Right heart failure (RHF) is a significant cause of morbidity and mortality from pulmonary hypertension (PH). To better understand the pathophysiology and implications of exercise-induced stress, we implemented an exercise regimen to further characterize this disease using our previously developed ovine model of chronic pulmonary hypertension-right ventricular failure (PH-RVF). To this end, eight Dorset cross sheep underwent the PH-RVF model via ligation of the left pulmonary artery (LPA) and progressive occlusion of the main pulmonary artery (MPA) with an inflatable cuff. The cuff and right ventricle (RV) pressure lines were subcutaneously tunneled to a port for access. Over eight weeks, each sheep underwent a weekly exercise regimen and cuff check (CC). The exercise regimen consisted of working speeds and recovery speeds for 10 min each. During exercise, we accessed both ports to transduce cuff pressure, hemodynamics, and take RV blood gases. At exercise conclusion, the PA cuff was inflated by 150-200 mmHg to increase RV afterload. A CC and RV blood gas were performed a few days after inflation to confirm cuff pressure and ensure compensation. Over the course of eight weeks, the SvO2 remained relatively stable at maximum intensity at week 1 vs week 8: 62.7 ± 4.5% vs 69.8 ± 4.0% despite the RVSP increasing from week 1 to week 8: 78 ± 8 mmHg vs 96 ± 8 mmHg. Additionally, the distance traveled increased from week 1 to week 8: 1390 ± 297 m vs 1834 ± 189 m, despite the PA cuff pressure at week 8 being increased to 777 ± 98 mmHg. This suggests that despite increased RV afterload, exercise may aid in an adaptive response and compensation to exercise in the setting of PH-RVF. This exercise regimen provides novel information about the effects of exercise in PH-RVF and enables complex studies of exercise physiology in a large animal model of PH-RVF.

Pulmonary arterial hypertension (PAH) is a complex progressive disease associated with high morbidity and mortality. Circulating serum biomarkers have the potential to optimize diagnosis and prognosis in PAH. The cellular communication network (CCN) protein family is a group of similarly structured matricellular proteins with many roles ranging from fibrosis to malignancy. Individual CCN proteins have been associated with PAH in previous studies, but no study has evaluated multiple CCN proteins as potentially relevant biomarkers in PAH. This study sought to establish associations using the circulating concentrations of measurable CCN proteins and PAH diagnosis, severity, outcomes, and other biomarkers. Serum levels of CCN1, CCN2, CCN3, and CCN6 were measured utilizing 225 patients from the PAH Biobank (PAHBiobank) with available hemodynamic data and 40 control samples. Serum levels of CCN1, 2, 3, and 6 proteins were significantly increased in PAH compared to controls. CCN1, CCN2, and CCN3 were associated with a lower 6-min walk distance. CCN2 and CCN3 were also associated with worse New York Heart Association Functional Class. Higher CCN2 and CCN3 levels correlated with higher levels of Endostatin and NT-proBNP. CCN6 was not significantly associated with any hemodynamic or clinical variables in the PAH cohort. Our results suggest that multiple CCN proteins are increased in PAH and that CCN2 and CCN3 have the most potential as novel biomarkers in PAH.

Pulmonary hypertension (BPD-PH) associated with bronchopulmonary dysplasia (BPD) in preterm infants associates with high morbidity and mortality within the first two years of life. In a previous unbiased study, we identified a panel miRNAs in tracheal aspirates (TA) that were differentially expressed in extremely low gestational age newborns (ELGANs) with BPD-PH compared to those with BPD but no PH. To explore the predictive potential of these miRNAs, we studied TA exosomes from 7 days old ELGANs and analysed a curated panel of 16 miRNAs through logistic regression and calculated the predictive AUROC to diagnose BPD-PH at 36 weeks PMA. AUROC of TA miRNAs was 0.76 with sensitivity and specificity of 53% and 93%, respectively. Adding sex and gestational age to the variables improved the AUROC to 0.78 with sensitivity and specificity of 61 and 87% respectively. Due to challenges of obtaining TA in non-invasively ventilated infants, we collected saliva samples from ELGANs at 7 days of age and compared the log expression of these 16 miRNAs in both biofluids and found significant correlation in their expression (pearson r=0.92, p<0.001). We calculated the predictive AUROC of the same miRNAs to diagnose BPD-PH at 36 weeks PMA. AUROC of these miRNAs in saliva was = 0.85 with sensitivity and specificity of 82% and 72%, respectively; addition of biological sex and gestational age improved AUROC to 0.86 with sensitivity and specificity of 79% and 76% respectively. Leave-one-sample-out sensitivity analysis demonstrated stable training performance with reduced performance in testing samples, supporting the need for validation in larger independent cohorts. In conclusion, early salivary miRNAs have great potential for risk stratification of ELGANs to develop BPD-PH, while also providing the opportunity to identify target molecules and mechanisms that modulate molecular function.

