BACKGROUND:Treprostinil for the treatment of bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH) has previously been described in small cohort studies, often used later in the course after failure to improve on other therapies. OBJECTIVE/METHODS:We retrospectively describe the clinical course and outcomes of 18 infants (gestational age 26.3 ± 2.6 weeks) from 2012 to 2025 who received parenteral treprostinil to treat BPD-PH, including changes in echocardiographic and cardiac catheterization parameters. RESULTS:All patients had moderate-to-severe BPD and PH, with a mean pulmonary arterial pressure of 45.6 ± 12.7 mmHg at cardiac catheterization prior to treprostinil. Treprostinil was initiated at a median postmenstrual age of 53.5 (IQR: 45.7, 62.6) weeks. Echocardiograms after 3 months of treatment showed improvement of PH severity. At repeat catheterization, mean pulmonary arterial pressure (delta -16.4 ± 12.2, p < 0.01) and indexed pulmonary vascular resistance (delta -4.2 ± 3.3, p < 0.01) significantly improved. Ten of 18 infants (55.6%) survived to discharge; BNP ≥ 35 pg/mL prior to treprostinil initiation demonstrates potential utility for mortality prediction with area under the receiver operator characteristic curve 0.87 ± 0.10 (95% CI: 0.67-1.00). CONCLUSION:Our study shows a potential benefit of treprostinil use in moderate-to-severe BPD-PH; larger studies are needed to validate our findings and guide decision-making around treprostinil initiation and duration.

BACKGROUND:Hypoxic-ischemic encephalopathy (HIE) is the leading cause of neonatal morbidity and mortality globally. An early tool to diagnose and prognosticate disease could aid in timely intervention and reduction in disease burden. We aimed to identify biomarkers for HIE and define whether these biomarkers are associated with neurologic outcome severity in our ovine model. METHODS:Study Cohort 1 (n = 46) included lambs with HIE induced via umbilical cord occlusion (UCO) and healthy controls (n = 19). Cohort 2 (n = 25 UCO lambs) served as a validation cohort for identified biomarkers. Blood samples collected at multiple early time points were analyzed using untargeted liquid chromatography-mass spectrometry. Biomarkers were considered significant at p < 1e-6. Neurological outcome biomarkers were identified using ordinal logistic regression. RESULTS:A total of 145 hypoxia biomarkers were identified, exhibiting a consistent and reproducible temporal pattern across both cohorts. Hypoxanthine level at 20 min of life showed a strong correlation with the severity of neurologic outcomes. CONCLUSIONS:Hypoxanthine emerged as a significant neurologic outcome biomarker in our study. Our findings support the potential utility of metabolomics for early diagnosis and prognostication in HIE. Further clinical validation is warranted to translate these biomarkers into accessible diagnostic and predictive tools, particularly for resource-limited settings. IMPACT:Our study identified plasma hypoxanthine as an early biomarker for hypoxic-ischemic encephalopathy (HIE) in an ovine model and uncovered a novel correlation between hypoxanthine levels and the severity of neurologic impairment in asphyxiated lambs. These findings highlight the potential of metabolomics in the early detection of HIE, paving the way for improved diagnostic and prognostic strategies. Translating these insights into clinical practice could enable the development of predictive tools for outcome assessment and resource allocation, ultimately enhancing early intervention and optimizing care for neonates at risk of HIE.

