Long-term safety and tolerability of ambrisentan treatment for pediatric patients with pulmonary arterial hypertension: An open-label extension study.

This open-label, extension study assessed long-term safety, tolerability, and efficacy of ambrisentan in a pediatric population (age 8- < 18 years) with pulmonary arterial hypertension (PAH). Following completion of a 6-month, randomized study, participants entered the long-term extension at individualized ambrisentan dosages (2.5/5/7.5 or 10 mg/day). Safety assessments included adverse events (AEs), AEs of special interest, and serious AEs (SAEs); efficacy outcomes included 6-min walking distance (6MWD) and World Health Organization functional class (WHO FC). Thirty-eight of 41 (93%) randomized study participants entered the extension; 21 (55%) completed (reaching age 18 years). Most participants received concomitant phosphodiesterase-5 inhibitors (n = 25/38, 66%). Median ambrisentan exposure was 3.5 years. Most participants experienced ≥ 1 AE (n = 34/38, 89%), and 21 (55%) experienced SAEs, most commonly worsening PAH (n = 3/38, 8%), acute cardiac failure, pneumonia, or anemia (n = 2/38; 5% each); none considered ambrisentan-related. Seven participants (18%) died, with recorded reasons (MedDRA preferred term): cardiac failure (n = 2), PAH (n = 2), COVID-19 (n = 1), acute right ventricular failure (n = 1), and failure to thrive (n = 1); median time to death: 5.2 years. Anemia and hepatotoxicity AEs were generally mild to moderate and did not require ambrisentan dose adjustment. Assessed at study end in 29 participants (76%), mean 6MWD improved by 17% (standard deviation: 34.3%), and all (29/29, 100%) had improved or unchanged WHO FC.    Conclusion: Long-term weight-based ambrisentan dosing, alone or combined with other PAH therapies in children with PAH aged 8- < 18 years, exhibited tolerability and clinical improvements consistent with prior randomized study results.    Trial registration: NCT01342952, April 27, 2011. What is Known: • The endothelin receptor antagonist, ambrisentan, is indicated for treatment of pulmonary arterial hypertension (PAH). Previous studies have shown similar efficacy and tolerability in pediatric patients as in adults. What is New: • This open-label extension study assessed the long-term use of ambrisentan in pediatric patients (8-<18 years) with PAH, most of whom were also receiving recommended background PAH treatment. • Weight-based dosing of ambrisentan, given alone or in combination with other PAH therapies, was well tolerated with clinical improvements consistent with prior randomized study results.

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.

Quantification of systemic-to-pulmonary collateral flow in univentricular physiology with 4D flow MRI.

PURPOSE: Systemic-to-pulmonary collateral flow is a well-recognised phenomenon in patients with single ventricle physiology, but remains difficult to quantify. The aim was to compare the reported formula's that have been used for calculation of systemic-to-pulmonary-collateral flow to assess their consistency and to quantify systemic-to-pulmonary collateral flow in patients with a Glenn and/or Fontan circulation using four-dimensional flow MRI (4D flow MR). METHODS: Retrospective case-control study of Glenn and Fontan patients who had a 4D flow MR study. Flows were measured at the ascending aorta, left and right pulmonary arteries, left and right pulmonary veins, and both caval veins. Systemic-to-pulmonary collateral flow was calculated using two formulas: 1) pulmonary veins - pulmonary arteries and 2) ascending aorta - caval veins. Anatomical identification of collaterals was performed using the 4D MR image set. RESULTS: Fourteen patients (n = 11 Fontan, n = 3 Glenn) were included (age 26 [22-30] years). Systemic-to-pulmonary collateral flow was significantly higher in the patients than the controls (n = 10, age 31.2 [15.1-38.4] years) with both formulas: 0.28 [0.09-0.5] versus 0.04 [-0.66-0.21] l/min/m2 (p = 0.036, formula 1) and 0.67 [0.24-0.88] versus -0.07 [-0.16-0.08] l/min/m2 (p < 0.001, formula 2). In patients, systemic-to-pulmonary collateral flow differed significantly between formulas 1 and 2 (13% versus 26% of aortic flow, p = 0.038). In seven patients, veno-venous collaterals were detected and no aortopulmonary collaterals were visualised. CONCLUSION: 4D flow MR is able to detect increased systemic-to-pulmonary collateral flow and visualise collaterals vessels in Glenn and Fontan patients. However, the amount of systemic-to-pulmonary collateral flow varies with the formula employed. Therefore, further research is necessary before it could be applied in clinical care.

