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

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

Role of KCNK3 Dysfunction in Dasatinib-associated Pulmonary Arterial Hypertension and Endothelial Cell Dysfunction.

Pulmonary arterial (PA) hypertension (PAH) is a severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (Potassium channel subfamily K member 3; coding for outward K+ channel) variant in a patient with dasatinib-associated PAH and investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control human PA smooth muscle cells (hPASMCs) and human pulmonary endothelial cells (hPECs), we evaluated the consequences of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp experiments revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to PA constriction by decreasing KCNK3 function and expression. In control hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that a loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

Serum and Pulmonary Expression Profiles of the Activin Signaling System in Pulmonary Arterial Hypertension.

BACKGROUND: Activins are novel therapeutic targets in pulmonary arterial hypertension (PAH). We therefore studied whether key members of the activin pathway could be used as PAH biomarkers. METHODS: Serum levels of activin A, activin B, α-subunit of inhibin A and B proteins, and the antagonists follistatin and follistatin-like 3 (FSTL3) were measured in controls and in patients with newly diagnosed idiopathic, heritable, or anorexigen-associated PAH (n=80) at baseline and 3 to 4 months after treatment initiation. The primary outcome was death or lung transplantation. Expression patterns of the inhibin subunits, follistatin, FSTL3, Bambi, Cripto, and the activin receptors type I (ALK), type II (ACTRII), and betaglycan were analyzed in PAH and control lung tissues. RESULTS: Death or lung transplantation occurred in 26 of 80 patients (32.5%) over a median follow-up of 69 (interquartile range, 50-81) months. Both baseline (hazard ratio, 1.001 [95% CI, 1.000-1.001]; P=0.037 and 1.263 [95% CI, 1.049-1.520]; P=0.014, respectively) and follow-up (hazard ratio, 1.003 [95% CI, 1.001-1.005]; P=0.001 and 1.365 [95% CI, 1.185-1.573]; P<0.001, respectively) serum levels of activin A and FSTL3 were associated with transplant-free survival in a model adjusted for age and sex. Thresholds determined by receiver operating characteristic analyses were 393 pg/mL for activin A and 16.6 ng/mL for FSTL3. When adjusted with New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-B-type natriuretic peptide, the hazard ratios for transplant-free survival for baseline activin A <393 pg/mL and FSTL3 <16.6 ng/mL were, respectively, 0.14 (95% CI, 0.03-0.61; P=0.009) and 0.17 (95% CI, 0.06-0.45; P<0.001), and for follow-up measures, 0.23 (95% CI, 0.07-0.78; P=0.019) and 0.27 (95% CI, 0.09-0.78, P=0.015), respectively. Prognostic values of activin A and FSTL3 were confirmed in an independent external validation cohort. Histological analyses showed a nuclear accumulation of the phosphorylated form of Smad2/3, higher immunoreactivities for ACTRIIB, ALK2, ALK4, ALK5, ALK7, Cripto, and FSTL3 in vascular endothelial and smooth muscle layers, and lower immunostaining for inhibin-α and follistatin. CONCLUSIONS: These findings offer new insights into the activin signaling system in PAH and show that activin A and FSTL3 are prognostic biomarkers for PAH.

Sotatercept for the Treatment of Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension is characterized by pulmonary vascular remodeling, cellular proliferation, and poor long-term outcomes. Dysfunctional bone morphogenetic protein pathway signaling is associated with both hereditary and idiopathic subtypes. Sotatercept, a novel fusion protein, binds activins and growth differentiation factors in the attempt to restore balance between growth-promoting and growth-inhibiting signaling pathways. METHODS: In this 24-week multicenter trial, we randomly assigned 106 adults who were receiving background therapy for pulmonary arterial hypertension to receive subcutaneous sotatercept at a dose of 0.3 mg per kilogram of body weight every 3 weeks or 0.7 mg per kilogram every 3 weeks or placebo. The primary end point was the change from baseline to week 24 in pulmonary vascular resistance. RESULTS: Baseline characteristics were similar among the three groups. The least-squares mean difference between the sotatercept 0.3-mg group and the placebo group in the change from baseline to week 24 in pulmonary vascular resistance was -145.8 dyn · sec · cm-5 (95% confidence interval [CI], -241.0 to -50.6; P = 0.003). The least-squares mean difference between the sotatercept 0.7-mg group and the placebo group was -239.5 dyn · sec · cm-5 (95% CI, -329.3 to -149.7; P<0.001). At 24 weeks, the least-squares mean difference between the sotatercept 0.3-mg group and the placebo group in the change from baseline in 6-minute walk distance was 29.4 m (95% CI, 3.8 to 55.0). The least-squares mean difference between the sotatercept 0.7-mg group and the placebo group was 21.4 m (95% CI, -2.8 to 45.7). Sotatercept was also associated with a decrease in N-terminal pro-B-type natriuretic peptide levels. Thrombocytopenia and an increased hemoglobin level were the most common hematologic adverse events. One patient in the sotatercept 0.7-mg group died from cardiac arrest. CONCLUSIONS: Treatment with sotatercept resulted in a reduction in pulmonary vascular resistance in patients receiving background therapy for pulmonary arterial hypertension. (Funded by Acceleron Pharma; PULSAR ClinicalTrials.gov number, NCT03496207.).