OBJECTIVES:The primary objective was to elucidate whether placental histopathology representative of intrauterine insults during pulmonary vascular development are associated with a diagnosis of persistent pulmonary hypertension of the newborn (PPHN). The secondary objective was to compare placental histopathologic lesions across PPHN etiologies. STUDY DESIGN:We conducted a case-control study of mother-infant dyads ≥35 weeks gestation who delivered at a tertiary care center between 2020 and 2025. Cases were infants diagnosed with PPHN and treated with inhaled nitric oxide; unaffected controls were infants without congenital anomalies. Placentas underwent blinded histopathologic review using standardized criteria. Multivariate logistic regression modeling was used to control for confounding maternal and infant factors. RESULTS:106 placentas were analyzed (53 PPHN, 53 controls). Placental lesions were significantly more common in PPHN placentas, including fetal vascular malperfusion (30.2% vs 9.4%, p < 0.01), placental inflammation (66.0% vs 37.7%, p < 0.01), chronic presence of meconium (43.4% vs 15.2%, p < 0.01), and chorangiosis (7.6% vs 0%, p = 0.04). In adjusted analyses compared to controls, among 41 placentas with fetal development etiologies (e.g. congenital anomalies) of PPHN, fetal vascular malperfusion, placental inflammation and fetal inflammatory response were more common. Among 12 placentas with typical causes of PPHN (e.g. meconium aspiration syndrome), placental inflammation, maternal and fetal inflammatory responses, and meconium were more common. CONCLUSIONS:PPHN placentas demonstrate lesions of malperfusion, inflammation, and chronic meconium, suggesting a complex interplay between intrauterine hypoxia and inflammation as a potential mechanism for the abnormal pulmonary vascular development and function seen in PPHN.

Lactate clearance is a key marker of oxygen delivery after neonatal cardiac surgery. While higher hematocrit (HCT) is often targeted to optimize oxygen delivery, its relationship with postoperative lactate clearance remains unclear. We hypothesized that supraphysiologic initial HCT may decrease lactate clearance. To assess whether higher initial postoperative HCT is associated with delayed lactate clearance. We performed a retrospective cohort study of 201 neonates requiring CPB. Clinical data were extracted from the EHR using standardized PC4 and STS definitions. The primary predictor was initial HCT analyzed as a continuous variable. Outcomes were lactate at 1, 6, 12, and 24 h and lactate trajectories over 24 h. Multivariable linear regression adjusted for demographics, surgical complexity, and CPB time. Linear mixed-effects models assessed longitudinal lactate trajectories. Initial postoperative HCT (median = 51, IQR = 47–56) was not associated with post-operative lactate after adjustment. However, each 10

INTRODUCTION:Neonatal hypoxic-ischemic (HI) brain injury is a major cause of mortality and long-term neurological disability, yet effective neuroprotective strategies remain limited. Microglia are central mediators of injury and repair, with arginase 1 (ARG1) marking anti-inflammatory, reparative states. However, the functional roles of ARG1+ microglia in tissue remodeling after HI are poorly understood. METHODS:Neonatal mice (P10) underwent HI using the Vannucci procedure. ARG1 activity was inhibited pharmacologically using N-omega-hydroxy-nor-L-arginine (nor-NOHA). ARG1 expression, microglial morphology, efferocytosis, tissue scar, and injury volume were assessed via immunohistochemistry, Western blotting, and arginase activity assays at 1 and 5 days post-injury. RESULTS:ARG1+ microglia rapidly engaged apoptotic neurons, exhibiting phagocytic activity confirmed by CD68 expression. Nor-NOHA treatment reduced ARG1 enzymatic activity, impaired microglial process extension, attenuated efferocytosis, and increased injury volume. ARG1+ microglia persisted in the glial scar and colocalized with collagen I alpha-1 (Col1a1), suggesting a role in extracellular matrix (ECM) deposition. Inhibition of ARG1 decreased Col1a1 expression, highlighting its contribution to tissue remodeling. CONCLUSIONS:ARG1+ microglia are pivotal in neonatal HI, mediating early efferocytosis and later ECM remodeling, thereby limiting injury and shaping scar architecture. Pharmacological blockade of ARG1 exacerbates injury, underscoring its reparative function. These findings establish ARG1 as a critical regulator of microglial-mediated neuroprotection and tissue repair, providing a potential therapeutic target for neonatal HI brain injury.