Intraoperative transit-time flow measurement of caval veins before and after bidirectional cavopulmonary anastomosis.

BACKGROUND: Haemodynamic changes in caval venous flow distribution occurring during bidirectional cavopulmonary anastomosis operation are still largely unknown. METHODS: Transit time flow measurements were performed in 15 cavopulmonary anastomosis operations. Superior and inferior caval vein flows were measured before and after the cavopulmonary anastomosis. Ratio of superior caval vein to overall caval veins flow was calculated. RESULTS: Mean superior caval vein flow ratio before cavopulmonary anastomosis was higher than previously reported for healthy children. Superior caval vein flow ratio decreased in 14/15 patients after cavopulmonary anastomosis: mean 0.63 ± 0.12 before versus 0.43 ± 0.14 after. No linear correlation between intraoperative superior caval vein pressure and superior caval vein flow after cavopulmonary anastomosis was found. Neither Nakata index nor pulmonary vascular resistance measured at preoperative cardiac catheterisation correlated with intraoperative flows. None of patients died or required a take down. CONCLUSIONS: The higher mean superior caval vein flow ratio before cavopulmonary anastomosis compared to healthy children suggests flow redistribution in univentricular physiology to protect brain and neurodevelopment. The decrease of superior caval vein flow ratio after cavopulmonary anastomosis may reflect the flow redistribution related to trans-pulmonary gradient. The lack of correlation between superior caval vein pressure and superior caval vein flow could be explained by limited sample size and multifactorial determinants of caval veins flow, although pressure remain essential. Larger sample of measurements are needed to find flow range potentially predictive for clinical failure. To authors' knowledge, this is the first intraoperative flow measurement of both caval veins during cavopulmonary operations.

Inhibition of the prolyl isomerase Pin1 improves endothelial function and attenuates vascular remodelling in pulmonary hypertension by inhibiting TGF-ß signalling.

Pulmonary arterial hypertension (PAH) is a devastating disease, characterized by obstructive pulmonary vascular remodelling ultimately leading to right ventricular (RV) failure and death. Disturbed transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) signalling, endothelial cell dysfunction, increased proliferation of smooth muscle cells and fibroblasts, and inflammation contribute to this abnormal remodelling. Peptidyl-prolyl isomerase Pin1 has been identified as a critical driver of proliferation and inflammation in vascular cells, but its role in the disturbed TGF-ß/BMP signalling, endothelial cell dysfunction, and vascular remodelling in PAH is unknown. Here, we report that Pin1 expression is increased in cultured pulmonary microvascular endothelial cells (MVECs) and lung tissue of PAH patients. Pin1 inhibitor, juglone significantly decreased TGF-ß signalling, increased BMP signalling, normalized their hyper-proliferative, and inflammatory phenotype. Juglone treatment reversed vascular remodelling through reducing TGF-ß signalling in monocrotaline + shunt-PAH rat model. Juglone treatment decreased Fulton index, but did not affect or harm cardiac function and remodelling in rats with RV pressure load induced by pulmonary artery banding. Our study demonstrates that inhibition of Pin1 reversed the PAH phenotype in PAH MVECs in vitro and in PAH rats in vivo, potentially through modulation of TGF-ß/BMP signalling pathways. Selective inhibition of Pin1 could be a novel therapeutic option for the treatment of PAH.

Female rats are less prone to clinical heart failure than male rats in a juvenile rat model of right ventricular pressure load.

