Pulmonary arterial hypertension (PAH) is a disease of abnormal pulmonary vascular remodeling and vascular obliteration that results in right heart failure and death. PAH pathogenesis is strongly associated with mutations of the Transforming Growth Factor Beta (TGF-β) superfamily signaling pathway, which has previously been challenging to target therapeutically. Sotatercept, a fusion protein of the extracellular portion of the activin type 2 receptor A (ACVR2A) and the human IgG1 Fc domain, is the first activin signaling inhibitor (ASI) FDA-approved for the treatment of PAH, demonstrating efficacy across the risk spectrum in PAH. This soluble protein binds to a range of circulating TGF-β superfamily ligands, including activins A and B, growth and differentiation factors (GDFs) 8 and 11, as well as some bone morphogenetic proteins (BMPs). Other ASIs have been developed primarily for hematologic indications, such as anemias and cytopenias associated with β-thalassemia, myelodysplastic syndromes, and myelodysplastic neoplasms. Fundamental questions remain regarding the basic biological mechanisms of ASIs, their short- and long-term side effect profiles, and their potential utility across the spectrum of different etiologies of pulmonary hypertension (PH). In this state-of-the-art review, we brought together basic and clinical researchers, and industry scientists under the umbrella of the Pulmonary Vascular Research Institute (PVRI) Innovative Drug Discovery Initiative (IDDI) to discuss the biology of ASIs, the importance of specific BMP ligands, efficacy and side effect profiles, and considerations in the development of next-generation ASIs.

Mutations of EIF2AK4 , which encodes the eIF2α kinase GCN2, cause a severe inherited form of pulmonary hypertension called pulmonary veno-occlusive disease (PVOD). Some pathogenic variants of GCN2 are amenable to pharmacological reactivation by low concentrations of ATP-pocket binding inhibitors. Kinase inhibition at modestly elevated concentrations limits the clinical utility of these drugs against PVOD. We therefore performed an in cellulo chemical screen for GCN2 activators and identified three structurally distinct compounds with low micromolar stimulatory activities. Unlike previously described GCN2 activators, one of these molecules activated GCN2 independently of GCN1. Modelling supported by structure activity screens suggested it binds within the ATP-pocket of GCN2, but unlike existing ligands does not protrude inward into the allosteric pocket or outward into the solvent. This overcomes a key requirement of other GCN2 activators.

Sophisticated prognostic scores have been proposed for SARS-CoV-2 but do not always perform consistently. We performed this systematic review to discover why and to investigate the impact of vaccination and viral variants. We searched the PubMed database for the keywords ‘SARS-CoV-2’ or ‘Covid19’ with ‘biomarker’ and ‘mortality’ for the baseline tranche (01/12/2019–30/06/2021) and either ‘SARS-CoV-2’ or ‘Covid19’ with ‘biomarker’ and either ‘vaccination’ or ‘variant’ from 01/12/2020 to 31/10/2023. To aggregate the data, the meta library in R was used, and a random effects model fitted to obtain pooled AUCs and 95

Background Non-communicable diseases such as coronary artery disease, atrial fibrillation, type 2 diabetes, hypertension, and others share endothelial dysfunction as one of their underlying features. The endothelium, as the interface between blood and vasculature, shapes disease onset and progression through its response to environmental cues. However, while the genetic component of these diseases has been captured by genome wide association studies (GWAS), which also highlighted a shared immune component, it remains unclear which of these disease loci exerts their effects through endothelial cells. This study identifies, and quantifies, the genetic determinants of endothelial cells molecular traits and their overlap to the common genetic variation component of these diseases. Methods We generated genotype, RNA-sequencing, H3K27ac ChIP-sequencing, ATAC-sequencing, and endothelial cells barrier stimuli response measurements for 100 samples of human umbilical vein endothelial cells. These were used to identify quantitative trait loci (QTL) for gene expression, transcriptional isoform usage, splice junction usage, chromatin activity and barrier response. We applied statistical colocalisation to identify the overlap between data layers, and to explain molecular QTLs contribution to GWAS disease loci. Results We used molecular QTLs to identify the regulatory features of 8,214 genes, representing 36% of all expressed genes in endothelial cells. We also identified the molecular mechanisms underlying 815 loci across 16 disease GWAS. These represent between 29% and 40% of all loci for each disease, compared to the previous average of 23%. This is due to the choice of a cell type often underrepresented in tissue level data, and the inclusion of isoform, splicing and chromatin activity datasets. Furthermore, we compared the endothelial cells molecular QTLs with similar datasets in monocytes, neutrophils and CD4 T lymphocytes to shed light on the interplay between the endothelial and the immune compartments in these diseases. We identified loci acting through both the endothelial and the immune compartment, mostly with the same directionality of effect, and endothelial specific ones. Conclusions This work expands the knowledge of the mechanisms and genes underlying the effect of common genetic variation on non-communicable diseases having endothelial dysfunction as a shared feature. It also illustrates the interplay between endothelial cells and immune cell types in these diseases, highlighting shared and unique pathways. ### Competing Interest Statement The authors have declared no competing interest.