Definition, classification and diagnosis of pulmonary hypertension.

Pulmonary hypertension (PH) is a haemodynamic condition characterised by elevation of mean pulmonary arterial pressure (mPAP) >20 mmHg, assessed by right heart catheterisation. Pulmonary arterial wedge pressure (PAWP) and pulmonary vascular resistance (PVR) distinguish pre-capillary PH (PAWP ≤15 mmHg, PVR >2 Wood Units (WU)), isolated post-capillary PH (PAWP >15 mmHg, PVR ≤2 WU) and combined post- and pre-capillary PH (PAWP >15 mmHg, PVR >2 WU). Exercise PH is a haemodynamic condition describing a normal mPAP at rest with an abnormal increase of mPAP during exercise, defined as a mPAP/cardiac output slope >3 mmHg/L/min between rest and exercise. The core structure of the clinical classification of PH has been retained, including the five major groups. However, some changes are presented herewith, such as the re-introduction of "long-term responders to calcium channel blockers" as a subgroup of idiopathic pulmonary arterial hypertension, the addition of subgroups in group 2 PH and the differentiation of group 3 PH subgroups based on pulmonary diseases instead of functional abnormalities. Mitomycin-C and carfilzomib have been added to the list of drugs with "definite association" with PAH. For diagnosis of PH, we propose a stepwise approach with the main aim of discerning those patients who need to be referred to a PH centre and who should undergo invasive haemodynamic assessment. In case of high probability of severe pulmonary vascular disease, especially if there are signs of right heart failure, a fast-track referral to a PH centre is recommended at any point during the clinical workup.

Characteristics and outcomes of patients developing pulmonary hypertension associated with proteasome inhibitors.

BACKGROUND: Pulmonary arterial hypertension (PAH) has been described in patients treated with proteasome inhibitors (PIs). Our objective was to evaluate the association between PIs and PAH. METHODS: Characteristics of incident PAH cases previously treated with carfilzomib or bortezomib were analysed from the French pulmonary hypertension registry and the VIGIAPATH programme from 2004 to 2023, concurrently with a pharmacovigilance disproportionality analysis using the World Health Organization (WHO) global database (VigiBase) and a meta-analysis of randomised controlled trials. RESULTS: 11 incident cases of PI-associated PAH were identified (six with carfilzomib and five with bortezomib) with a female:male ratio of 2.7:1, a median age of 61 years, and a median delay between PI first exposure and PAH of 6 months. Four patients died (two from right heart failure, one from respiratory distress and one from an unknown cause). At diagnosis, six were in New York Heart Association Functional Class III/IV with severe haemodynamic impairment (median mean pulmonary arterial pressure 39 mmHg, cardiac index 2.45 L·min-1·m-2 and pulmonary vascular resistance 7.2 WU). In the WHO pharmacovigilance database, 169 cases of PH associated with PI were reported since 2013 with significant signals of disproportionate reporting (SDR) for carfilzomib, regardless of the definition of cases or control group. However, SDR for bortezomib were inconsistent. The systematic review identified 17 clinical trials, and carfilzomib was associated with a significantly higher risk of dyspnoea, severe dyspnoea and PH compared with bortezomib. CONCLUSION: PIs may induce PAH in patients undergoing treatment, with carfilzomib emitting a stronger signal than bortezomib, and these patients should be monitored closely.

Pulmonary vascular phenotype identified in patients with GDF2 (BMP9) or BMP10 variants: an international multicentre study.