Sex is increasingly emerging as determinant of right ventricular (RV) adaptation to abnormal loading conditions. It is unknown, however, whether sex-related differences already occur in childhood. Therefore, this study aimed to assess sex differences in a juvenile model of early RV pressure load by pulmonary artery banding (PAB) during transition from pre to postpuberty. Rat pups (n = 57, 3 wk old, 30-45 g) were subjected to PAB or sham surgery. Animals were euthanized either before or after puberty (4 and 8 wk postsurgery, respectively). Male PAB rats demonstrated failure to thrive already after 4 wk, whereas females did not. After 8 wk, female PAB rats showed less clinical symptoms of RV failure than male PAB rats. RV pressure-volume analysis demonstrated increased end-systolic elastance after 4 wk in females only, and a trend toward preserved end-diastolic elastance in female PAB rats compared with males (P = 0.055). Histology showed significantly less RV myocardial fibrosis in female compared with male PAB rats 8 wk after surgery. Myosin heavy chain 7-to-6 ratio switch and calcineurin signaling were less pronounced in female PAB rats compared with males. In this juvenile rat model of RV pressure load, female rats appeared to be less prone to clinical heart failure compared with males. This was driven by increased RV contractility before puberty, and better preservation of diastolic function with less RV myocardial fibrosis after puberty. These findings show that RV adaptation to increased loading differs between sexes already before the introduction of pubertal hormones.NEW & NOTEWORTHY In this study, we describe sex differences in our unique weanling rat model of increased RV pressure load by pulmonary artery banding. We are the first to assess temporal sex-related differences in RV adaptation during pubertal development. Female rats show superior RV function and less diastolic dysfunction and fibrosis compared with male rats. These differences are already present before puberty, indicating that the differences in RV adaptation are not only determined by sex hormones.

Clinical Significance of Early Pulmonary Hypertension in Preterm Infants.

OBJECTIVE: To characterize different phenotypes of early pulmonary hypertension (PH) in preterm infants and their respective associations with bronchopulmonary dysplasia (BPD) and survival. STUDY DESIGN: A prospective cohort study in a tertiary university medical center from June 2016 until March 2019. Infants with a gestational age <30 weeks and/or a birth weight <1000 g were included. Echocardiographic assessment for PH was performed at 3-10 days after birth. Subsequent development of BPD at 36 weeks postmenstrual age and mortality were assessed. RESULTS: Early PH was identified in 55% of 104 included infants, including 21% with persistent PH of the newborn (PPHN), 61% with flow-associated PH, and 18% PH without shunt. Only PPHN was associated with placental fetal vascular malperfusion, lower gestational age, and low Apgar score. Both PPHN and flow PH were associated with the development of BPD. Early PH was associated with poorer survival, driven by PPHN. CONCLUSIONS: Early PH is highly prevalent (55%) in preterm infants and associated with the development of BPD, independent of the phenotype of PH. Infants with PPHN had the poorest survival. Early PH presents in various phenotypes characterized by differences in etiology, pathophysiology, and associated long-term sequelae.

Neonatal pulmonary hypertension after severe early-onset fetal growth restriction: post hoc reflections on the Dutch STRIDER study.

The aim was to reflect on the unexpected finding of persistent pulmonary hypertension of the neonate (PPHN) and pulmonary hypertension in infants born within the Dutch STRIDER trial, its definition and possible pathophysiological mechanisms. The trial randomly assigned pregnant women with severe early-onset fetal growth restriction to sildenafil 25 mg three times a day versus placebo. Sildenafil use did not reduce perinatal mortality and morbidity, but did result in a higher rate of neonatal pulmonary hypertension (PH). The current paper reflects on the used definition, prevalence, and possible pathophysiology of the data on pulmonary hypertension. Twenty infants were diagnosed with pulmonary hypertension (12% of 163 live born infants). Of these, 16 infants had PPHN shortly after birth, and four had pulmonary hypertension associated with sepsis or bronchopulmonary dysplasia. Four infants with PPHN in the early neonatal period subsequently developed pulmonary hypertension associated with bronchopulmonary dysplasia in later life. Infants with pulmonary hypertension were at lower gestational age at delivery, had a lower birth weight and a higher rate of neonatal co-morbidity. The infants in the sildenafil group showed a significant increase in pulmonary hypertension compared to the placebo group (relative risk 3.67; 95% confidence interval 1.28 to 10.51, P = 0.02). CONCLUSION: Pulmonary hypertension occurred more frequent among infants of mothers allocated to antenatal sildenafil compared with placebo. A possible pathophysiological mechanism could be a "rebound" vasoconstriction after cessation of sildenafil. Additional studies and data are necessary to understand the mechanism of action. WHAT IS KNOWN: • In the Dutch STRIDER trial, persistent pulmonary hypertension in the neonate (PPHN) was more frequent among infants after antenatal sildenafil exposure versus placebo. WHAT IS NEW: • The current analysis focuses on the distinction between PPHN and pulmonary hypertension associated with sepsis or bronchopulmonary dysplasia and on timing of diagnosis and aims to identify the infants at risk for developing pulmonary hypertension. • The diagnosis pulmonary hypertension is complex, especially in infants born after severe early-onset fetal growth restriction. The research field could benefit from an unambiguous consensus definition and standardized screening in infants at risk is proposed.

