Using the Plasma Proteome for Risk Stratifying Patients with Pulmonary Arterial Hypertension.

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.

Mining the Plasma Proteome for Insights into the Molecular Pathology of Pulmonary Arterial Hypertension.

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.

First Genotype-Phenotype Study in TBX4 Syndrome: Gain-of-Function Mutations Causative for Lung Disease.

Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV1, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

SUR1 As a New Therapeutic Target for Pulmonary Arterial Hypertension.

Mutations in ABCC8 have been identified in pulmonary arterial hypertension (PAH). ABCC8 encodes SUR1, a regulatory subunit of the ATP-sensitive potassium channel Kir6.2. However, the pathophysiological role of the SUR1/Kir6.2 channel in PAH is unknown. We hypothesized that activation of SUR1 could be a novel potential target for PAH. We analyzed the expression of SUR1/Kir6.2 in the lungs and pulmonary artery (PA) in human PAH or experimental pulmonary hypertension (PH). The contribution of SUR1 in human or rat PA tone was evaluated, and we measured the consequences of in vivo activation of SUR1 in control and PH rats. SUR1 and Kir6.2 protein expression was not reduced in the lungs or human pulmonary arterial endothelial cells and smooth muscle cells from PAH or experimentally induced PH. We showed that pharmacological activation of SUR1 by three different SUR1 activators (diazoxide, VU0071063, and NN414) leads to PA relaxation. Conversely, the inhibition of SUR1/Kir6.2 channels causes PA constriction. In vivo, long- and short-term activation of SUR1 with diazoxide reversed monocrotaline-induced PH in rats. In addition, in vivo diazoxide application (short protocol) reduced the severity of PH in chronic-hypoxia rats. Moreover, 3 weeks of diazoxide exposure in control rats had no cardiovascular effects. Finally, in vivo, activation of SUR1 with NN414 reduced monocrotaline-induced PH in rats. In PAH and experimental PH, the expression of SUR1/Kir6.2 was still present. In vivo pharmacological SUR1 activation by two different molecules alleviated experimental PH, providing proof of concept that SUR1 activation should be considered for PAH and evaluated more thoroughly.

An emerging phenotype of pulmonary arterial hypertension patients carrying SOX17 variants.

BACKGROUND: The phenotype of pulmonary arterial hypertension (PAH) patients carrying SOX17 pathogenic variants remains mostly unknown. METHODS: We report the genetic analysis findings, characteristics and outcomes of patients with heritable PAH carrying SOX17 variants from the French Pulmonary Hypertension Network. RESULTS: 20 patients and eight unaffected relatives were identified. The median (range) age at diagnosis was 17 (2-53) years, with a female:male ratio of 1.5. At diagnosis, most of the patients (74%) were in New York Heart Association Functional Class III or IV with severe haemodynamic compromise, including a median pulmonary vascular resistance of 14.0 (4.2-31.5) WU. An associated congenital heart disease (CHD) was found in seven PAH patients (35%). Patients with CHD-associated PAH were significantly younger at diagnosis than PAH patients without CHD. Four patients (20%) suffered from recurrent haemoptysis requiring repeated arterial embolisations. 13 out of 16 patients (81%) for whom imaging was available displayed chest computed tomography abnormalities, including dilated, tortuous pulmonary vessels, ground-glass opacities as well as anomalies of the bronchial and nonbronchial arteries. After a median (range) follow-up of 47 (1-591) months, 10 patients underwent lung transplantation and one patient benefited from a heart-lung transplantation due to associated CHD. Histopathological analysis of lung explants showed a congested lung architecture with severe pulmonary arterial remodelling, subpleural vessel dilation and numerous haemorrhagic foci. CONCLUSIONS: PAH due to SOX17 pathogenic variants is a severe phenotype, frequently associated with CHD, haemoptysis and radiological abnormalities. Pathological assessment reveals severe pulmonary arterial remodelling and malformations affecting pulmonary vessels and thoracic systemic arteries.

Single-Cell Study of Two Rat Models of Pulmonary Arterial Hypertension Reveals Connections to Human Pathobiology and Drug Repositioning.