Background: Hypoxic–ischemic (HI) brain injury triggers a dynamic, multi-phase response involving early microglial efferocytosis followed by extracellular matrix (ECM) deposition and scar formation. Arginase-1 (ARG1), a key enzyme in tissue repair, is implicated in both processes, yet its role in neonatal microglia remains poorly defined. We characterize ARG1-linked pathways in neonatal microglia, identifying distinct efferocytic and fibrotic phases post-HI. Methods: HI was induced in P9 mice using the Vannucci model, and brains were collected at 24 h (D1) and 5 days (D5). Spatially resolved single-cell transcriptomics (seqFISH) was performed using a targeted panel enriched for microglial, ARG1-pathway, efferocytosis, and profibrotic genes. Cell segmentation, clustering, and spatial mapping were conducted using Navigator and Seurat. Differential expression, GSEA, and enrichment analyses were used to identify time- and injury-dependent pathways. Results: Spatial transcriptomics identified 12 transcriptionally distinct cell populations with preserved neuroanatomical organization. HI caused the expansion of microglia and astrocytes and the loss of glutamatergic neurons by D5. Microglia rapidly activated regenerative and profibrotic programs—including TGF-β, PI3K–Akt, cytoskeletal remodeling, and migration—driven by early DEGs such as Cd44, Reln, TGF-β1, and Col1a2. By D5, microglia adopted a collagen-rich fibrotic state with an upregulation of Bgn, Col11a1, Anxa5, and Npy. Conclusion: Neonatal microglia transition from early efferocytic responses to later fibrotic remodeling after HI, driven by the persistent activation of PI3K–Akt, TGF-β, and Wnt/FZD4 pathways. These findings identify microglia as central regulators of neonatal scar formation and highlight therapeutic targets within ARG1-linked signaling.

Pediatric pulmonary vein stenosis (PVS) is associated with high morbidity and mortality. We describe a case series of pediatric patients with advanced multivessel PVS who underwent cardiac catheterization with veno-arterial extracorporeal life support (VA-ECLS) to prevent cardiac arrest around high-risk pulmonary vein angioplasty and/or stent placement. VA-ECLS provided hemodynamic and respiratory stabilization, allowing completion of technically complex interventions in critically-ill pediatric patients who may not have otherwise tolerated catheterization. Using VA-ECLS in this setting requires a thorough assessment of risks versus benefits and specific patient criteria for consideration. This series highlights the potential role of VA-ECLS as an adjunctive strategy during high-risk catheter-based interventions for pediatric PVS to improve quality of life and lengthen lifespan.

Pulmonary arterial hypertension (PAH) is a rare vascular disorder characterized by elevated pressure in pulmonary arteries, eventually leading to right ventricular failure. Approximately 50% of pediatric disease and 20% of adult disease can be linked to a genetic mutation, with nearly 70% of these cases involving mutations in the bone morphogenetic protein receptor type 2 (BMPR2) locus. Investigations using rodent models have made substantial advances in our understanding of BMPR2 signaling; however, limited data exist regarding the onset and course of PAH, and etiologies for phenotypic expression in these patients remain unknown. In this work, we describe the development of an ovine model of heritable PAH. Because homozygous disruption of BMPR2 is embryonic lethal, we developed heterozygous BMPR2-edited (BMPR2+/-) sheep by using a PAM-disrupting synonymous single-stranded oligodeoxyribonucleotide alongside a single guide RNA and Cas9-mediated gene editing strategy. The resulting BMPR2+/- lambs demonstrated cardiac and pulmonary vascular pathology that are consistent with BMPR2 mutation-driven PAH observed in humans. Given the genetic and physiological similarities of BMPR2+/- sheep to humans with heritable PAH, this large animal model will serve as a vital platform for mechanistic molecular studies and will provide a much-needed preclinical model for extensive treatment evaluations.