BACKGROUND: Bone morphogenetic proteins 9 and 10 (BMP9 and BMP10), encoded by GDF2 and BMP10, respectively, play a pivotal role in pulmonary vascular regulation. GDF2 variants have been reported in pulmonary arterial hypertension (PAH) and hereditary haemorrhagic telangiectasia (HHT). However, the phenotype of GDF2 and BMP10 carriers remains largely unexplored. METHODS: We report the characteristics and outcomes of PAH patients in GDF2 and BMP10 carriers from the French and Dutch pulmonary hypertension registries. A literature review explored the phenotypic spectrum of these patients. RESULTS: 26 PAH patients were identified: 20 harbouring heterozygous GDF2 variants, one homozygous GDF2 variant, four heterozygous BMP10 variants, and one with both GDF2 and BMP10 variants. The prevalence of GDF2 and BMP10 variants was 1.3% and 0.4%, respectively. Median age at PAH diagnosis was 30 years, with a female/male ratio of 1.9. Congenital heart disease (CHD) was present in 15.4% of the patients. At diagnosis, most of the patients (61.5%) were in New York Heart Association Functional Class III or IV with severe haemodynamic compromise (median (range) pulmonary vascular resistance 9.0 (3.3-40.6) WU). Haemoptysis was reported in four patients; none met the HHT criteria. Two patients carrying BMP10 variants underwent lung transplantation, revealing typical PAH histopathology. The literature analysis showed that 7.6% of GDF2 carriers developed isolated HHT, and identified cardiomyopathy and developmental disorders in BMP10 carriers. CONCLUSIONS: GDF2 and BMP10 pathogenic variants are rare among PAH patients, and occasionally associated with CHD. HHT cases among GDF2 carriers are limited according to the literature. BMP10 full phenotypic ramifications warrant further investigation.

Deep phenotyping of unaffected carriers of pathogenic BMPR2 variants screened for pulmonary arterial hypertension.

INTRODUCTION: Pathogenic variants in the gene encoding for BMPR2 are a major genetic risk factor for heritable pulmonary arterial hypertension. Owing to incomplete penetrance, deep phenotyping of unaffected carriers of a pathogenic BMPR2 variant through multimodality screening may aid in early diagnosis and identify susceptibility traits for future development of pulmonary arterial hypertension. METHODS: 28 unaffected carriers (44±16 years, 57% female) and 21 healthy controls (44±18 years, 48% female) underwent annual screening, including cardiac magnetic resonance imaging, transthoracic echocardiography, cardiopulmonary exercise testing and right heart catheterisation. Right ventricular pressure-volume loops were constructed to assess load-independent contractility and compared with a healthy control group. A transgenic Bmpr2Δ71Ex1/+ rat model was employed to validate findings from humans. RESULTS: Unaffected carriers had lower indexed right ventricular end-diastolic (79.5±17.6 mL·m-2 versus 62.7±15.3 mL·m-2; p=0.001), end-systolic (34.2±10.5 mL·m-2 versus 27.1±8.3 mL·m-2; p=0.014) and left ventricular end-diastolic (68.9±14.1 mL·m-2 versus 58.5±10.7 mL·m-2; p=0.007) volumes than control subjects. Bmpr2Δ71Ex1/+ rats were also observed to have smaller cardiac volumes than wild-type rats. Pressure-volume loop analysis showed that unaffected carriers had significantly higher afterload (arterial elastance 0.15±0.06 versus 0.27±0.08 mmHg·mL-1; p<0.001) and end-systolic elastance (0.28±0.07 versus 0.35±0.10 mmHg·mL-1; p=0.047) in addition to lower right ventricular pulmonary artery coupling (end-systolic elastance/arterial elastance 2.24±1.03 versus 1.36±0.37; p=0.006). During the 4-year follow-up period, two unaffected carriers developed pulmonary arterial hypertension, with normal N-terminal pro-brain natriuretic peptide and transthoracic echocardiography indices at diagnosis. CONCLUSION: Unaffected BMPR2 mutation carriers have an altered cardiac phenotype mimicked in Bmpr2Δ71Ex1/+ transgenic rats. Future efforts to establish an effective screening protocol for individuals at risk for developing pulmonary arterial hypertension warrant longer follow-up periods.

Risk stratification refinements with inclusion of haemodynamic variables at follow-up in patients with pulmonary arterial hypertension.

