooth muscle cell ST2 expression and secretion were assayed in vitro. RESULTS: In an adjusted (age and sex) analysis in the PAHB, ST2 was significantly associated with shorter 6-min walk distance (P = .03) and increased PVR index (P = .02). In adjusted longitudinal regression in the Children's Hospital of Colorado cohort, ST2 was significantly associated with higher PVR index (P < .001), shorter 6-min walk distance (P = .01), and higher mean pulmonary artery pressure (P < .001). Although the REVEAL Risk Score Calculator 2.0 was predictive of clinical worsening in the PAHB (hazard ratio, 1.88), addition of ST2 significantly improved the model (hazard ratio, 2.05). In cell culture, ST2 was produced and secreted predominately by endothelial cells as opposed to smooth muscle cells (P < .0001). INTERPRETATION: In two pediatric PAH cohorts, elevated ST2 was associated with unfavorable pulmonary hemodynamics and functional measures, clinical worsening, and significantly improved prediction of clinical worsening. Pulmonary artery endothelial cellular expression of ST2 suggests that ST2 is a more pulmonary vascular-specific marker for pulmonary hypertension.

United States Pulmonary Hypertension Scientific Registry: Baseline Characteristics.

BACKGROUND: The treatment, genotyping, and phenotyping of patients with World Health Organization Group 1 pulmonary arterial hypertension (PAH) have evolved dramatically in the last decade. RESEARCH QUESTION: The United States Pulmonary Hypertension Scientific Registry was established as the first US PAH patient registry to investigate genetic information, reproductive histories, and environmental exposure data in a contemporary patient population. STUDY DESIGN AND METHODS: Investigators at 15 US centers enrolled consecutively screened adults diagnosed with Group 1 PAH who had enrolled in the National Biological Sample and Data Repository for PAH (PAH Biobank) within 5 years of a cardiac catheterization demonstrating qualifying hemodynamic criteria. Exposure and reproductive histories were collected by using a structured interview and questionnaire. The biobank provided genetic data. RESULTS: Between 2015 and 2018, a total of 499 of 979 eligible patients with clinical diagnoses of idiopathic PAH (IPAH) or familial PAH (n = 240 [48%]), associated PAH (APAH; n = 256 [51%]), or pulmonary venoocclusive disease/pulmonary capillary hemangiomatosis (n = 3 [1%]) enrolled. The mean age was 55.8 years, average BMI was 29.2 kg/m2, and 79% were women. Mean duration between symptom onset and diagnostic catheterization was 1.9 years. Sixty-six percent of patients were treated with more than one PAH medication at enrollment. Past use of prescription weight loss drugs (16%), recreational drugs (27%), and oral contraceptive pills (77%) was common. Women often reported miscarriage (37%), although PAH was rarely diagnosed within 6 months of pregnancy (1.9%). Results of genetic testing identified pathogenic or suspected pathogenic variants in 13% of patients, reclassifying 18% of IPAH patients and 5% of APAH patients to heritable PAH. INTERPRETATION: Patients with Group 1 PAH remain predominately middle-aged women diagnosed with IPAH or APAH. Delays in diagnosis of PAH persist. Treatment with combinations of PAH-targeted medications is more common than in the past. Women often report pregnancy complications, as well as exposure to anorexigens, oral contraceptives, and/or recreational drugs. Results of genetic tests frequently identify unsuspected heritable PAH.

Bayesian Inference Associates Rare KDR Variants with Specific Phenotypes in Pulmonary Arterial Hypertension.

Background - Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. Methods - We analyzed 13,037 participants enrolled in the NIHR BioResource - Rare Diseases (NBR) study, of which 1,148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension (PH), we used the Bayesian rare-variant association method BeviMed. Results - Heterozygous, high impact, likely loss-of-function variants in the Kinase Insert Domain Receptor (KDR) gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (KCO, posterior probability (PP)=0.989) and older age at diagnosis (PP=0.912). We also provide evidence for familial segregation of a rare nonsense KDR variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the five patients harboring these predicted deleterious variants in KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions - The Bayesian inference approach allowed us to independently validate KDR, which encodes for the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for KDR being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.