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Rationale: NT-proBNP (N-terminal pro-brain natriuretic peptide), a biomarker of cardiac origin, is used to risk stratify patients with pulmonary arterial hypertension (PAH). Its limitations include poor sensitivity to early vascular pathology. Other biomarkers of vascular or systemic origin may also be useful in the management of PAH. Objectives: Identify prognostic proteins in PAH that complement NT-proBNP and clinical risk scores. Methods: An aptamer-based assay (SomaScan version 4) targeting 4,152 proteins was used to measure plasma proteins in patients with idiopathic, heritable, or drug-induced PAH from the UK National Cohort of PAH (n = 357) and the French EFORT (Evaluation of Prognostic Factors and Therapeutic Targets in PAH) study (n = 79). Prognostic proteins were identified in discovery-replication analyses of UK samples. Proteins independent of 6-minute-walk distance and NT-proBNP entered least absolute shrinkage and selection operator modeling, and the best combination in a single score was evaluated against clinical targets in EFORT. Measurements and Main Results: Thirty-one proteins robustly informed prognosis independent of NT-proBNP and 6-minute-walk distance in the UK cohort. A weighted combination score of six proteins was validated at baseline (5-yr mortality; area under the curve [AUC], 0.73; 95% confidence interval [CI], 0.63-0.85) and follow-up in EFORT (AUC, 0.84; 95% CI, 0.75-0.94; P = 9.96 × 10-6). The protein score risk stratified patients independent of established clinical targets and risk equations. The addition of the six-protein model score to NT-proBNP improved prediction of 5-year outcomes from AUC 0.762 (0.702-0.821) to 0.818 (0.767-0.869) by receiver operating characteristic analysis (P = 0.00426 for difference in AUC) in the UK replication and French samples combined. Conclusions: The plasma proteome informs prognosis beyond established factors in PAH and may provide a more sensitive measure of therapeutic response.

Pulmonary arterial hypertension (PAH) is an unmet clinical need. The lack of models of human disease is a key obstacle to drug development. We present a biomimetic model of pulmonary arterial endothelial-smooth muscle cell interactions in PAH, combining natural and induced bone morphogenetic protein receptor 2 (BMPR2) dysfunction with hypoxia to induce smooth muscle activation and proliferation, which is responsive to drug treatment. BMPR2- and oxygenation-specific changes in endothelial and smooth muscle gene expression, consistent with observations made in genomic and biochemical studies of PAH, enable insights into underlying disease pathways and mechanisms of drug response. The model captures key changes in the pulmonary endothelial phenotype that are essential for the induction of SMC remodelling, including a BMPR2-SOX17-prostacyclin signalling axis and offers an easily accessible approach for researchers to study pulmonary vascular remodelling and advance drug development in PAH.

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

Background: Autoimmunity may play a role in idiopathic pulmonary arterial hypertension (IPAH). It is not clear if this is causative, or as a bystander of disease and if it carries any prognostic or treatment significance. Methods: Comprehensive immune phenotyping was performed in IPAH patients, utilising patient samples from the UK National cohort study of Idiopathic and Heritable PAH. Standardised flow cytometric analysis of peripheral blood mononuclear cell types was assessed in IPAH patients and healthy controls. Serum autoantibody biomarkers were assayed using a Protoplex™ autoimmune panel in 473 type I PAH patients and 946 matched controls. Finally, a GST-fusion human proteomic screen was undertaken to identify novel circulating autoantibodies in PAH, and 350 PAH sera samples ELISA assayed for a putative antibody to BMPR2. Findings: Flow cytometric immune profiling demonstrates IPAH is associated with a signature consistent with altered humoral immune response. Increased autoantibody positivity was detected in PAH and patient subgroups associated with worse haemodynamic parameters but better overall survival. We identify a small subset of PAH patients who demonstrate immunoglobulin reactivity to the extracellular domain of the TGFbeta superfamily receptor, BMPR2, a protein which is strongly associated with hereditary causes of the disease. Interpretation: This evidence strongly suggests a proportion of patients with IPAH have an autoimmune component to the disease and that the BMPR2 pathway may be a target. Future trials targeting inflammation need to consider biomarker stratification. Funding Information: NIHR BRC, The Dinosaur Trust, MRC (MC_UU_00002/4) and the Wellcome Trust [WT107881]. Declarations of Interest: Gerry Coghlan reports grants and personal fees for conference attendance from Johnson & Johnson, personal fees for lectures from GlaxoSmithKline, Bayer and MSD, outside the submitted work. Paul Corris reports grants and personal fees for lectures and consultations from Actelion and Bayer, and personal fees for lectures and consultations from MSD, outside the submitted work. Luke Howard reports grants, personal fees for lectures, steering committee and advisory board work, and non-financial support for meeting attendance from Actelion, personal fees for lectures and advisory board work from Bayer and MSD, personal fees for advisory board work from GSK, outside the submitted work. Martin Johnson reports grants and personal fees for meeting attendance, lectures, and advisory board work from Actelion and MSD, outside the submitted work. David Kiely reports grants, personal fees, and other support from Actelion, Bayer and GSK, and personal fees and other support from MSD, outside the submitted work. Allan Lawrie reports grants from British Heart Foundation and Medical Research Council, grants, personal fees, and travel support from Actelion Pharmaceuticals Ltd, grants and personal fees from GlaxoSmithKline, outside the submitted work. James Lordan has received modest honoraria from Actelion and modest support to attend conference from Merck Sharp & Dohme, outside the submitted work. Joanna Pepke Zaba received fees for research grant, support to travel to meetings and honoraria from Actelion, Bayer, GlaxoSmithKline and Merck Sharp & Dohme, outside the submitted work. S.John Wort has received modest honoraria from GlaxoSmithKline, Actelion and Bayer and has received significant research grants from Actelion and Bayer, outside the submitted work. Nicholas Morrell reports personal fees from GSK and Johnson & Johnson/Actelion, outside the submitted work. Mark Toshner reports personal fees from GSK, grants and personal fees from J&J/Actelion, grants from Merck and Bayer, outside the submitted work. All other authors have nothing to disclose Ethics Approval Statement: Ethical approval was obtained, and informed written consent was given. For collection and processing of samples for autoantibody analysis, including controls ethical approval was obtained (An integrated approach to dissect the determinants, risk factors and pathways of ageing in the immune system 15/EE/0327, IRAS # 188383 and Genetics and epidemiology of aging-associated conditions in the Sardinian population, ProgeNIA PROT 2171/CE).