Biallelic mutations in eukaryotic translation initiation factor 2 α kinase 4, EIF2AK4 (which encodes general control nonderepressible 2, GCN2) underpin heritable forms of pulmonary veno-occlusive disease (PVOD), a rare and fatal form of pulmonary hypertension. The mechanisms linking these are mostly uncharacterised. We demonstrate for the first time that homozygous loss of gcn2 is sufficient to cause mild pulmonary hypertension in mice. Single-cell transcriptomics of mouse lungs identified adventitial fibroblasts as having the greatest GCN2-dependent transcriptional differences, implicating them as key players in this model of PVOD. The most significantly upregulated pathways in gcn2-/- adventitial fibroblasts were inflammatory. Therefore, we went on to demonstrate a pro-inflammatory phenotype in gcn2-/- mouse embryonic fibroblasts and gcn2-/- mice. In a novel murine model of pulmonary hypertension induced by exposure to mitomycin C, deletion of interleukin-6 rescued the pulmonary vascular phenotype. When chronically exposed to lipopolysaccharide, the pulmonary hypertensive phenotype of gcn2-/- mice is exaggerated. Genetic ablation of interleukin-6 completely rescues both the baseline and LPS-exaggerated pulmonary hypertensive phenotype. Targeting Il6 -dependent pathways may be useful in treating this deadly disease. ### Competing Interest Statement The authors have declared no competing interest.

Background:Genetic diagnosis and precision medicine are rapidly advancing, driven by innovations in next-generation sequencing and omic methods. The UK's collaboration between national research initiatives and the National Health Service facilitates translation of research into clinical practice. This rapid transition impacts family dynamics and family planning, and raises ethical concerns, compounded by limited public and practitioner awareness of the long-term consequences of genetic diagnosis. Our objective is to explore the impact of genetic diagnosis on family dynamics and the ethical considerations of genetic testing at different life stages in patients with pulmonary arterial hypertension (PAH) and their at-risk relatives. Methods:Stakeholders from the National Institute for Health Research BioResource Rare Diseases Study and the National Cohort Study of Idiopathic and Heritable Pulmonary Arterial Hypertension were recruited using purposive sampling. 53 interviews and focus groups with 63 participants were recorded, transcribed and thematically analysed using MAXQDA data analysis software. Results:The study revealed three main themes: the impact of diagnosis on family dynamics, considerations for family planning, and genetic testing of relatives. Two attitudes toward testing offspring emerged: proactive advocates and gatekeepers. The gatekeeper stance was driven by three key factors: shielding children from genetic risk awareness, feelings of guilt or a desire to avoid blame for disease transmission, and limited family connections. Each theme highlighted various moral and ethical dilemmas faced by individuals. Conclusions:A PAH diagnosis reshapes family roles and responsibilities. Genetic risk awareness strengthens bonds but also introduces challenges such as disclosing information and deciding on testing for at-risk relatives. Our research highlights the need for comprehensive genetic counselling and support systems to enhance patient care and familial wellbeing.