Introduction: Mutations in the ACVRL1 gene are associated with hereditary hemorrhagic telangiectasia (HHT). Patients with HHT due to ACVRL1 mutations who develop pulmonary arterial hypertension (PAH) have worse outcomes than those who do not, but the etiology of this variable penetrance is not well understood. Elucidating the modifiers of PAH penetrance in HHT patients could improve early detection and treatment in at-risk patients and improve morbidity and mortality outcomes. Case Report: In 2016, during a workup for murmur, a 4.5-month-old male term infant (9% for weight; 5% for height) had a transthoracic echocardiogram (TTE) notable for mild to moderately depressed right ventricular (RV) systolic function, severely increased RV pressure estimate, and a small atrial septal defect with right to left shunt concerning for pulmonary hypertension (PH). A Chest CT angiogram showed PH features but with dilated and tortuous peripheral arteries. Cardiac catheterization was notable for a mean pulmonary artery pressure (mPAP) of 76 mmHg, mean systemic pressure of 79 mmHg, pulmonary vascular resistance index (PVRi) 28 wU x m2, normal pulmonary venous pressures, and no response to acute vasodilator testing; the distal pulmonary vasculature had an engorged and mildly tortuous appearance but no obvious arteriovenous connections. A diagnosis of precapillary PH, most consistent with PAH, was assigned and the patient was managed using up-front prostacyclin infusion as well as sildenafil and bosentan. Genetic testing detected a heterozygous pathogenic germline mutation in the gene ACVRL1 (also called ALK1): c.1450C>T, p.Arg484Trp, as well as a heterozygous germline variant in the SMAD9 gene (c.1270G>A, p.Ala424Thr), reported in patients with PAH but of uncertain significance at a highly conserved site across species. Around his 4th birthday, he underwent a modified Pott's shunt procedure to reduce RV strain. At age 5.5 years, treprostinil was transitioned to selexipag. Today, he is 8.5 years old, continues on triple PAH-specific therapy, and is active but functional class III with mildly depressed RV function and systemic-suprasystemic RV pressure estimates. Summary: This case highlights an early presentation in infancy of a patient with HHT and PAH associated with an ACVRL1 mutation The concurrent presence of a SMAD9 mutation, at an important DNA locus may indicate a genetic ‘second hit’ that contributes to early PAH emergence in patients with HHT.

Rationale: Transcription factor TBX4 rare variants associate with pulmonary arterial hypertension (PAH), particularly in children, and are the second most common cause of heritable PAH. However, both molecular and physiologic mechanisms by which TBX4 results in PAH are still largely unknown. Methods: Mice carrying two floxed TBX4 alleles had knockout induced at postnatal days 3 and 5 using tamoxifen injections, with hemodynamic phenotyping after week 8. Lung structure was assessed by mean linear intercept, and molecular effects were assessed by whole lung RNA-seq compared to RNA-seq on fibroblasts and pericytes. Results: TBX4 knockout mice developed elevated right ventricular systolic pressures with strong correlation to the degree of knockout, as well as simplified alveoli. RNA-seq suggested this was associated with defects in extracellular matrix, pericyte differentiation and movement, and Wnt signaling. Conclusions: Loss of TBX4 in alveologenesis can reproduce many of the salient physiologic features of human patients, suggesting that this may be the stage of development in which its loss is most important. The mechanism seems to be through alteration in pericyte and fibroblast movement and adhesion, which impacts alveologenesis along with accompanying vessels. This bears hope for patients, because although this implies that the problem is fundamentally developmental, it occurs at such a late stage that intervention is still plausible.