Upfront triple combination therapy in severe paediatric pulmonary arterial hypertension.

Treatment strategies in paediatric pulmonary arterial hypertension (PAH) have evolved over the last years, but survival is still poor. Recently, in adults with severe PAH, upfront triple combination therapy (uTCT) from diagnosis has been reported to show significant clinical improvement and excellent long-term outcome. This retrospective, observational study aimed to assess the efficacy of uTCT in paediatric PAH.Children diagnosed with PAH between 2010 and 2019 and started with uTCT were included. World Health Organization Functional Class (WHO-FC), haemodynamics, echocardiography, 6-min walking distance and serum level of N-terminal pro-brain-natriuretic-peptide were assessed at baseline, after 3 and 6 months and at last available follow-up. Events were defined as death, lung transplantation or Potts shunt.21 children (median age 4.8 years (2.5-12.8), 57% females) were included. All children except one were in WHO-FC III or IV (28% and 67%, respectively). After 3 months, one child had died and one child had received a Potts shunt. The remaining 19 children showed clinical and echocardiographic improvement, which persisted at 6 months. Children with idiopathic and heritable PAH showed one-, two- and three-year transplant-free survival estimates of 100%, 94% and 87%, albeit 47% of them receiving a Potts shunt during follow-up.Children with severe PAH, but not pulmonary veno-occlusive disease, improved significantly with uTCT and showed beneficial up to 3-year survival rates, albeit 47% of them receiving a Potts shunt during follow-up. The role of a Potts shunt in conjunction to uTCT in paediatric PAH needs to be further established.

Pulmonary hypertension in extremely preterm infants: a call to standardize echocardiographic screening and follow-up policy.

Pulmonary hypertension (PH) is a frequent complication in extremely preterm born infants that seriously affects outcome. We aimed to describe the prevalence of PH in extremely preterm infants and the policy on screening and follow-up in the ten Dutch intensive care units (NICUs). We performed a retrospective cohort study at the University Medical Centre Groningen on infants with gestational age < 30 weeks and/or birthweight < 1000 g, born between 2012 and 2013. Additionally, we carried out a survey among the Dutch NICUs covering questions on the awareness of PH, the perceived prevalence, and policy regarding screening and following PH in extremely preterm infants. Prevalence of early-onset PH in our study was 26% and 5% for late-onset PH. PH was associated with poor survival and early-onset PH was associated with subsequent development of bronchopulmonary dysplasia (BPD). All the NICUs completed the questionnaire and we found that no standardized policy existed regarding screening and following PH in extremely preterm infants.Conclusion: Despite the frequent occurrence of PH and its clinically important consequences, (inter-)national standardized guidelines regarding screening and following of PH in extremely preterm infants are lacking. Standardizing screening and follow-up will enable early identification of infants with late-onset PH and allow for earlier treatment. Additionally, greater clarity is required regarding the prevalence of early PH as are new preventive treatment strategies to combat BPD. What is known? • Pulmonary hypertension (PH) substantially impairs the survival of extremely preterm infants. • PH is associated with bronchopulmonary dysplasia (BPD): Early-onset PH predicts the development of BPD. Late-onset PH is prevalent in infants with severe BPD. What is new? • Pulmonary hypertension (PH) is prevalent in preterm infants. Its consequences for morbidity and mortality justify a standardized policy aimed at early detection to improve prevention and treatment. • No structured policy exists in the Netherlands regarding screening/follow-up for PH in extremely preterm infants.

Evaluation of Macitentan in Patients With Eisenmenger Syndrome.