Pulmonary arterial hypertension (PAH) is characterized by increased lung vascular stiffness and impaired vessel relaxation, primarily due to reduced nitric oxide (NO) production in endothelial cells. Recent studies indicate that chloroquine, an autophagy inhibitor, may help lower pulmonary arterial pressure and enhance lung vascular function. This study investigates the mechanisms underlying the chloroquine-mediated restoration of NO bioavailability in endothelial cells derived from aortopulmonary shunt lambs, a relevant model for congenital heart defect (CHD)-associated PAH. We found that NO production was significantly reduced in shunt pulmonary artery endothelial cells (PAECs), attributable to decreased levels of tetrahydrobiopterin (BH4) and diminished expression of GTP cyclohydrolase 1 (GCH1), despite a slight increase in endothelial nitric oxide synthase (eNOS) levels. Chloroquine robustly restored endothelial NO production, which correlated with increased BH4 levels and restored GCH1 expression. The mechanistically upregulated carboxyl terminus of Hsp70-interacting protein (CHIP) in shunt PAECs is responsible for heightened GCH1 degradation, and chloroquine disrupted the assembly of the GCH1-HSP70-CHIP complex to preserve cellular GCH1. Similarly, another autophagy inhibitor, bafilomycin A1, demonstrated comparable effects. These findings suggest that autophagy inhibition can effectively enhance NO synthesis in endothelial cells experiencing depleted NO bioavailability, presenting a potential therapeutic strategy for managing PAH.

Background: Persistent pulmonary hypertension of the newborn (PPHN) is a cause of neonatal hypoxic respiratory failure due to the failed transition of the pulmonary vasculature after birth. Mechanisms of disease are unknown, but we hypothesize they are directly related to insults in the intrauterine environment. The objective was to describe and compare placentas of PPHN infants to understand significant preceding factors from the maternal fetal environment. Methods: We conducted a case control study of mother infant dyads;35 weeks gestation who delivered at a tertiary care center between 2020 to 2025. Cases were infants diagnosed with PPHN and treated with inhaled nitric oxide; controls were infants without congenital anomalies. Placentas underwent blinded histopathologic review using standardized criteria. Results: 106 placentas were analyzed (53 PPHN, 53 controls). Placental lesions were significantly more common in PPHN, including maternal vascular malperfusion (30.2% vs 9.4%, p<0.01), fetal vascular malperfusion (34.0% vs 17.0%, p=0.05), placental inflammation (66.0% vs 37.7%, p<0.01), meconium (43.4% vs 15.2%, p<0.01), and chorangiosis (7.6% vs 0%, p=0.04). Conclusion: PPHN placentas demonstrate lesions of malperfusion, inflammation, and chronic meconium exposure, suggesting a complex interplay between intrauterine hypoxia and inflammation as a mechanism for the abnormal pulmonary vascular reactivity see in PPHN. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by T32HL160508-01A1 [SMT] ### 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: IRB of University of California, San Francisco gave 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

OBJECTIVETo describe the course and outcomes of children under 18 with left-to-right (LR) shunts and pulmonary arterial hypertension (PAH) undergoing one of two management approaches: PAH treatment prior to LR shunt repair (Treat First) and LR shunt repair first, with or without subsequent PAH treatment (Repair First).METHODSRetrospective single center study, conducted from September 2015 to September 2021, of children LR shunts and PAH (defined as indexed pulmonary vascular resistance (PVRi) ≥ 4WU*m2) but without Eisenmenger physiology. Patient characteristics, longitudinal hemodynamics data, PAH management, LR shunt repair, and outcomes were reviewed.RESULTSOf 768 patients evaluated for LR shunt closure, 51 (6.8%) had LR shunt associated PAH [median age 1.1 (0.37,5) years, median PVRi 6 (5.2,8.7) WU*m2]. Of the "Treat First" group (n=33, 65%), 27 (82%) underwent LR shunt closure and 6 (18%) did not respond to PAH therapy and did not undergo LR shunt closure. In the "Repair First" group (n=18, 35%), 12 (67%) received PAH therapy and 6 (33%) did not. Mortality rates were 6% in "Treat First" and 11% in "Repair First", follow-up of 3.4 and 2.5 years, respectively. After LR shunt closure, there was no significant change in PVRi over a median follow-up of 2 years after surgery (p=0.77).CONCLUSIONSIn children with LR shunts and associated PAH, treatment with PAH-targeted therapy before defect repair does not appear to endanger the subjects and may have some benefit. The response to PAH-targeted therapy before shunt closure persists 2-3 years post-closure, providing valuable insights into the long-term management of these patients.