Rationale: The cellular and molecular landscape and translational value of commonly used models of pulmonary arterial hypertension (PAH) are poorly understood. Single-cell transcriptomics can enhance molecular understanding of preclinical models and facilitate their rational use and interpretation.Objectives: To determine and prioritize dysregulated genes, pathways, and cell types in lungs of PAH rat models to assess relevance to human PAH and identify drug repositioning candidates.Methods: Single-cell RNA sequencing was performed on the lungs of monocrotaline (MCT), Sugen-hypoxia (SuHx), and control rats to identify altered genes and cell types, followed by validation using flow-sorted cells, RNA in situ hybridization, and immunofluorescence. Relevance to human PAH was assessed by histology of lungs from patients and via integration with human PAH genetic loci and known disease genes. Candidate drugs were predicted using Connectivity Map.Measurements and Main Results: Distinct changes in genes and pathways in numerous cell types were identified in SuHx and MCT lungs. Widespread upregulation of NF-κB signaling and downregulation of IFN signaling was observed across cell types. SuHx nonclassical monocytes and MCT conventional dendritic cells showed particularly strong NF-κB pathway activation. Genes altered in SuHx nonclassical monocytes were significantly enriched for PAH-associated genes and genetic variants, and candidate drugs predicted to reverse the changes were identified. An open-access online platform was developed to share single-cell data and drug candidates (http://mergeomics.research.idre.ucla.edu/PVDSingleCell/).Conclusions: Our study revealed the distinct and shared dysregulation of genes and pathways in two commonly used PAH models for the first time at single-cell resolution and demonstrated their relevance to human PAH and utility for drug repositioning.

Screening for pulmonary arterial hypertension in adults carrying a BMPR2 mutation.

BACKGROUND: Heritable pulmonary arterial hypertension (PAH) is most commonly due to heterozygous mutations of the BMPR2 gene. Based on expert consensus, guidelines recommend annual screening echocardiography in asymptomatic BMPR2 mutation carriers. The main objectives of this study were to evaluate the characteristics of asymptomatic BMPR2 mutation carriers, assess their risk of occurrence of PAH and detect PAH at an early stage in this high-risk population. METHODS: Asymptomatic BMPR2 mutation carriers underwent screening at baseline and annually for a minimum of 2 years (DELPHI-2 study; ClinicalTrials.gov: NCT01600898). Annual screening included clinical assessment, ECG, pulmonary function tests, 6-min walk distance, cardiopulmonary exercise testing, chest radiography, echocardiography and brain natriuretic peptide (BNP) or N-terminal (NT)-proBNP level. Right heart catheterisation (RHC) was performed based on predefined criteria. An optional RHC at rest and exercise was proposed at baseline. RESULTS: 55 subjects (26 males; median age 37 years) were included. At baseline, no PAH was suspected based on echocardiography and NT-proBNP levels. All subjects accepted RHC at inclusion, which identified two mild PAH cases (3.6%) and 12 subjects with exercise pulmonary hypertension (21.8%). At long-term follow-up (118.8 patient-years of follow-up), three additional cases were diagnosed, yielding a PAH incidence of 2.3% per year (0.99% per year in males and 3.5% per year in females). All PAH cases remained at low-risk status on oral therapy at last follow-up. CONCLUSIONS: Asymptomatic BMPR2 mutation carriers have a significant risk of developing incident PAH. International multicentre studies are needed to confirm that refined multimodal screening programmes with regular follow-up allow early detection of PAH.

Involvement of CFTR in the pathogenesis of pulmonary arterial hypertension.

INTRODUCTION: A reduction in pulmonary artery relaxation is a key event in the pathogenesis of pulmonary arterial hypertension (PAH). Cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction in airway epithelial cells plays a central role in cystic fibrosis; CFTR is also expressed in pulmonary arteries and has been shown to control endothelium-independent relaxation. AIM AND OBJECTIVES: We aimed to delineate the role of CFTR in PAH pathogenesis through observational and interventional experiments in human tissues and animal models. METHODS AND RESULTS: Reverse-transcriptase quantitative PCR, confocal imaging and electron microscopy showed that CFTR expression was reduced in pulmonary arteries from patients with idiopathic PAH (iPAH) and in rats with monocrotaline-induced pulmonary hypertension (PH). Moreover, using myography on human, pig and rat pulmonary arteries, we demonstrated that CFTR activation induces pulmonary artery relaxation. CFTR-mediated pulmonary artery relaxation was reduced in pulmonary arteries from iPAH patients and rats with monocrotaline- or chronic hypoxia-induced PH. Long-term in vivo CFTR inhibition in rats significantly increased right ventricular systolic pressure, which was related to exaggerated pulmonary vascular cell proliferation in situ and vessel neomuscularisation. Pathologic assessment of lungs from patients with severe cystic fibrosis (F508del-CFTR) revealed severe pulmonary artery remodelling with intimal fibrosis and medial hypertrophy. Lungs from homozygous F508delCftr rats exhibited pulmonary vessel neomuscularisation. The elevations in right ventricular systolic pressure and end diastolic pressure in monocrotaline-exposed rats with chronic CFTR inhibition were more prominent than those in vehicle-exposed rats. CONCLUSIONS: CFTR expression is strongly decreased in pulmonary artery smooth muscle and endothelial cells in human and animal models of PH. CFTR inhibition increases vascular cell proliferation and strongly reduces pulmonary artery relaxation.

Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension.

RATIONALE: Pulmonary arterial hypertension is a severe lethal cardiopulmonary disease. Loss of function mutations in KCNK3 (potassium channel subfamily K member 3) gene, which encodes an outward rectifier K+ channel, have been identified in pulmonary arterial hypertension patients. OBJECTIVE: We have demonstrated that KCNK3 dysfunction is common to heritable and nonheritable pulmonary arterial hypertension and to experimental pulmonary hypertension (PH). Finally, KCNK3 is not functional in mouse pulmonary vasculature. METHODS AND RESULTS: Using CRISPR/Cas9 technology, we generated a 94 bp out of frame deletion in exon 1 of Kcnk3 gene and characterized these rats at the electrophysiological, echocardiographic, hemodynamic, morphological, cellular, and molecular levels to decipher the cellular mechanisms associated with loss of KCNK3. Using patch-clamp technique, we validated our transgenic strategy by demonstrating the absence of KCNK3 current in freshly isolated pulmonary arterial smooth muscle cells from Kcnk3-mutated rats. At 4 months of age, echocardiographic parameters revealed shortening of the pulmonary artery acceleration time associated with elevation of the right ventricular systolic pressure. Kcnk3-mutated rats developed more severe PH than wild-type rats after monocrotaline exposure or chronic hypoxia exposure. Kcnk3-mutation induced a lung distal neomuscularization and perivascular extracellular matrix activation. Lungs of Kcnk3-mutated rats were characterized by overactivation of ERK1/2 (extracellular signal-regulated kinase1-/2), AKT (protein kinase B), SRC, and overexpression of HIF1-α (hypoxia-inducible factor-1 α), survivin, and VWF (Von Willebrand factor). Linked with plasma membrane depolarization, reduced endothelial-NOS expression and desensitization of endothelial-derived hyperpolarizing factor, Kcnk3-mutated rats presented predisposition to vasoconstriction of pulmonary arteries and a severe loss of sildenafil-induced pulmonary arteries relaxation. Moreover, we showed strong alteration of right ventricular cardiomyocyte excitability. Finally, Kcnk3-mutated rats developed age-dependent PH associated with low serum-albumin concentration. CONCLUSIONS: We established the first Kcnk3-mutated rat model of PH. Our results confirm that KCNK3 loss of function is a key event in pulmonary arterial hypertension pathogenesis. This model presents new opportunities for understanding the initiating mechanisms of PH and testing biologically relevant therapeutic molecules in the context of PH.

Phenotype and Outcomes of Pulmonary Hypertension Associated with Neurofibromatosis Type 1.

Rationale: Pulmonary hypertension (PH) associated with neurofibromatosis type 1 (NF1) is a rare and largely unknown complication of NF1.Objectives: To describe characteristics and outcomes of PH-NF1.Methods: We reported the clinical, functional, radiologic, histologic, and hemodynamic characteristics, response to pulmonary arterial hypertension (PAH)-approved drugs, and transplant-free survival of patients with PH-NF1 from the French PH registry.Measurements and Main Results: We identified 49 PH-NF1 cases, characterized by a female/male ratio of 3.9 and a median (minimum-maximum) age at diagnosis of 62 (18-82) years. At diagnosis, 92% were in New York Heart Association functional class III or IV. The 6-minute-walk distance was 211 (0-460) m. Pulmonary function tests showed low DlCO (30% [12-79%]) and severe hypoxemia (PaO2 56 [38-99] mm Hg). Right heart catheterization showed severe precapillary PH with a mean pulmonary artery pressure of 45 (10) mm Hg and a pulmonary vascular resistance of 10.7 (4.2) Wood units. High-resolution computed tomography images revealed cysts (76%), ground-glass opacities (73%), emphysema (49%), and reticulations (39%). Forty patients received PAH-approved drugs with a significant improvement in functional class and hemodynamic parameters. Transplant-free survival at 1, 3, and 5 years was 87%, 54%, and 42%, respectively, and four patients were transplanted. Pathologic assessment showed nonspecific interstitial pneumonia and major pulmonary vascular remodeling.Conclusions: PH-NF1 is characterized by a female predominance, a low DlCO, and severe functional and hemodynamic impairment. Despite a potential benefit of PAH treatment, prognosis remains poor, and double-lung transplantation is an option for eligible patients.