Transcription factor TBX4 rare variants associate with pulmonary arterial hypertension (PAH), particularly in children, and are the second most common cause of heritable PAH. However, TBX4's down-stream targets and the molecular and cellular pathways these targets regulate remain largely unknown in PAH. We combined RNA-seq and ChIP-seq results to identify TBX4 direct targets in lung fibroblasts and pericytes, respectively. There were 555 genes with altered expression with TBX4 knockdown in both fibroblasts and pericytes by RNA-seq, and which also were found to be bound by TBX4 by ChIP-seq. Gene ontology analysis found that these were dominated by genes related to extracellular matrix, actin organization, and migration guidance, although there were also significant groups related to serine/threonine kinase signaling, GTPase mediated signaling, and glycoprotein metabolism. Migration and proliferation studies using TBX4 knockdown fibroblasts confirmed functional effects. These studies provide the first insights into how genes and pathways regulated by TBX4 are impacted and inform future studies about the key biological processes that lead to PAH in patients who carry pathologic TBX4 rare variants.

ABSTRACT Female sex increases risk of Group I pulmonary arterial hypertension by roughly threefold, but the mechanism is unclear. Low expression of Cyp1b1, an enzyme that metabolizes estrogens, is associated with disease penetrance, particularly in women. We previously found that lower Pparγ levels in murine PAH models, which may drive disease, are rescued by estrogen blockade. The goal of the current studies was to examine interaction of estrogen, Cyp1b1, and energy metabolism in cell culture and in knockout mice. We found that both estrogen and siRNA to Cyp1b1 resulted in reduction of Pparγ at a protein, but not transcript level, in addition to regulating Pparγ cofactors. siCyp1b1 reduced both basal and maximal respiration rates in a fatty acid oxidation Seahorse protocol. This Pparγ inhibition could be eliminated by blocking ubiquitination. RNA‐seq suggested that Cyp1b1 may be having important pulmonary hypertension effects both in concert with and independently of its effect on estrogen. Cyp1b1 knockout mice have lower Pparγ levels than WT mice both in normoxia and hypoxia, and develop mild pulmonary hypertension on a high fat diet. RNA‐seq on their lungs reflected similar pathways to those altered in endothelial cells alone – lipid metabolism, cytokines, and vasoreactivity‐associated genes, among others, but added genes associated with circadian rhythm. These data suggest multiple potential points for intervention in estrogen and Cyp1b1 mediated etiology of PAH, in particular Pparγ ubiquitination, but also suggests that both the difference between E2 and 16aOHE and the impact of Cyp1b1 is more complex than simply “degree of estrogenicity”.

Background: Patients with idiopathic pulmonary arterial hypertension (IPAH), a progressive pulmonary vasculopathy, may be clinically similar, yet have different responses to therapy and outcomes. Understanding proteomic profiles that distinguish amongst clinically similar patients could improve disease classification to better target treatment. Research Question: Can proteomic profiling identify molecular sub-phenotypes amongst otherwise clinically similar patients with IPAH? Methods: A cross-sectional, prevalent cohort of patients with IPAH (N=120, 60 survivors/60 non-survivors) was selected from the PAH Biobank to have similar clinical, genetic hemodynamic and risk profiles. Plasma protein levels of 11,000 proteins (SomaScan 11K Platform) were assayed. The top 10% of proteins with highest variance were used for spectral clustering. Survival by cluster was evaluated by Kaplan-Meier analysis and Cox proportional hazards model, adjusted for enrollment age and sex. Clinical differences between clusters were assessed with Chi-squared, Kruskal-Wallis and Fisher’s exact tests. Pathway analysis identified major proteomic pathways differentiating each cluster. Results: Spectral clustering identified 3 clusters, with 36%, 45% and 68% events over 5-years in clusters 1, 2 and 3 respectively (Figure 1, p=0.0025). The adjusted hazard ratio for death was 1.40 and 2.78 in clusters 2, 3 comparing to cluster 1. Subjects in cluster 3 were older than cluster 1 and 2. Medications, six-minute walk test and hemodynamics were similar across clusters and most subjects were intermediate risk (ERS criteria) and low or average risk (REVEAL 2.0)(Table 1).Differentially expressed proteins showed enrichment of Vascular Endothelial Growth Factor (VEGF) signaling (p=9.30E-04) between clusters 1 and 2, the Wnt signaling between clusters 1 and 3 (p=0.0014), and Neurotrophin signaling (p=4.82E-09) and VEGF signaling (p=5.93E-09) between clusters 2 and 3. Conclusions: This study identified differential expression of 3 pathways across clusters of similar IPAH patients with differences in outcomes not predicted by clinical risk assessment. All three of these pathways, Wnt, VEGF and Neurotrophin, have been implicated in pulmonary vascular and right ventricular dysfunction and may identify patients at higher risk or at different stages of the IPAH disease process. Understanding the proteome may identify molecular phenotypes of IPAH associated with outcomes but missed by clinical classification.