BACKGROUND: Eisenmenger syndrome describes congenital heart disease-associated severe pulmonary hypertension accompanied by right-to-left shunting. The multicenter, double-blind, randomized, placebo-controlled, 16-week, phase III MAESTRO study (Macitentan in Eisenmenger Syndrome to Restore Exercise Capacity) evaluated the efficacy and safety of the endothelin receptor antagonist macitentan in patients with Eisenmenger syndrome. METHODS: Patients with Eisenmenger syndrome aged ≥12 years and in World Health Organization functional class II-III were randomized 1:1 to placebo or macitentan 10 mg once daily for 16 weeks. Patients with complex cardiac defects, Down syndrome and background PAH therapy were eligible. The primary end point was change from baseline to week 16 in 6-minute walk distance. Secondary end points included change from baseline to week 16 in World Health Organization functional class. Exploratory end points included NT-proBNP (N-terminal pro-B-type natriuretic peptide) at end of treatment expressed as a percentage of baseline. In a hemodynamic substudy, exploratory end points included pulmonary vascular resistance index (PVRi) at week 16 as a percentage of baseline. RESULTS: Two hundred twenty six patients (macitentan n=114; placebo n=112) were randomized. At baseline, 60% of patients were in World Health Organization functional class II and 27% were receiving phosphodiesterase type-5 inhibitors. At week 16, the mean change from baseline in 6-minute walk distance was 18.3 m and 19.7 m in the macitentan and placebo groups (least-squares mean difference, -4.7 m; 95% confidence limit (CL), -22.8, 13.5; P=0.612). World Health Organization functional class improved from baseline to week 16 in 8.8% and 14.3% of patients in the macitentan and placebo groups (odds ratio, 0.53; 95% CL, 0.23, 1.24). NT-proBNP levels decreased with macitentan versus placebo (ratio of geometric means, 0.80; 95% CL, 0.68, 0.94). In the hemodynamic substudy (n=39 patients), macitentan decreased PVRi compared with placebo (ratio of geometric means, 0.87; 95% CL, 0.73, 1.03). The most common adverse events with macitentan versus placebo were headache (11.4 versus 4.5%) and upper respiratory tract infection (9.6 versus 6.3%); a hemoglobin decrease from baseline of ≥2 g/dL occurred in 36.0% versus 8.9% of patients. Five patients (3 macitentan; 2 placebo) prematurely discontinued treatment and 1 patient died (macitentan group). CONCLUSIONS: Macitentan did not show superiority over placebo on the primary end point of change from baseline to week 16 in exercise capacity in patients with Eisenmenger syndrome. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT01743001.

Early angiogenic proteins associated with high risk for bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.

Early pulmonary vascular disease in preterm infants is associated with the subsequent development of bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH); however, mechanisms that contribute to or identify infants with increased susceptibility for BPD and/or PH are incompletely understood. Therefore, we tested if changes in circulating angiogenic peptides during the first week of life are associated with the later development of BPD and/or PH. We further sought to determine alternate peptides and related signaling pathways with the risk for BPD or PH. We prospectively enrolled infants with gestational age <34 wk and collected blood samples during their first week of life. BPD and PH were assessed at 36 wk postmenstrual age. Samples were assayed for each of the 1,121 peptides included in the SOMAscan scan technology, with subsequent pathway analysis. Of 102 infants in the study, 82 had BPD, and 13 had PH. Multiple angiogenic proteins (PF-4, VEGF121, ANG-1, bone morphogenetic protein 10 [BMP10], hepatocyte growth factor (HGF), ANG-2) were associated with the subsequent diagnosis of BPD; and FGF-19, PF-4, connective tissue activating peptide (CTAP)-III, and PDGF-AA levels were associated with BPD severity. Early increases in BMP10 was strongly associated with the late risk for BPD and PH. We found that early alterations of circulating angiogenic peptides and others were associated with the subsequent development of BPD. We further identified peptides that were associated with BPD severity and BPD-associated PH, including BMP10. We speculate that proteomic biomarkers during the first week of life may identify infants at risk for BPD and/or PH to enhance care and research.

Serial Measurements of N-Terminal Pro-B-Type Natriuretic Peptide Serum Level for Monitoring Pulmonary Arterial Hypertension in Children.