BACKGROUND: Haemodynamic variables are prognostic factors in pulmonary arterial hypertension (PAH). However, right heart catheterisation (RHC) is not systematically recommended to assess the risk status during follow-up. This study aimed to assess the added value of haemodynamic variables in prevalent patients to predict the risk of death or lung transplantation according to their risk status assessed by the non-invasive four-strata model as recommended by the European guidelines. METHODS: We evaluated incident patients with PAH enrolled in the French pulmonary hypertension registry between 2009 and 2020 who had a first follow-up RHC. Cox regression identified, in each follow-up risk status, haemodynamic variables significantly associated with transplant-free survival. Optimal thresholds were determined by time-dependent receiver operating characteristics. Several multivariable Cox regression models were performed to identify the haemodynamic variables improving the non-invasive risk stratification model. RESULTS: We analysed 1240 incident patients reassessed within 1 year by RHC. None of the haemodynamic variables were significantly associated with transplant-free survival among low-risk (n=386) or high-risk (n=71) patients. Among patients at intermediate (intermediate-low, n=483 and intermediate-high, n=300) risk at first follow-up, multivariable models including either stroke volume index (SVI) or mixed venous oxygen saturation (S vO2 ) were the best. The prognostic performance of a refined six-strata risk stratification model including the non-invasive four-strata model and SVI >37 mL·m-2 and/or S vO2 >65% for patients at intermediate risk (area under the curve (AUC) 0.81; c-index 0.74) was better than that of the four-strata model (AUC 0.79, p=0.009; c-index 0.72). CONCLUSION: Cardiopulmonary haemodynamics may improve risk stratification at follow-up in patients at intermediate risk.

Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. METHODS: Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). RESULTS: Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. CONCLUSIONS: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.

Radiological pulmonary sequelae after COVID-19 and correlation with clinical and functional pulmonary evaluation: results of a prospective cohort.

OBJECTIVES: Whether COVID-19 leads to long-term pulmonary sequelae or not remains unknown. The aim of this study was to assess the prevalence of persisting radiological pulmonary fibrotic lesions in patients hospitalized for COVID-19. MATERIALS AND METHODS: We conducted a prospective single-center study among patients hospitalized for COVID-19 between March and May 2020. Patients with residual symptoms or admitted into intensive care units were investigated 4 months after discharge by a chest CT (CCT) and pulmonary function tests (PFTs). The primary endpoint was the rate of persistent radiological fibrotic lesions after 4 months. Secondary endpoints included further CCT evaluation at 9 and 16 months, correlation of fibrotic lesions with clinical and PFT evaluation, and assessment of predictive factors. RESULTS: Among the 1151 patients hospitalized for COVID-19, 169 patients performed a CCT at 4 months. CCTs showed pulmonary fibrotic lesions in 19% of the patients (32/169). These lesions were persistent at 9 months and 16 months in 97% (29/30) and 95% of patients (18/19) respectively. There was no significant clinical difference based on dyspnea scale in patients with pulmonary fibrosis. However, PFT evaluation showed significantly decreased diffusing lung capacity for carbon monoxide (p < 0.001) and total lung capacity (p < 0.001) in patients with radiological lesions. In multivariate analysis, the predictive factors of radiological pulmonary fibrotic lesions were pulmonary embolism (OR = 9.0), high-flow oxygen (OR = 6.37), and mechanical ventilation (OR = 3.49). CONCLUSION: At 4 months, 19% of patients investigated after hospitalization for COVID-19 had radiological pulmonary fibrotic lesions; they persisted up to 16 months. CLINICAL RELEVANCE STATEMENT: Whether COVID-19 leads to long-term pulmonary sequelae or not remains unknown. The aim of this study was to assess the prevalence of persisting radiological pulmonary fibrotic lesions in patients hospitalized for COVID-19. The prevalence of persisting lesions after COVID-19 remains unclear. We assessed this prevalence and predictive factors leading to fibrotic lesions in a large cohort. The respiratory clinical impact of these lesions was also assessed. KEY POINTS: • Nineteen percent of patients hospitalized for COVID-19 had radiological fibrotic lesions at 4 months, remaining stable at 16 months. • COVID-19 fibrotic lesions did not match any infiltrative lung disease pattern. • COVID-19 fibrotic lesions were associated with pulmonary function test abnormalities but did not lead to clinical respiratory manifestation.

Chronic interstitial lung disease associated with systemic lupus erythematosus: A multicentric study of 89 cases.