As with adult pulmonary hypertension (PH), high morbidity and mortality persist with diverse types of paediatric PH. Despite major advances in pharmacologic therapies based on extensive studies in adult PH, few drugs have been comprehensively studied in neonates, infants, and children, leaving current paediatric PH care largely dependent on small observational studies and extrapolation of evidence from adult clinical trials. Challenges in developing successful clinical trials in children include the need to define distinct disease phenotypes with well-characterised natural history and outcomes, the lack of established age- and disease-specific study endpoints, small and heterogeneous paediatric populations, and the common off-label use of PH-targeted drug therapies without regulatory approval. From a regulatory perspective, sufficient studies of safety, pharmacokinetics, and pharmacodynamics in neonates and young children are often lacking, and the potential role for bridging biomarkers has been underexplored. Additional opportunities include developing innovative trial designs, employing real-world data from existing registries, and fostering collaborations among sponsors, regulatory authorities, physicians, patients, and their families. By reducing reliance on off-label drug use and leveraging paediatric PH registry data, this approach offers a path toward more effective and evidence-based treatment protocols for paediatric patients. This review provides an overview of integrated international perspectives from an interprofessional platform that includes academia, the pharmaceutical industry, and regulatory agencies surrounding the future design of clinical trials for paediatric PH. Ongoing evaluation and adaptation of these strategies will be essential for ensuring that paediatric PH patients receive the highest standard of care.

Cellular communication network 2 (CCN2) is a secreted matricellular protein associated with pulmonary arterial hypertension (PAH) but has not been studied relative to PAH severity, outcomes, or right ventricle (RV) structure and function in a large human cohort and preclinical animal model. This study assessed the associations between CCN2 and PAH severity, survival, hemodynamic measurements, and cardiovascular dysfunction. Serum CCN2 levels were compared in 2548 adults with PAH and 216 controls. CCN2 levels in PAH patients were compared to functional and hemodynamic measurements, and survival outcomes. RV-pulmonary artery coupling and RV morphology were also assessed in a small subset of patients via pressure-volume loops and cardiac magnetic resonance imaging. In a preclinical PAH model, plasma CCN2 levels were compared between ventricles with PAH progression. CCN2 mRNA levels in both ventricles in the preclinical model were measured to compare with morphologic histologic variables. CCN2 serum levels were significantly higher in PAH compared to controls (p < 0.0001). Higher CCN2 levels were associated with reduced RV contractility (p = 0.003). Higher CCN2 levels were associated with worse 6MWD (p = 0.035), and higher risk of mortality or transplant (p = 0.025). In the preclinical model, prepulmonary CCN2 plasma levels increased with the progression of disease. CCN2 mRNA levels in the RV were associated with decreased RV capillary density (p = 0.015) and increased RV fibrosis (p = 0.045). Though more investigation is needed, it appears that CCN2 plays a role in the development of PAH and potentially in RV maladaptation in PAH.

OBJECTIVES:To characterize an observational cohort of ventilator-dependent infants and children with bronchopulmonary dysplasia (BPD) with or without tracheobronchomalacia (TBM) and determine the impact of TBM on the need for ventilator support, liberation from the ventilator and tracheostomy decannulation. METHODS:Demographics and clinical outcomes were obtained by retrospective review from 12 centers participating in the outpatient BPD Collaborative registry. The cohort consisted of infants born between 2016 and 2021 who were dependent on invasive mechanical ventilation at home. The respiratory outcomes of those infants with TBM were compared to those who did not have TBM. RESULTS:There were 154 subjects included and about half (48.7%) had documented TBM. Both the TBM and non-TBM groups had similar demographic characteristics and respiratory outcomes. However, the non-TBM were found to have lower mean birth weight (673 vs. 832 grams; p = 0.006), higher likelihood of having Nissen fundoplication (34.2% vs. 12.2%; p = 0.006) and higher use of diuretics (59.2% vs. 37.3%; p = 0.007). Both groups were similar in terms of ventilator requirements, timing of liberation from the ventilator, and rate of decannulation. CONCLUSIONS:The presence of TBM in ventilator-dependent infants with BPD did not affect ventilator support needs, liberation from the ventilator and the rate of tracheostomy decannulation. We speculate that the relative contributions of the other components of BPD disease may play critical roles in determining the need for tracheostomy and their ultimate respiratory outcomes. A prospective multicenter study to assess the impact of TBM in severe BPD is urgently needed.

