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.

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.

Allele-specific control of rodent and human lncRNA KMT2E-AS1 promotes hypoxic endothelial pathology in pulmonary hypertension.

Hypoxic reprogramming of vasculature relies on genetic, epigenetic, and metabolic circuitry, but the control points are unknown. In pulmonary arterial hypertension (PAH), a disease driven by hypoxia inducible factor (HIF)-dependent vascular dysfunction, HIF-2α promoted expression of neighboring genes, long noncoding RNA (lncRNA) histone lysine N-methyltransferase 2E-antisense 1 (KMT2E-AS1) and histone lysine N-methyltransferase 2E (KMT2E). KMT2E-AS1 stabilized KMT2E protein to increase epigenetic histone 3 lysine 4 trimethylation (H3K4me3), driving HIF-2α-dependent metabolic and pathogenic endothelial activity. This lncRNA axis also increased HIF-2α expression across epigenetic, transcriptional, and posttranscriptional contexts, thus promoting a positive feedback loop to further augment HIF-2α activity. We identified a genetic association between rs73184087, a single-nucleotide variant (SNV) within a KMT2E intron, and disease risk in PAH discovery and replication patient cohorts and in a global meta-analysis. This SNV displayed allele (G)-specific association with HIF-2α, engaged in long-range chromatin interactions, and induced the lncRNA-KMT2E tandem in hypoxic (G/G) cells. In vivo, KMT2E-AS1 deficiency protected against PAH in mice, as did pharmacologic inhibition of histone methylation in rats. Conversely, forced lncRNA expression promoted more severe PH. Thus, the KMT2E-AS1/KMT2E pair orchestrates across convergent multi-ome landscapes to mediate HIF-2α pathobiology and represents a key clinical target in pulmonary hypertension.

Low-affinity insulin-like growth factor binding protein 7 and its association with pulmonary arterial hypertension severity and survival.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) are a family of growth factor modifiers, some of which are known to be independently associated with pulmonary arterial hypertension (PAH) survival. IGF factor binding protein 7 (IGFBP7) is a unique low-affinity IGFBP that, independent of IGF, stimulates prostacyclin production. This study proposed to establish associations between IGFBP7 and PAH severity and survival, using enrollment and longitudinal samples. Serum IGFBP7 levels were significantly elevated in patients with PAH compared to controls. After adjusting for age and sex, logarithmic increases in IGFBP7 were associated with a 20 m shorter six-minute walk distance (6MWD; p < 0.001), a 2-3 mmHg higher mean right atrial pressure (p < 0.001 and 0.02), and a higher likelihood of a greater REVEAL 2.0 risk category placement (p < 0.001). Kaplan-Meier analysis demonstrated significantly decreased survival with IGFBP7 above the median and Cox multivariable analysis adjusted for age and sex, demonstrated higher serum IGFBP7 was an independent predictor of survival. Though the exact mechanism is still unknown, given IGFBP7's role as a prostacyclin stimulant, it has potential use as a therapeutic target for disease modulation.

Human liver single nuclear RNA sequencing implicates BMPR2, GDF15, arginine, and estrogen in portopulmonary hypertension.

Portopulmonary hypertension (PoPH) is a type of pulmonary vascular disease due to portal hypertension that exhibits high morbidity and mortality. The mechanisms driving disease are unknown, and transcriptional characteristics unique to the PoPH liver remain unexplored. Here, we apply single nuclear RNA sequencing to compare cirrhotic livers from patients with and without PoPH. We identify characteristics unique to PoPH in cells surrounding the central hepatic vein, including increased growth differentiation factor signaling, enrichment of the arginine biosynthesis pathway, and differential expression of the bone morphogenic protein type II receptor and estrogen receptor type I genes. These results provide insight into the transcriptomic characteristics of the PoPH liver and mechanisms by which PoPH cellular dysfunction might contribute to pulmonary vascular remodeling.

Deriving Convergent and Divergent Metabolomic Correlates of Pulmonary Arterial Hypertension.

High-dimensional metabolomics analyses may identify convergent and divergent markers, potentially representing aligned or orthogonal disease pathways that underly conditions such as pulmonary arterial hypertension (PAH). Using a comprehensive PAH metabolomics dataset, we applied six different conventional and statistical learning techniques to identify analytes associated with key outcomes and compared the results. We found that certain conventional techniques, such as Bonferroni/FDR correction, prioritized metabolites that tended to be highly intercorrelated. Statistical learning techniques generally agreed with conventional techniques on the top-ranked metabolites, but were also more inclusive of different metabolite groups. In particular, conventional methods prioritized sterol and oxylipin metabolites in relation to idiopathic versus non-idiopathic PAH, whereas statistical learning methods tended to prioritize eicosanoid, bile acid, fatty acid, and fatty acyl ester metabolites. Our findings demonstrate how conventional and statistical learning techniques can offer both concordant or discordant results. In the case of a rare yet morbid condition, such as PAH, convergent metabolites may reflect common pathways to shared disease outcomes whereas divergent metabolites could signal either distinct etiologic mechanisms, different sub-phenotypes, or varying stages of disease progression. Notwithstanding the need to investigate the mechanisms underlying the observed results, our main findings suggest that a multi-method approach to statistical analyses of high-dimensional human metabolomics datasets could effectively broaden the scientific yield from a given study design.

Insulin-like growth factor binding Protein-4: A novel indicator of pulmonary arterial hypertension severity and survival.