OBJECTIVE: To assess the association between serially measured N-terminal pro-B-type natriuretic peptide (NT-proBNP) serum levels and disease severity in children with pulmonary arterial hypertension (PAH), and to assess its predictive value for death or (heart-)lung transplantation. STUDY DESIGN: This was a longitudinal cohort study of the Dutch National Network for Pediatric Pulmonary Hypertension conducted between 2003 and 2017. Data on NT-proBNP and disease severity markers (World Health Organization Functional Class [WHO-FC], 6-minute walking distance [6MWD], and tricuspid annular plane systolic excursion [TAPSE]) were collected every 3 to 6 months from 82 children with PAH. The outcome measure was death or (heart-)lung transplantation. Also, NT-proBNP levels over time were compared between survivors and nonsurvivors. RESULTS: The median patient age was 8.8 years (IQR, 4.6-13.5 years), and 61% were female. The median duration of follow-up was 4.8 years (IQR, 1.9-10.0 years). At all times during the course of disease, higher NT-proBNP levels were associated with higher WHO-FC (ß = 0.526; 95% CI, 0.451-0.600), lower 6MWD z-score (ß = -0.587; 95% CI, -0.828 to -0.346), lower TAPSE z-score (ß = -0.783; 95% CI, -1.016 to -0.549), and elevated risk of death or (heart-)lung transplantation (hazard ratio 16.61; 95% CI, 7.81-35.33). Compared with survivors, nonsurvivors had NT-proBNP levels that were higher at first measurement and increased exponentially over time (P = .005). Changes in NT-proBNP serum level over time were predictive of outcome. CONCLUSIONS: Throughout the disease course of pediatric PAH, serial measurements of NT-proBNP are associated with disease severity and transplant-free survival. Monitoring NT-proBNP levels over time provides important prognostic information that can support clinical decision making in combination with other established prognostic markers.

The Genetic Epidemiology of Pediatric Pulmonary Arterial Hypertension.

OBJECTIVE: To describe the prevalence of pulmonary arterial hypertension (PAH)-associated gene mutations, and other genetic characteristics in a national cohort of children with PAH from the Dutch National registry and to explore genotype-phenotype associations and outcomes. STUDY DESIGN: Children (n = 70) diagnosed with idiopathic PAH, heritable PAH, PAH associated with congenital heart disease with coincidental shunt (PAH-congenital heart disease group 3), PAH after closure of a cardiac shunt (PAH-congenital heart disease group 4), or PAH associated with other noncardiac conditions were enrolled. Targeted next-generation sequencing was performed on PAH-associated genes (BMPR2, ACVRL1, EIF2AK4, CAV1, ENG, KCNK3, SMAD9, and TBX4). Also, children were tested for specific genetic disorders in case of clinical suspicion. Additionally, children were tested for copy number variations. RESULTS: Nineteen children (27%) had a PAH-associated gene mutation/variant: BMPR2 n = 7, TBX4 n = 8, ACVRL1 n = 1, KCNK3 n = 1, and EIF2AK4 n = 2. Twelve children (17%) had a genetic disorder with an established association with PAH (including trisomy 21 and cobalamin C deficiency). In another 16 children (23%), genetic disorders without an established association with PAH were identified (including Noonan syndrome, Beals syndrome, and various copy number variations). Survival rates differed between groups and was most favorable in TBX4 variant carriers. CONCLUSIONS: Children with PAH show a high prevalence of genetic disorders, not restricted to established PAH-associated genes. Genetic architecture could play a role in risk-stratified care management in pediatric PAH.

TBX4 variants and pulmonary diseases: getting out of the 'Box'.

PURPOSE OF REVIEW: In 2013, the association between T-Box factor 4 (TBX4) variants and pulmonary arterial hypertension (PAH) has first been described. Now - in 2020 - growing evidence is emerging indicating that TBX4 variants associate with a wide spectrum of lung disorders. RECENT FINDINGS: TBX4 variants are enriched in both children and adults with PAH. The clinical phenotype associated with a TBX4 variant seems to be milder than that in other PAH-associated gene mutations. Further, TBX4 variants have increasingly been associated with a variety of clinical and histopathological phenotypes, including lethal developmental parenchymal lung diseases such as not only acinar dysplasia in neonates, but also less outspoken parenchymal lung diseases in children and adults. SUMMARY: The clinical phenotype of a TBX4 variant has recently been recognised to expand from bone disorders to different types of lung diseases. Recent data suggest that variants of TBX4, a transcription factor known to be an important regulator in embryonic development, are not rare in both children and adults with PAH and/or developmental parenchymal lung diseases.