The BMPR2 gene encodes the BMPR-II (bone morphogenetic protein receptor type-II) and is a known regulator of endothelial proliferation, apoptosis, and translational stress responses. While these effects are generally attributed to the actions of BMPR-II protein, we used circular RNA profiling to identify circ3218 and circ5078 as new BMPR2-derived functional RNAs. Circular RNAs were profiled by ultradeep RNA sequencing of human pulmonary artery endothelial cells. Novel BMPR2-derived circular RNAs were assessed for their effects on endothelial proliferation, apoptosis, and translational stress responses in human pulmonary artery endothelial cells and endothelial cells from patients with pulmonary arterial hypertension with heterozygous loss-of-function BMPR2 mutations. circ5078 to linear BMPR2 mRNA ratios were quantified in cultured lymphocytes from patients with pulmonary arterial hypertension with BMPR2 mutations versus unaffected mutation carriers. Depletion of circ3218 enhanced human pulmonary artery endothelial cell apoptosis, whereas circ5078 silencing increased proliferation. Enhanced proliferation with circ5078 depletion was eliminated by cosilencing of circ5078 with either linear BMPR2 mRNA or the stress granule protein, Caprin-1, indicating a potential interdependence of BMPR2 transcripts in the regulation of endothelial function. Patients with pulmonary arterial hypertension with BMPR2 mutations exhibited increased circ5078 to linear BMPR2 mRNA ratios, alongside impaired stress responses in patient endothelial cells that were deficient in linear BMPR2 transcripts but not circ5078. circ5078 depletion enhanced stress responses in human pulmonary artery endothelial cells and rescued stress granule formation in patient endothelial cells, independent of BMPR-II protein levels. Assessment of translational regulation by polysome profiling did not identify any impact of linear or circular BMPR2 transcript loss on global protein synthesis or stress-induced eIF2α (eukaryotic initiation factor 2α) phosphorylation but did identify the enhanced translational efficiency of select nuclear-encoded mitochondrial ribosome proteins with circ5078 depletion, offering a link between mitochondrial function and the circ5078-deficient endothelial phenotype. The identification of circ3218 and circ5078 as novel BMPR2-derived gene products reveals interdependent roles for coding and noncoding BMPR2 transcripts as regulators of endothelial function.

BACKGROUND:A history of atopy is associated with respiratory morbidities in term-born children; however, little is known about how allergies/atopy affect respiratory outcomes in children with bronchopulmonary dysplasia (BPD). This study aims to describe the prevalence of reported allergies/atopy in young children with BPD and assess whether allergies/atopy are associated with outpatient outcomes. METHODS:A retrospective longitudinal cohort study of children between 0 and 36 months of age followed at outpatient BPD clinics was performed using data from questionnaires administered during routine clinical encounters. The presence of allergy/atopy was defined by caregiver questionnaires. Generalized estimating equations were used to adjust associations between allergy/atopy and respiratory outcomes. RESULTS:Rates of reported allergy/atopy in a cohort of infants and children with BPD (21.6%) were similar to previously published rates in healthy children. Children with atopy/allergy were more likely to be born at earlier gestational ages, have pulmonary hypertension, and be non-white and non-Hispanic compared to their non-atopic peers and to experience trouble breathing, nighttime symptoms, activity limitations, and rescue medication use during the first three years of life. CONCLUSIONS:In children with BPD, allergy/atopy was more common among those born at earlier gestational ages and was significantly associated with increased respiratory symptoms during the first 3 years of life. Further studies are needed to assess whether the association between allergy/atopy and increased respiratory morbidity persists throughout childhood and affects later lung function and whether potential interventions, including inhaled steroids, may modify this risk. IMPACT:There is limited data on the prevalence of atopy/allergy in children with bronchopulmonary dysplasia (BPD) and the association between allergy/atopy and respiratory outcomes in this population. Our findings demonstrate that in children with BPD, allergy/atopy was more common among those born at earlier gestational ages and was associated with increased respiratory symptoms and rescue medication use during the first 3 years of life. Further studies are needed to determine whether this association persists throughout childhood and affects later lung function and whether potential interventions may modify this risk.