Characterization of GDF2 Mutations and Levels of BMP9 and BMP10 in Pulmonary Arterial Hypertension.

Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor.Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH.Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260).Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations.Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

Comparison of Human and Experimental Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

Bmpr2 Mutant Rats Develop Pulmonary and Cardiac Characteristics of Pulmonary Arterial Hypertension.

BACKGROUND: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. METHODS: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. RESULTS: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. CONCLUSIONS: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

Familial pulmonary arterial hypertension by KDR heterozygous loss of function.

Beyond the major gene BMPR2, several new genes predisposing to PAH have been identified during the last decade. Recently, preliminary evidence of the involvement of the KDR gene was found in a large genetic association study.We prospectively analysed the KDR gene by targeted panel sequencing in a series of 311 PAH patients referred to a clinical molecular laboratory for genetic diagnosis of PAH.Two index cases with severe PAH from two different families were found to carry a loss-of-function mutation in the KDR gene. These two index cases were clinically characterised by low diffusing capacity for carbon monoxide adjusted for haemoglobin (D LCOc) and interstitial lung disease. In one family, segregation analysis revealed that variant carriers are either presenting with PAH associated with low D LCOc, or have only decreased D LCOc, whereas non-carrier relatives have normal D LCOc. In the second family, a single affected carrier was alive. His carrier mother was unaffected with normal D LCOc.We provided genetic evidence for considering KDR as a newly identified PAH-causing gene by describing the segregation of KDR mutations with PAH in two families. In our study, KDR mutations are associated with a particular form of PAH characterised by low D LCOc and radiological evidence of parenchymal lung disease including interstitial lung disease and emphysema.

Phenotype and outcome of pulmonary arterial hypertension patients carrying a TBX4 mutation.

INTRODUCTION: TBX4 mutation causes small patella syndrome (SPS) and/or pulmonary arterial hypertension (PAH). The characteristics and outcomes of PAH associated with TBX4 mutations are largely unknown. METHODS: We report the clinical, functional, radiologic, histologic and haemodynamic characteristics and outcomes of heritable PAH patients carrying a TBX4 mutation from the French pulmonary hypertension (PH) network. RESULTS: 20 patients were identified in 17 families. They were characterised by a median age at diagnosis of 29 years (0-76 years) and a female to male ratio of three. Most of the patients (70%) were in New York Heart Association (NYHA) functional class III or IV with a severe haemodynamic impairment (median pulmonary vascular resistance (PVR) of 13.6 (6.2-41.8) Wood units). Skeletal signs of SPS were present in 80% of cases. Half of the patients had mild restrictive or obstructive limitation and diffusing capacity of the lung for carbon monoxide (D LCO) was decreased in all patients. High-resolution computed tomography (HRCT) showed bronchial abnormalities, peri-bronchial cysts, mosaic distribution and mediastinal lymphadenopathies. PAH therapy was associated with significant clinical improvement. At follow-up (median 76 months), two patients had died and two had undergone lung transplantation. One-year, three-year and five-year event-free survival rates were 100%, 94% and 83%, respectively. Histologic examination of explanted lungs revealed alveolar growth abnormalities, major pulmonary vascular remodelling similar to that observed in idiopathic pulmonary arterial hypertension (IPAH) and accumulation of cholesterol crystals within the lung parenchyma. CONCLUSION: PAH due to TBX4 mutations may occur with or without skeletal abnormalities across a broad age range from birth to late adulthood. PAH is usually severe and associated with bronchial and parenchymal abnormalities.

Understanding the Similarities and Differences between Hepatic and Pulmonary Veno-Occlusive Disease.

Hepatic veno-occlusive disease (HVOD), alias sinusoidal obstruction syndrome, may develop as a complication of chemotherapy in the setting of hematopoietic stem cell transplantation. HVOD is less frequently described after exposure to chemotherapy in the nontransplant setting and can also be a complication after ingestion of toxins, such as pyrrolizidine alkaloids. Veno-occlusive disease may also affect the lungs, and it is therefore termed pulmonary veno-occlusive disease (PVOD). Similarly, PVOD can develop after exposure to chemotherapeutic agents in the treatment of solid and hematological malignancies. In addition, PVOD has also been linked to autoimmune disorders and occupational solvent exposure. Finally, the heritable form of PVOD is due to biallelic mutations of the EIF2AK4 gene. Both HVOD and PVOD share common histopathological features and pathophysiologic mechanisms. Both clinical disorders are rare complications that can appear after exposure to the common inciting trigger of chemotherapeutic agents. The present review aims to summarize the current knowledge of HVOD and PVOD and to describe both similarities as well as differences regarding both conditions.

Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells.

The physiopathology of pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) and endothelial cell (PAEC) dysfunction, contributing to pulmonary arterial obstruction and PAH progression. KCNK3 loss of function mutations are responsible for the first channelopathy identified in PAH. Loss of KCNK3 function/expression is a hallmark of PAH. However, the molecular mechanisms involved in KCNK3 dysfunction are mostly unknown. To identify the pathological molecular mechanisms downstream of KCNK3 in human PASMCs (hPASMCs) and human PAECs (hPAECs), we used a Liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach to identify the molecular pathways regulated by KCNK3. KCNK3 loss of expression was induced in control hPASMCs or hPAECs by specific siRNA targeting KCNK3. We found that the loss of KCNK3 expression in hPAECs and hPASMCs leads to 326 and 222 proteins differentially expressed, respectively. Among them, 53 proteins were common to hPAECs and hPASMCs. The specific proteome remodeling in hPAECs in absence of KCNK3 was mostly related to the activation of glycolysis, the superpathway of methionine degradation, and the mTOR signaling pathways, and to a reduction in EIF2 signaling pathways. In hPASMCs, we found an activation of the PI3K/AKT signaling pathways and a reduction in EIF2 signaling and the Purine Nucleotides De Novo Biosynthesis II and IL-8 signaling pathways. Common to hPAECs and hPASMCs, we found that the loss of KCNK3 expression leads to the activation of the NRF2-mediated oxidative stress response and a reduction in the interferon pathway. In the hPAECs and hPASMCs, we found an increased expression of HO-1 (heme oxygenase-1) and a decreased IFIT3 (interferon-induced proteins with tetratricopeptide repeats 3) (confirmed by Western blotting), allowing us to identify these axes to understand the consequences of KCNK3 dysfunction. Our experiments, based on the loss of KCNK3 expression by a specific siRNA strategy in control hPAECs and hPASMCs, allow us to identify differences in the activation of several signaling pathways, indicating the key role played by KCNK3 dysfunction in the development of PAH. Altogether, these results allow us to better understand the consequences of KCNK3 dysfunction and suggest that KCNK3 loss of expression acts in favor of the proliferation and migration of hPASMCs and promotes the metabolic shift and apoptosis resistance of hPAECs.

Widening the landscape of heritable pulmonary hypertension mutations in paediatric and adult cases.

BACKGROUND: Heritable forms of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease/pulmonary capillary haemangiomatosis (PVOD/PCH) diverge by lung histopathological lesions, clinical and para-clinical presentation, their responsible genes, and mode of transmission. Since the identification of the BMPR2 gene in families affected by PAH, mutations in several other genes have been discovered for both forms. The mutation landscape in these new genes is not yet well known. METHODS: We set up a next-generation sequencing-based targeted sequencing gene panel allowing known genes for PAH and PVOD/PCH to be analysed simultaneously. Genetic analysis was prospectively performed on 263 PAH and PVOD/PCH patients (adult and paediatric cases). RESULTS: Pathogenic mutations were identified in 19.5% of sporadic PAH patients (n=180), 54.5% of familial PAH patients and 13.3% of PVOD/PCH patients. BMPR2 was the most frequently mutated gene, followed by TBX4 in both paediatric and adult PAH. BMP9 mutations were identified in 1.2% of adult PAH cases. EIF2AK4 biallelic mutations were restricted to PVOD/PCH. A truncating mutation and a predicted loss-of-function variant were also identified in BMP10 in two severely affected sporadic PAH female patients. CONCLUSION: Our results confirm that mutations are found in genes beyond BMPR2 in heritable PAH, emphasise the role of TBX4 and BMP9, and designate BMP10 as a new PAH gene.

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation.

Ion Channels in Pulmonary Hypertension: A Therapeutic Interest?

Pulmonary arterial hypertension (PAH) is a multifactorial and severe disease without curative therapies. PAH pathobiology involves altered pulmonary arterial tone, endothelial dysfunction, distal pulmonary vessel remodeling, and inflammation, which could all depend on ion channel activities (K⁺, Ca2+, Na⁺ and Cl-). This review focuses on ion channels in the pulmonary vasculature and discusses their pathophysiological contribution to PAH as well as their therapeutic potential in PAH.