Background: Pulmonary vascular disease in patients with single ventricular heart disease (SVHD) following the partial cavalpulmonary connection (Glenn) is a significant source of morbidity and shortened lifespan. Pulmonary vascular endothelial cell (EC) dysfunction, an established precursor to pulmonary vascular disease, is well characterized following the Fontan, but data are limited following the Glenn procedure. The role of potential drivers of this pathology, which include hypoxemia, polycythemia, and aberrant pulmonary blood flow patterns, are unclear. Hypothesis: Non-pulsatile blood flow is the primary driver of pulmonary EC dysfunction following the Glenn procedure. To study this hypothesis, we utilized an ovine classic Glenn model which induces passive, non-pulsatile blood flow to the right lung, independent of hypoxemia and polycythemia. Methods: Seven lambs (6-8 weeks old) underwent a classic Glenn procedure in which the right pulmonary artery (PA) and superior vena cava were anastomosed in an end-to-end fashion. 8 weeks after surgery, 7 Glenn and 7 age-matched controls were studied. The response to acetylcholine (Ach, an endothelium-dependent vasodilator) was determined in 5 th generation isolated PAs. Right lung tissues were obtained to measure nitric oxide (NO) metabolites (NOx, chemiluminescence), endothelin-1 (ET-1, ELISA) levels, endothelial nitric oxide synthase (eNOS) protein and Pre-pro ET-1 protein (Western blot). Pulmonary artery endothelial cells (PAECs) were cultured to determine cell proliferation, angiogenesis (Matrigel Assay), and apoptosis (ELISA). Comparisons were made by unpaired t-test and ANOVA. A p<0.05 was considered significant. Results: Baseline demographics were similar between the groups. Mean PA pressure (12.1±2.4vs. 16.7±3.1 mmHg) and left PA flow (0.039±0.01 vs. 0.064±0.01 L/kg/min) were higher in Glenn lambs (p<0.05). Glenn PAs had impaired relaxation to Ach (Fig. 1). Glenn PAECs had higher rates of proliferation (201%) and angiogenesis (164%), and decreased apoptosis (-14%) (p < 0.05). Glenn lung tissue eNOS protein expression was 1.7-fold lower; Prepro-ET-1 protein was 6.6-fold higher (p<0.05). Glenn lung tissue NOx was lower (Fig. 2) and ET-1 was higher than controls (Fig. 3). Conclusions: The initiation of non-pulsatile pulmonary blood flow following the Glenn procedure induces early EC dysfunction. Further investigation of these mechanisms could lead to important therapeutic targets for patients with SVHD.

Excessive mitochondrial fission and a shift to a Warburg phenotype are hallmarks of pulmonary hypertension (PH), although the mechanistic link between these processes remains unclear. We show that in pulmonary arterial endothelial cells (PAEC), Drp1 overexpression induces mitochondrial fission and increases glycolytic ATP production and glycolysis. This is due to mitochondrial reactive oxygen species (mito-ROS)-mediated activation of hypoxia-inducible factor-1α (HIF-1α) signaling, and this is linked to hydrogen peroxide (H2O2)-mediated inhibition of prolyl hydroxylase domain-2 (PHD2) due to its cysteine 326 oxidation and dimerization. Furthermore, these findings are validated in PAEC isolated from a lamb model of PH, which are glycolytic (Shunt PAEC), exhibit increases in both H2O2 and PHD2 dimer levels. The overexpression of catalase reversed the PHD2 dimerization, decreased HIF-1α levels, and attenuated glycolysis in Shunt PAEC. Our data suggest that reducing PHD2 dimerization could be a potential therapeutic target for PH.