Multicenter Preclinical Validation of BET Inhibition for the Treatment of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is a degenerative arteriopathy that leads to right ventricular (RV) failure. BRD4 (bromodomain-containing protein 4), a member of the BET (bromodomain and extra-terminal motif) family, has been identified as a critical epigenetic driver for cardiovascular diseases.Objectives: To explore the therapeutic potential in PAH of RVX208, a clinically available BET inhibitor.Methods: Microvascular endothelial cells, smooth muscle cells isolated from distal pulmonary arteries of patients with PAH, rats with Sugen5416 + hypoxia- or monocrotaline + shunt-induced PAH, and rats with RV pressure overload induced by pulmonary artery banding were treated with RVX208 in three independent laboratories.Measurements and Main Results: BRD4 is upregulated in the remodeled pulmonary vasculature of patients with PAH, where it regulates FoxM1 and PLK1, proteins implicated in the DNA damage response. RVX208 normalized the hyperproliferative, apoptosis-resistant, and inflammatory phenotype of microvascular endothelial cells and smooth muscle cells isolated from patients with PAH. Oral treatment with RVX208 reversed vascular remodeling and improved pulmonary hemodynamics in two independent trials in Sugen5416 + hypoxia-PAH and in monocrotaline + shunt-PAH. RVX208 could be combined safely with contemporary PAH standard of care. RVX208 treatment also supported the pressure-loaded RV in pulmonary artery banding rats.Conclusions: RVX208, a clinically available BET inhibitor, modulates proproliferative, prosurvival, and proinflammatory pathways, potentially through interactions with FoxM1 and PLK1. This reversed the PAH phenotype in isolated PAH microvascular endothelial cells and smooth muscle cells in vitro, and in diverse PAH rat models. RVX208 also supported the pressure-loaded RV in vivo. Together, these data support the establishment of a clinical trial with RVX208 in patients with PAH.

Hypoxic/ischemic hits predispose to necrotizing enterocolitis in (near) term infants with congenital heart disease: a case control study.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease that is relatively frequently diagnosed in term infants with congenital heart disease (CHD), compared with term infants without CHD, in whom NEC is rare. The exact pathogenesis of NEC in term infants with CHD is unknown, but it is hypothesized that ischemia of the intestines plays a pivotal role. We aimed to explore whether (near) term CHD infants, who develop NEC, exhibit more clinical signs of hypoxia/ischemia and low body perfusion directly after birth and during the first 48 hours after admission to the neonatal intensive care unit, when compared with (near) term CHD infants who did not develop NEC. METHODS: 956 infants with CHD born after ≥ 35 weeks of gestational age were retrospectively reviewed for this case-control study between January 1999 and February 2020. We included infants with radiographically confirmed pneumatosis intestinalis and controls matched by type of CHD. Seven infants were diagnosed with transposition of the great arteries, six with left and four with right ventricular outflow tract obstruction. Several parameters suggestive of (relative) hypoxia/ischemia were used for analyses. RESULTS: We included sixteen CHD infants with NEC and selected sixteen controls. There were no significant demographic differences between both groups. Apgar score at one and five minutes (median [IQR]) were lower in infants who developed NEC compared with control infants (8 [7-8]) vs. (9 [8-9], P = .011) and (8 [8-9]) vs. (9 [9-10], P = .009). A higher proportion of infants with NEC required respiratory support in the delivery room (11(69) vs. 2(13), P = .001). The (median [IQR]) diastolic blood pressure on the second day after admission (39 mmHg [34-42], vs. 43 mmHg [37-51], P = .112) and lowest (median [IQR]) pH in the 48 hours after admission (7.24 [7.17-7.35] vs. 7.38 ([7.27-7.43], P = .157) were not significantly lower in NEC infants but both demonstrated a similar direction towards (relative) hypoxia/ischemia in NEC infants. CONCLUSIONS: Our clinical results support a hypoxic/ischemic pathophysiology of NEC in (near) term CHD infants, with lower Apgar scores, more respiratory support in the delivery room and a tendency towards a lower diastolic blood pressure and pH in CHD infants who develop NEC.

N-terminal pro-brain natriuretic peptide serum levels reflect attrition of the Fontan circulation.