Proteomic analysis of patients with pulmonary arterial hypertension (PAH) has demonstrated significant abnormalities in the insulin-like growth factor axis (IGF). This study proposed to establish associations between a specific binding protein, insulin-like growth factor binding protein 4 (IGFBP4), and PAH severity as well as survival across varying study cohorts. In all cohorts studied, serum IGFBP4 levels were significantly elevated in PAH compared to controls (p < 0.0001). IGFBP4 concentration was also highest in the connective tissue-associated PAH (CTD-PAH) and idiopathic PAH subtypes (876 and 784 ng/mL, median, respectively). After adjustment for age and sex, IGFBP4 was significantly associated with worse PAH severity as defined by a decreased 6-min walk distance (6MWD), New York heart association functional class (NYHA-FC), REVEAL 2.0 score and higher right atrial pressures. In longitudinal analysis provided by one of the study cohorts, IGFBP4 was prospectively significantly associated with a shorter 6MWD, worse NYHA-FC classification, and decreased survival. Cox multivariable analysis demonstrated higher serum IGFBP4 as an independent predictor of survival in the overall PAHB cohort. Therefore, this study established that higher circulating IGFBP4 levels were significantly associated with worse PAH severity, decreased survival and disease progression. Dysregulation of IGF metabolism/growth axis may play a significant role in PAH cardio-pulmonary pathobiology.

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.

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.

Germline and Somatic Mutations in DNA Methyltransferase 3A (DNMT3A) Predispose to Pulmonary Arterial Hypertension (PAH) in Humans and Mice: Implications for Associated PAH.

Background: Mutations are found in 10-20% of idiopathic PAH (IPAH) patients, but none are consistently identified in connective tissue disease-associated PAH (APAH), which accounts for ∼45% of PAH cases. TET2 mutations, a cause of clonal hematopoiesis of indeterminant potential (CHIP), predispose to an inflammatory type of PAH. We now examine mutations in another CHIP gene, DNMT3A , in PAH. Methods: We assessed DNMT3A mutation prevalence in PAH Biobank subjects as compared with controls, first using whole exome sequencing (WES)-derived CHIP calls in 1832 PAH Biobank patients versus 7509 age-and sex-matched gnomAD controls. We then performed deep, targeted panel sequencing of CHIP genes on a subset of 710 PAH Biobank patients and compared the prevalence of DNMT3A mutations therein to an independent pooled control cohort (N = 3645). In another cohort of 80 PAH patients and 41 controls, DNMT3A mRNA expression was studied in peripheral blood mononuclear cells (PBMCs). Finally, we evaluated the development of PAH in a conditional, hematopoietic, Dnmt3a knockout mouse model. Results: DNMT3A mutations were more frequent in PAH cases versus control subjects in the WES dataset (OR 2.60, 95% CI: 1.71-4.27). Among PAH patients, 33 had DNMT3A variants, most of whom had APAH (21/33). While 21/33 had somatic mutations (female:male 17:4), germline variants occurred in 12/33 (female:male 11:1). Hemodynamics were comparable with and without DNMT3A mutations (mPAP=58±21 vs. 52±18 mmHg); however, patients with DNMT3A mutations were unresponsive to acute vasodilator testing. Targeted panel sequencing identified that 14.6% of PAH patients had CHIP mutations (104/710), with DNMT3A accounting for 49/104. There was a significant association between all CHIP mutations and PAH in analyses adjusted for age and sex (OR 1.40, 95% CI: 1.09-1.80), though DNMT3A CHIP alone was not significantly enriched (OR:1.15, 0.82-1.61). DNMT3A expression was reduced in patient-derived versus control PAH-PBMCs. Spontaneous PAH developed in Dnmt3a -/- mice, and it was exacerbated by 3 weeks of hypoxia. Dnmt3a -/- mice had increased lung macrophages and elevated plasma IL-13. The IL-1ß antibody canakinumab attenuated PAH in Dnmt3a -/- mice. Conclusions: Germline and acquired DNMT3A variants predispose to PAH in humans. DNMT3A mRNA expression is reduced in human PAH PBMCs. Hematopoietic depletion of Dnmt3a causes inflammatory PAH in mice. DNMT3A is a novel APAH gene and may be a biomarker and therapeutic target.

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.

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.

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.

Subthalamic Peak Beta Ratio Is Asymmetric in Glucocerebrosidase Mutation Carriers With Parkinson's Disease: A Pilot Study.

Introduction: Up to 27% of individuals undergoing subthalamic nucleus deep brain stimulation (STN-DBS) have a genetic form of Parkinson's disease (PD). Glucocerebrosidase (GBA) mutation carriers, compared to sporadic PD, present with a more aggressive disease, less asymmetry, and fare worse on cognitive outcomes with STN-DBS. Evaluating STN intra-operative local field potentials provide the opportunity to assess and compare symmetry between GBA and non-GBA mutation carriers with PD; thus, providing insight into genotype and STN physiology, and eligibility for and programming of STN-DBS. The purpose of this pilot study was to test differences in left and right STN resting state beta power in non-GBA and GBA mutation carriers with PD. Materials and Methods: STN (left and right) resting state local field potentials were recorded intraoperatively from 4 GBA and 5 non-GBA patients with PD while off medication. Peak beta power expressed as a ratio to total beta power (peak beta ratio) was compared between STN hemispheres and groups while co-varying for age, age of disease onset, and disease severity. Results: Peak beta ratio was significantly different between the left and the right STN for the GBA group (p < 0.01) but not the non-GBA group (p = 0.56) after co-varying for age, age of disease onset, and disease severity. Discussion: Peak beta ratio in GBA mutation carriers was more asymmetric compared with non-mutation carriers and this corresponded with the degree of clinical asymmetry as measured by rating scales. This finding suggests that GBA mutation carriers have a physiologic signature that is distinct from that found in sporadic PD.

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.