In patients with congenital heart disease, the development of pulmonary arterial hypertension (PAH) is based on vascular exposure to abnormal hemodynamic forces. In our work using a large animal model of increased pulmonary blood flow and pressure, we have previously described a pattern of alterations to vascular cell metabolism, mitochondrial function, and mitochondrial redox signaling, paralleling changes in advanced pulmonary vasculopathy states. Based on our findings and emerging literature, we believe that endothelial mitochondria play a central role in integrating and relaying pathologic mechanotransductive signals in abnormal pulmonary hemodynamics. In this manuscript, we demonstrate that exposure of the pulmonary vascular endothelium to aberrant mechanical forces increases production of mitochondrial reactive oxygen species (ROS) and stabilizes the transcription factor Hypoxia Inducible Factor-1α (HIF-1α), and that these changes are associated with impaired endothelial production of Nitric Oxide (NO). We validate that the mitochondrial antioxidant 10-(6′-ubiquinonyl)decyltriphenylphosphonium bromide (MitoQ) can reverse these alterations in vitro, and evaluate the effects of MitoQ treatment in vivo utilizing our large animal shunt model. We find that MitoQ therapy in pulmonary overcirculation decreases the production of mitochondrial ROS, diminishes the mechanically-induced stabilization of HIF-1α, and partially restores vascular reactivity by rescuing endothelial NO production. These findings raise exciting prospects concerning shared pathophysiologic mechanisms and possible common therapeutic targets amongst PAH etiologies.

Juvenile systemic sclerosis (jSSc) associated pulmonary hypertension (PH) is rare, but, the leading cause of morbidity and mortality in jSSc. This is a case of a 10-year-old girl whose initial presentation of positive U3-RNP antibody jSSc included diffuse skin findings, severe pulmonary arterial hypertension, and right ventricular failure. Veno-arterial extracorporeal membranous oxygenation (VA-ECMO) and atrial stent placement facilitated treatment with pulse-dose steroids, mycophenolate mofetil, and B-cell depleting therapy to treat the underlying autoimmune inflammation and triple therapy with treprostinil, ambrisentan, and tadalafil for her pulmonary hypertension. At 9-month follow-up, her jSSc is well-controlled with complete resolution of her PH. This case demonstrates that multi-disciplinary treatment, including upfront multi-drug therapy for jSSC and PAH, that included VA-ECMO, may improve outcomes, particularly when treatment for underlying causes (in this case, jSSc) is just being initiated.

Endothelial cell (EC) dysfunction is key in initiating and progressing pulmonary hypertension (PH). EC dysfunction in PH leads to hyperproliferation and vascular remodeling of the pulmonary blood vessels. Increased glutaminolysis and altered cellular metabolism are pivotal in hyperproliferative cancer cells. However, whether a similar enhancement in glutamine metabolism is involved in the EC hyperproliferation and if this contributes to vascular remodeling during PH development is unresolved and was the focus of our study. Metabolic flux analysis showed elevated glutaminolysis and enhanced metabolic flux through the reductive tricarboxylic acid (TCA) cycle in pulmonary arterial ECs isolated from an ovine experimental model of PH (PH-PAECs). PH-PAECs also exhibited increased c-Myc protein levels, a master regulator of glutaminolysis. Therefore, we assessed the effect of increased c-Myc expression on metabolic reprogramming, glutaminolysis, and proliferation in control PAECs. Results from a comprehensive snapshot metabolomics investigation and metabolic flux analysis confirmed the reprogramming of mitochondrial metabolism, enhanced glutamine metabolism, and increased glycolysis in c-Myc overexpressing PAECs. Additionally, c-Myc overexpression impacted the ATP production rate, disrupted mitochondrial respiration, increased reactive oxygen species production, induced cell proliferation, and suppressed apoptosis. Functionally, these metabolic changes suppressed nitric oxide (NO) production. We also demonstrate that a small-molecule c-Myc inhibitor, 10058-F4, attenuates glutaminolysis, suppresses the reverse TCA cycle and glycolysis, and reverses the hyperproliferative phenotype, thereby restoring NO levels in PH-PAECs. We also demonstrate that directly targeting HIF-1α reverses the hyper-proliferative, anti-apoptotic phenotype in PH-PAECs. Thus, targeting c-Myc signaling and suppressing glutaminolysis or glycolysis could be a novel therapy for PH.