Introduction: Pulmonary arterial hypertension (PAH) describes diseases characterised by increased pulmonary vessel pressures that lead to right ventricular failure and death if left untreated. Dysregulated metabolic function has been reported in endothelial cells from PAH patients. Healthy endothelial cells can rapidly shift from quiescence to proliferative and apoptotic states, partly due to a greater utilization of glycolysis for ATP production, rather than the slower oxidative phosphorylation more commonly relied on by other cell types. However, in PAH patients, pulmonary artery endothelial cells demonstrate an even greater shift towards glycolytic ATP production. This phenomenon, which was first described in cancer as the "Warburg effect", is an adaptation for optimizing both energy production and biosynthetic processes for maximum proliferative potential. Hypothesis and Methods: We hypothesize that targeting the PKM2 axis in endothelial cells may shift the balance to favour oxidative phosphorylation, in effect reversing the Warburg effect; and that this would have beneficial effects in cell and rodent models of PAH. To this end we have tested TEPP-46, an allosteric PKM2 activator that stabilizes the tetrameric form, blocking its translocation into the nucleus and increasing canonical enzymatic activity, in blood outgrowth endothelial cells (BOECs) and in a rat Sugen-hypoxia model. Results: TEPP-46 treatment reduced levels of nuclear PKM2 (the homodimer form which promotes anabolic effects and proliferation). Cytoplasmic levels of PKM2 and levels of PKM1 were unaffected. Exposure to TEPP-46 lowered levels of polypyrimidine tract-binding protein 1 (PTPB1), which controls alternative splicing of the PKM gene to promote PKM2 expression; and reduced LDHA, which converts pyruvate to lactate, preventing its utilization for oxidative phosphorylation. In the Sugen-hypoxia rat model, administration of TEPP-46 significantly ameliorated the elevated right ventricular systolic pressures, reduced the loss of body weight and increased survival. The increased muscularisation of the smaller blood arteries and arterioles in the lungs of rats due to Sugen-hypoxia were also improved by concurrent administration of TEPP-46. Conclusion: We have shown that TEPP-46 treatment reduces nuclear PKM2 and represses PKM2-driven gene expression, with the functional effect of ameliorating the Sugen-hypoxia phenotype. This suggests that antagonizing the Warburg effect may offer a novel therapeutic avenue for PAH. ### Competing Interest Statement The authors have declared no competing interest. British Heart Foundation, https://ror.org/02wdwnk04 Medical Research Council, https://ror.org/03x94j517

Pulmonary arterial hypertension (PAH) is characterised by pulmonary vascular remodelling causing premature death from right heart failure. Established DNA variants influence PAH risk, but susceptibility from epigenetic changes is unknown. We addressed this through epigenome-wide association study (EWAS), testing 865,848 CpG sites for association with PAH in 429 individuals with PAH and 1226 controls. Three loci, at Cathepsin Z (CTSZ, cg04917472), Conserved oligomeric Golgi complex 6 (COG6, cg27396197), and Zinc Finger Protein 678 (ZNF678, cg03144189), reached epigenome-wide significance (p < 10(-7)) and are hypermethylated in PAH, including in individuals with PAH at 1-year follow-up. Of 16 established PAH genes, only cg10976975 in BMP10 shows hypermethylation in PAH. Hypermethylation at CTSZ is associated with decreased blood cathepsin Z mRNA levels. Knockdown of CTSZ expression in human pulmonary artery endothelial cells increases caspase-3/7 activity (p < 10(-4)). DNA methylation profiles are altered in PAH, exemplified by the pulmonary endothelial function modifier CTSZ, encoding protease cathepsin Z.

Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKTSer473, ERK 1/2Thr202/Tyr204 and PDH-E1αSer293, indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKTSer473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1αSer293), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology.

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–β/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.

AimsBone morphogenetic protein-9 (BMP9) is critical for bone morphogenetic protein receptor type-2 (BMPR2) signalling in pulmonary vascular endothelial cells. Furthermore, human genetics studies support the central role of disrupted BMPR2 mediated BMP9 signalling in vascular endothelial cells in the initiation of pulmonary arterial hypertension (PAH). In addition, loss-of-function mutations in BMP9 have been identified in PAH patients. BMP9 is considered to play an important role in vascular homeostasis and quiescence.Methods and resultsWe identified a novel BMP9 target as the class-3 semaphorin, SEMA3G. Although originally identified as playing a role in neuronal development, class-3 semaphorins may have important roles in endothelial function. Here we show that BMP9 transcriptional regulation of SEMA3G occurs via ALK1 and the canonical Smad pathway, requiring both Smad1 and Smad5. Knockdown studies demonstrated redundancy between type-2 receptors in that BMPR2 and ACTR2A were compensatory. Increased SEMA3G expression by BMP9 was found to be regulated by the transcription factor, SOX17. Moreover, we observed that SEMA3G regulates VEGF signalling by inhibiting VEGFR2 phosphorylation and that VEGF, in contrast to BMP9, negatively regulated SEMA3G transcription. Functional endothelial cell assays of VEGF-mediated migration and network formation revealed that BMP9 inhibition of VEGF was abrogated by SEMA3G knockdown. Conversely, treatment with recombinant SEMA3G partially mimicked the inhibitory action of BMP9 in these assays.ConclusionsThis study provides further evidence for the anti-angiogenic role of BMP9 in microvascular endothelial cells and these functions are mediated at least in part via SOX17 and SEMA3G induction.

Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1½ GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.

Vitamin D (vitD) deficiency is frequently observed in patients with pulmonary arterial hypertension (PAH) and, in these patients, low levels of vitD correlate with worse prognosis. The aim of this study was to examine the expression and the antiproliferative role of vitD receptor (VDR) and its signalling pathway in the human pulmonary vasculature. VDR presence and expression was analyzed in lungs, pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) from controls and PAH-patients. VDR expression and VDR-target genes were examined in PASMC treated with calcitriol. The antiproliferative effect of 48 h-calcitriol was studied in PASMC by MTT and BrdU assays. VDR is expressed in PASMC. It is downregulated in lungs and in PASMC, but not in PAEC, from PAH-patients compared to non-hypertensive controls. Calcitriol strongly upregulated VDR expression in PASMC and the VDR target genes KCNK3 (encoding TASK1), BIRC5 (encoding survivin) and BMP4. Calcitriol produced an antiproliferative effect which was diminished by silencing or by pharmacological inhibition of survivin or BMPR2, but not of TASK1. In conclusion, the expression of VDR is low in PAH-patients and can be rescued by calcitriol. VDR exerts an antiproliferative effect in PASMC by modulating survivin and the BMP signalling pathway.

INTRODUCTION:Establishing a diagnosis is paramount in medical practice as it shapes patients' experiences and guides treatment. Patients grappling with rare diseases face a triple challenge: prolonged diagnostic journeys, limited responses to existing therapies, and the absence of effective monitoring tools. Genetic diagnosis often provides crucial diagnostic and prognostic information, opening up possibilities for genotype-targeted treatments and facilitating counselling and relative testing. The NIHR BioResource - Rare Diseases (NBR) Study and the Cohort Study in Idiopathic and Hereditary Pulmonary Arterial Hypertension (PAH Cohort study) aimed to enhance diagnosis and treatment for PAH, successfully identifying the genetic cause in 25% of idiopathic cases. However, the diagnostic and therapeutic odyssey in patients with PAH remains largely unexplored. METHODS:Stakeholders from the NBR and PAH Cohort studies were recruited using purposive sampling. In-depth interviews and focus groups were recorded, transcribed, anonymised, and analysed thematically using MAXQDA software. RESULTS:The study involved 53 interviews and focus groups with 63 participants, revealing key themes across five stages of the diagnostic odyssey: initial health concerns and interactions with general practitioners, experiences of misdiagnosis, relief upon receiving the correct diagnosis, and mixed emotions regarding genetic results and the challenges of living with the disease. Following the diagnosis, participants embarked on a therapeutic journey, facing various challenges, including the disease's impact on professional and social lives, the learning curve associated with understanding the disease, shifts in communication dynamics with healthcare providers, therapeutic hurdles, and insurance-related issues. Building on these insights, we identified areas of unmet needs, such as improved collaboration with primary care providers and local hospitals, the provision of psychological support and counselling, and the necessity for ongoing patient education in the ever-evolving realms of research and therapy. CONCLUSIONS:The study highlights the significant challenges encountered throughout the diagnostic and therapeutic journey in PAH. To enhance patient outcomes, it is crucial to raise awareness of the disease, establish clear diagnostic pathways, and seamlessly integrate genetic diagnostics into clinical practice. Streamlining the diagnostic process can be achieved by utilising existing clinical infrastructure to support research and fostering better communication within the NHS. Moreover, there is an urgent need for more effective therapies alongside less burdensome drug delivery methods.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension arising from EIF2AK4 gene mutations or mitomycin C (MMC) administration. The lack of effective PVOD therapies is compounded by a limited understanding of the mechanisms driving the vascular remodeling in PVOD. We show that the administration of MMC in rats mediates the activation of protein kinase R (PKR) and the integrated stress response (ISR), which lead to the release of the endothelial adhesion molecule VE-Cadherin in the complex with Rad51 to the circulation, disruption of endothelial barrier, and vascular remodeling. Pharmacological inhibition of PKR or ISR attenuates the depletion of VE-Cadherin, elevation of vascular permeability, and vascular remodeling instigated by MMC, suggesting potential clinical intervention for PVOD. Finally, the severity of PVOD phenotypes was increased by a heterozygous BMPR2 mutation that truncates the carboxyl tail of BMPR2, underscoring the role of deregulated BMP signal in the development of PVOD.