BACKGROUND AND OBJECTIVE: Chronic interstitial lung disease (ILD) occurs rarely with systemic lupus erythematosus (SLE) as compared with other connective tissue diseases. This multicentric retrospective study of patients with SLE-ILD from the OrphaLung and French SLE networks during 2005-2020 aimed to describe the characteristics of patients with SLE-ILD and analyse factors associated with prognosis. METHODS: We analysed data for 89 patients with SLE-ILD (82 women, 92.1%) (median age at SLE diagnosis: 35 years [interquartile range 27-47]). All patients met the 2019 EULAR/ACR criteria for the diagnosis of SLE. RESULTS: Forty two (47.2%) patients were positive for anti-ribonuclear protein antibodies and 45 (50.6%) for anti SSA/Ro antibodies. A total of 58 (65.2%) patients had another connective tissue disease: Sjögren's syndrome (n = 33, 37.1%), systemic sclerosis (n = 14, 15.7%), inflammatory myopathy (n = 6, 6.7%), or rheumatoid arthritis (n = 6, 6.7%). ILD was diagnosed along with SLE in 25 (28.1%) patients and at a median of 6 (0-14) years after the SLE diagnosis. The most frequent CT pattern was suggestive of non-specific interstitial pneumonia (n = 41, 46.0%) with or without superimposed organizing pneumonia. After a median follow-up of 86.5 [39.5-161.2] months, 18 (20.2%) patients had died and 6 (6.7%) underwent lung transplantation. The median 5-year and 10-year transplantation-free survival were 96% (92-100) and 87% (78-97). In total, 44 (49.4%) patients showed ILD progression. Cutaneous manifestations and Raynaud's phenomenon were associated with better survival. Only forced vital capacity was significantly associated with survival and ILD progression. CONCLUSION: ILD is a rare manifestation of SLE with good overall prognosis but with possible risk of ILD progression. Patients with SLE-ILD frequently have another connective tissue disease.

Clinical Phenotype and Outcomes of Pulmonary Hypertension Associated with Myeloproliferative Neoplasms: A Population-based Study.

Rationale: Precapillary pulmonary hypertension (PH) is a rare and largely unrecognized complication of myeloproliferative neoplasms (MPNs), including polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (MF). Objectives: To describe characteristics and outcomes of MPN-associated PH. Methods: We report clinical, functional, and hemodynamic characteristics, classification, and outcomes of patients with PV, ET, or primary MF in the French PH registry. Measurements and Main Results: Ninety patients with MPN (42 PV, 35 ET, 13 primary MF) presented with precapillary PH with severe hemodynamic impairment, with a median mean pulmonary arterial pressure and pulmonary vascular resistance of 42 mm Hg and 6.7 Wood units, respectively, and impaired clinical conditions, with 71% in New York Heart Association functional classes III/IV and having a median 6-minute-walk distance of 310 m. Half of the patients were diagnosed with chronic thromboembolic PH (CTEPH); the other half were considered to have group 5 PH. MF was preferentially associated with group 5 PH, whereas PV and ET were generally related to CTEPH. Proximal lesions were diagnosed in half of the patients with CTEPH. Thromboendarterectomy was performed in 18 selected patients with high risk of complications (5 early deaths). Overall survival at 1, 3, and 5 years was 67%, 50%, and 34% in group 5 PH and 81%, 66%, and 42% in CTEPH, respectively. Conclusions: PH is a life-threatening condition potentially occurring in MPN. There are multiple mechanisms, with equal diagnoses of CTEPH and group 5 PH. Physicians should be aware that PH strongly affects the burden of patients with MPN, especially in group 5 PH, with unknown pathophysiological mechanisms.

Molecular Function and Contribution of TBX4 in Development and Disease.

Over the past decade, recognition of the profound impact of the TBX4 (T-box 4) gene, which encodes a member of the evolutionarily conserved family of T-box-containing transcription factors, on respiratory diseases has emerged. The developmental importance of TBX4 is emphasized by the association of TBX4 variants with congenital disorders involving respiratory and skeletal structures; however, the exact role of TBX4 in human development remains incompletely understood. Here, we discuss the developmental, tissue-specific, and pathological TBX4 functions identified through human and animal studies and review the published TBX4 variants resulting in variable disease phenotypes. We also outline future research directions to fill the gaps in our understanding of TBX4 function and of how TBX4 disruption affects development.

Unraveling the Impact of miR-146a in Pulmonary Arterial Hypertension Pathophysiology and Right Ventricular Function.