Single-center pilot study suggests a threefold increase in self-reported history of adverse pregnancy outcomes (APO) among women with subsequent diagnosis of pulmonary arterial hypertension (PAH). Females with PAH and a history of APO were younger and had higher mean pulmonary artery pressures at diagnosis compared to those without a history of APO.

Background: Pulmonary arterial hypertension (PAH) is a devastating disease that affects women more often than men; recent U.S. cohorts demonstrate a female:male ratio of 3 to 4:1. Paradoxically, males have worse survival. The differential effects of sex hormones, particularly estrogen, on the pulmonary vasculature and right ventricle likely account for some of these differences. The role of female-specific risk factors, such as reproductive exposures, are poorly understood in the pathophysiology of PAH. Research Question: To investigate the role of reproductive factors and exogenous estrogen exposures in PAH onset and severity in women enrolled in the United States Pulmonary Hypertension Scientific Registry (USPHSR). Study Design and Methods: Using questionnaires from 390 women with PAH, enrolled in both the PAH Biobank and USPHSR, we conducted linear regression analyses to assess the association between patient reported reproductive variables and PAH disease severity variables, as well as REVEAL Lite 2.0 scores. We adjusted for potential confounders including age, race, BMI, and PAH sub-group (idiopathic, heritable, associated). Results: Younger menopause age (< 40 years) associates with a lower cardiac index (CI) at diagnosis, even when controlling for use of hormone replacement therapy (HRT). There was a trend toward lower CI in women with menopause age of 41-50 years. Women who had ever used HRT were diagnosed with PAH an average of 13.4 years later and 'ever use' of HRT associates with higher pulmonary vascular resistance and lower CI at diagnosis. Interpretation: Premature menopause (age < 40 years) and ever use of HRT associate with worse hemodynamics, including lower CI, at diagnosis in women with PAH. Further investigations into reproductive history and estrogen exposures may offer an opportunity for more comprehensive risk factor screening and modification by physicians treating patients with PAH. ### Competing Interest Statement R. D. Stapleton has NIH grants; royalties from Elsevier; DSMB honoraria and travel. D. B. Badesch has contracts with Acceleron/Merck, United Therapeutics, Liquidia, AI Therapeutics, Altavant, and Pfizer. H. W. Farber is a non-commercial speaker for Bayer; participates on DSMB or advisory board for Acceleron, Actelion, Aerovate, Aerami, United Therapeutics, Altavant, and Keros. A. E. Frost is on study monitoring committees for Actelion, Gossamer Bio, and United Therapeutics. W. C. Nichols has NIH grants. C. G. Elliott participates on DSMB for Gossamer Bio, Insmed, and Respira. E. D. Austin has NIH grants; participates on advisory board for Merck and adjudication committee for ACI, Inc. The remaining authors have no potential conflicts of interest to disclose. ### Funding Statement The PAH Biobank study was funded from HL105333 (W.C.N.) and is presently supported by HL160941 (W.C.N.). Financial support for statistical analysis was provided by the University of Vermont Larner College of Medicine, Department of Medicine Pilot Grant Award (IRB 00002284). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Institutional Review Board of the University of Vermont waived ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

The six-minute walk test (6MWT) is a common method to assess submaximal exercise capacity in children and adults with pulmonary arterial hypertension (PAH) and other chronic diseases. There is no guideline specifically for 6MWT in children. In this observational pilot study, we evaluated the impact of procedural variations on the outcome of the 6MWT in the real-world clinical setting at pediatric PAH programs. We collected 6MWT data from 33 children with PAH participating in a multicenter, prospective, non-interventional study. Data range/quantiles and standard deviation (SD) were used to describe distribution of the six-minute walk distance (6MWD) and data variability. Levene’s test was used to test for heterogeneity of variance with the two sites of similar altitude and their age/height/weight-matched Panama Function Class II participants. We analyzed all 33 eligible participants and their qualified first walks at five centers (A-E) with 6MWD ranges of 420–570, 357–683, 418–481, 400–700, 377–549 m, respectively. Site D performed the 6MWT in a busy hallway and allowed parental/caregiver’s cheering, while Site E performed the 6MWT in a secluded area with no parental/caregiver involvement. Mean 6MWD and SD for Sites D and E were 547 (125) and 432 (67.5) meters, respectively (p = 0.03). In conclusion, procedural variations seem to associate with 6MWD data variability. Although interpretation of our results is limited by the small sample size, our findings suggest that standardizing pediatric 6MWT procedures are needed. Graphical Abstract