OBJECTIVE: N-terminal pro-brain natriuretic peptide has an established role in the diagnosis and prognosis of heart failure. In Fontan patients, this peptide is often increased, but its diagnostic value in this particular non-physiologic, univentricular circulation is unclear. We investigated whether N-terminal pro-brain natriuretic peptide represents ventricular function or other key variables in Fontan patients. METHODS AND RESULTS: Ninety-five consecutive Fontan patients ≥10 years old who attended the outpatient clinic of the Center for Congenital Heart Diseases in 2012-2013 were included. Time since Fontan completion was 16 ± 9 years. Median N-terminal pro-brain natriuretic peptide was 114 (61-264) ng/l and was higher than gender-and age-dependent normal values in 54% of the patients. Peptide Z-scores were higher in patients in NYHA class III/IV compared to those in class I/II, but did not correlate with ventricular function assessed by MRI and echocardiography, nor with peak exercise capacity. Instead, peptide Z-scores significantly correlated with follow-up duration after Fontan completion (p < 0.001), right ventricular morphology (p = 0.004), indexed ventricular mass (p = 0.001), and inferior caval vein diameter (p < 0.001) (adjusted R2 = 0.615). CONCLUSIONS: N-terminal pro-brain natriuretic peptide levels in Fontan patients correlate with functional class, but do not necessarily indicate ventricular dysfunction. Increased peptide levels were associated with a longer existence of the Fontan circulation, morphologic ventricular characteristics, and signs of increased systemic venous congestion. Since the latter are known to be key determinants of the performance of the Fontan circulation, these findings suggest increase in N-terminal pro-brain natriuretic peptide levels to indicate attrition of the Fontan circulation, independent of ventricular function.

Converging Paths of Pulmonary Arterial Hypertension and Cellular Senescence.

Cellular senescence is recognized as a crucial contributor to the pathobiology of various degenerative and cardiovascular diseases, such as idiopathic pulmonary fibrosis and atherosclerosis. We describe the potential link between cellular senescence and the degenerative character of neointimal pulmonary vascular disease in pulmonary arterial hypertension (PAH). Senescence markers have been described in remodeled pulmonary arteries, and PAH and senescence share common triggers and pathogenic pathways, such as transforming growth factor-ß/bone morphogenetic protein and TNF-α. In addition, interventions that target a senescence phenotype also target pulmonary vascular remodeling in vivo. These data provide a basis for further exploration of the role of senescence in the pathobiology of PAH and for preclinical trials with a senolytic class of drugs.

A novel method optimizing the normalization of cardiac parameters in small animal models: the importance of dimensional indexing.

For indexing cardiac measures in small animal models, tibia length (TL) is a recommended surrogate for body weight (BW) that aims to avoid biases because of disease-induced BW changes. However, we question if indexing by TL is mathematically correct. This study aimed to investigate the relation between TL and BW, heart weight, ventricular weights, and left ventricular diameter to optimize the current common practice of indexing cardiac parameters in small animal models. In 29 healthy Wistar rats (age 5-34 wk) and 116 healthy Black 6 mice (age 3-17 wk), BW appeared to scale nonlinearly to TL1 but linearly to TL3. Formulas for indexing cardiac weights were derived. To illustrate the effects of indexing, cardiac weights between the 50% with highest BW and the 50% with lowest BW were compared. The nonindexed cardiac weights differed significantly between groups, as could be expected (P < 0.001). However, after indexing by TL1, indexed cardiac weights remained significantly different between groups (P < 0.001). With the derived formulas for indexing, indexed cardiac weights were similar between groups. In healthy rats and mice, BW and heart weights scale linearly to TL3. This indicates that not TL1 but TL3 is the optimal surrogate for BW. New formulas for indexing heart weight and isolated ventricular weights are provided, and we propose a concept in which cardiac parameters should not all be indexed to the same measure but one-dimensional measures to BW1/3 or TL1, two-dimensional measures to BW2/3 or TL2, and three-dimensional measures to BW or TL3. NEW & NOTEWORTHY In healthy rats and mice, body weight (BW) scales linearly to tibia length (TL) to the power of three (TL3). This indicates that for indexing cardiac parameters, not TL1 but TL3 is the optimal surrogate for BW. New formulas for indexing heart weight and isolated ventricular weights are provided, and we propose a concept of dimensionally consistent indexing. This concept is proposed to be widely applied in small animal experiments.