Pulmonary arterial hypertension (PAH) is a chronic disorder characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary vascular resistance and right ventricle (RV) overload and failure. MicroRNA-146a (miR-146a) promotes vascular smooth muscle cell proliferation and vascular neointimal hyperplasia, both hallmarks of PAH. This study aimed to investigate the effects of miR-146a through pharmacological or genetic inhibition on experimental PAH and RV pressure overload animal models. Additionally, we examined the overexpression of miR-146a on human pulmonary artery smooth muscle cells (hPASMCs). Here, we showed that miR-146a genic expression was increased in the lungs of patients with PAH and the plasma of monocrotaline (MCT) rats. Interestingly, genetic ablation of miR-146a improved RV hypertrophy and systolic pressures in Sugen 5415/hypoxia (SuHx) and pulmonary arterial banding (PAB) mice. Pharmacological inhibition of miR-146a improved RV remodeling in PAB-wild type mice and MCT rats, and enhanced exercise capacity in MCT rats. However, overexpression of miR-146a did not affect proliferation, migration, and apoptosis in control-hPASMCs. Our findings show that miR-146a may play a significant role in RV function and remodeling, representing a promising therapeutic target for RV hypertrophy and, consequently, PAH.

Physiological and pathophysiological roles of the KCNK3 potassium channel in the pulmonary circulation and the heart.

Potassium channel subfamily K member 3 (KCNK3), encoded by the KCNK3 gene, is part of the two-pore domain potassium channel family, constitutively active at resting membrane potentials in excitable cells, including smooth muscle and cardiac cells. Several physiological and pharmacological mediators, such as intracellular signalling pathways, extracellular pH, hypoxia and anaesthetics, regulate KCNK3 channel function. Recent studies show that modulation of KCNK3 channel expression and function strongly influences pulmonary vascular cell and cardiomyocyte function. The altered activity of KCNK3 in pathological situations such as atrial fibrillation, pulmonary arterial hypertension and right ventricular dysfunction demonstrates the crucial role of KCNK3 in cardiovascular homeostasis. Furthermore, loss of function variants of KCNK3 have been identified in patients suffering from pulmonary arterial hypertension and atrial fibrillation. This review focuses on current knowledge of the role of the KCNK3 channel in pulmonary circulation and the heart, in healthy and pathological conditions.

T-cell dysregulation and inflammatory process in Gcn2 (Eif2ak4-/-)-deficient rats in basal and stress conditions.

Hereditary pulmonary veno-occlusive disease (hPVOD) is a severe form of autosomal recessive pulmonary hypertension and is due to biallelic loss of function of the EIF2AK4 gene (alias GCN2) coding for GCN2. GCN2 is a stress kinase that belongs to the integrated stress response pathway (ISR). Three rat lines carrying biallelic Gcn2 mutation were generated and found phenotypically normal and did not spontaneously develop a PVOD-related disease. We submitted these rats to amino acid deprivation to document the molecular and cellular response of the lungs and to identify phenotypic changes that could be involved in PVOD pathophysiology. Gcn2-/- rat lungs were analyzed under basal conditions and 3 days after a single administration of PEG-asparaginase (ASNase). Lung mRNAs were analyzed by RNAseq and single-cell RNAseq (scRNA-seq), flow cytometry, tissue imaging, and Western blots. The ISR was not activated after ASNase treatment in Gcn2-/- rat lungs, and apoptosis was increased. Several proinflammatory and innate immunity genes were overexpressed, and inflammatory cells infiltration was also observed in the perivascular area. Under basal conditions, scRNA-seq analysis of Gcn2-/- rat lungs revealed increases in two T-cell populations, a LAG3+ T-cell population and a proliferative T-cell population. Following ASNase administration, we observed an increase in calprotectin expression involved in TLR pathway activation and neutrophil infiltration. In conclusion, under basal and asparagine and glutamine deprivation induced by asparaginase administration, Gcn2-/- rats display molecular and cellular signatures in the lungs that may indicate a role for Gcn2 in immune homeostasis and provide further clues to the mechanisms of hPVOD development.

Contribution of transient receptor potential canonical channels in human and experimental pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is due to progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated store-operated Ca2+ entry (SOCE) contributes to PAH pathogenesis, mediating human PA smooth muscle cell (hPASMC) abnormalities. The transient receptor potential canonical channels (TRPC family) are Ca2+-permeable channels contributing to SOCE in different cell types, including PASMCs. However, the properties, signaling pathways, and contribution to Ca2+ signaling of each TRPC isoform are unclear in human PAH. We studied in vitro the impact of TRPC knockdown on control and PAH-hPASMCs function. In vivo, we analyzed the consequences of pharmacological TRPC inhibition using the experimental model of pulmonary hypertension (PH) induced by monocrotaline (MCT) exposure. Compared with control-hPASMCs cells, in PAH-hPASMCs, we found a decreased TRPC4 expression, overexpression of TRPC3 and TRPC6, and unchanged TRPC1 expression. Using the siRNA strategy, we found that the knockdown of TRPC1-C3-C4-C6 reduced the SOCE and the proliferation rate of PAH-hPASMCs. Only TRPC1 knockdown decreased the migration capacity of PAH-hPASMCs. After PAH-hPASMCs exposure to the apoptosis inducer staurosporine, TRPC1-C3-C4-C6 knockdown increased the percentage of apoptotic cells, suggesting that these channels promote apoptosis resistance. Only TRPC3 function contributed to exacerbated calcineurin activity. In the MCT-PH rat model, only TRPC3 protein expression was increased in lungs compared with control rats, and in vivo "curative" administration of a TRPC3 inhibitor attenuated PH development in rats. These results suggest that TRPC channels contribute to PAH-hPASMCs dysfunctions, including SOCE, proliferation, migration, and apoptosis resistance, and could be considered as therapeutic targets in PAH.NEW & NOTEWORTHY TRPC3 is increased in human and experimental pulmonary arterial hypertension (PAH). In PAH pulmonary arterial smooth muscle cells, TRPC3 participates in the aberrant store-operated Ca2+ entry contributing to their pathological cell phenotypes (exacerbated proliferation, enhanced migration, apoptosis resistance, and vasoconstriction). Pharmacological in vivo inhibition of TRPC3 reduces the development of experimental PAH. Even if other TRPC acts on PAH development, our results prove that TRPC3 inhibition could be considered as an innovative treatment for PAH.

The evolving landscape of pulmonary arterial hypertension clinical trials.

Although it is a rare disease, the number of available therapeutic options for treating pulmonary arterial hypertension has increased since the late 1990s, with multiple drugs developed that are shown to be effective in phase 3 randomised controlled trials. Despite considerable advancements in pulmonary arterial hypertension treatment, prognosis remains poor. Existing therapies target pulmonary endothelial dysfunction with vasodilation and anti-proliferative effects. Novel therapies that target proliferative vascular remodelling and affect important outcomes are urgently needed. There is need for additional innovations in clinical trial design so that all emerging candidate therapies can be rigorously studied. Pulmonary arterial hypertension trial design has shifted from short-term submaximal exercise capacity as a primary endpoint, to larger clinical event-driven trial outcomes. Event-driven pulmonary arterial hypertension trials could face feasibility and efficiency issues in the future because increasing sample sizes and longer follow-up durations are needed, which would be problematic in such a rare disease. Enrichment strategies, innovative and alternative trial designs, and novel trial endpoints are potential solutions that could improve the efficiency of future pulmonary arterial hypertension trials while maintaining robustness and clinically meaningful evidence.

Genetic counselling and testing in pulmonary arterial hypertension: a consensus statement on behalf of the International Consortium for Genetic Studies in PAH.

Pulmonary arterial hypertension (PAH) is a rare disease that can be caused by (likely) pathogenic germline genomic variants. In addition to the most prevalent disease gene, BMPR2 (bone morphogenetic protein receptor 2), several genes, some belonging to distinct functional classes, are also now known to predispose to the development of PAH. As a consequence, specialist and non-specialist clinicians and healthcare professionals are increasingly faced with a range of questions regarding the need for, approaches to and benefits/risks of genetic testing for PAH patients and/or related family members. We provide a consensus-based approach to recommendations for genetic counselling and assessment of current best practice for disease gene testing. We provide a framework and the type of information to be provided to patients and relatives through the process of genetic counselling, and describe the presently known disease causal genes to be analysed. Benefits of including molecular genetic testing within the management protocol of patients with PAH include the identification of individuals misclassified by other diagnostic approaches, the optimisation of phenotypic characterisation for aggregation of outcome data, including in clinical trials, and importantly through cascade screening, the detection of healthy causal variant carriers, to whom regular assessment should be offered.