Right ventricular (RV) failure is the primary cause of death among patients with pulmonary arterial hypertension (PAH). Patients with congenital heart disease-associated PAH (CHD-PAH) demonstrate improved outcomes compared with patients with other forms of PAH, which is related to the maintenance of an adaptively hypertrophied RV. In an ovine model of CHD-PAH, we aimed to elucidate the cellular, microvascular, and transcriptional adaptations to congenital pressure overload that support RV function. Fetal surgery was performed on late gestation lambs to insert an aortopulmonary graft, leading to a persistent congenital left-right shunt and RV pressure load. At 3 days and 4-6 wk of life, shunt RV myocardial structure, growth mechanisms, and transcriptomes were compared with age-matched control and unoperated fetal RV. At 4-6 wk of age, shunt lambs demonstrate significant RV enlargement (shunt 37.1 ± 7.6 g vs. control 15.9 ± 1.9 g, P < 0.001) but maintain stable microvascular density (fetal 3.0 ± 1.2 vs. shunt 2.9 ± 0.5 vs. control 3.1 ± 1.2 capillaries per 1,000 µm2, P > 0.05). Shunt RV cardiomyocytes are significantly more numerous and smaller by cross-sectional area than age-matched controls (shunt 73.3 ± 11.0 µm2 vs. control 99.2 ± 9.8 µm2, P = 0.013). At 3 days, shunt RV cardiomyocytes show evidence of increased proliferative capacity and ongoing hyperplasia compared with controls. RNA sequencing analyses reveal a distinct transcriptomic profile in shunt RV consistent with a delay in terminal differentiation and metabolic adaptations to support adaptive function. This study provides novel insights into the roles of microvascular preservation and cardiomyocyte hyperplasia in the development of adaptive RV hypertrophy in CHD-PAH.NEW & NOTEWORTHY In this study, we utilize an ovine model of congenital heart disease-associated pulmonary arterial hypertension (CHD-PAH) to unveil the structural and transcriptional changes that underlie the maintenance of an adaptively hypertrophied right ventricle (RV). In CHD-PAH, the RV hypertrophies while maintaining microvascular density and expanding its cardiomyocyte population, structural and cellular adaptations that are critical to supporting RV function. These findings provide novel insight into myocardial growth mechanisms that are triggered by congenital pressure overload.

Background:Pulmonary vascular disease (PVD) in patients with single ventricular heart disease following the partial cavalpulmonary connection (Glenn) is a significant source of morbidity. However, the etiology of pulmonary vascular endothelial cell (EC) dysfunction, an established precursor to PVD, is incompletely understood but may involve abnormal blood flow patterns, hypoxemia, and polycythemia. Hypothesis:Utilizing an ovine Glenn model, we hypothesized that non-pulsatile pulmonary blood flow (PBF) induces pulmonary vascular EC dysfunction, independent of hypoxemia or polycythemia. Methods:Seven lambs (6-8 weeks old) underwent a Glenn procedure. Eight weeks later, Glenn and age-matched controls were studied. The response to the endothelium-dependent vasodilator acetylcholine (Ach) was determined in isolated pulmonary arteries (PA). Nitric oxide (NO) and endothelin-1 (ET-1) signaling was determined in right lung tissues. Indices of cell proliferation, angiogenesis, and apoptosis were determined in PA endothelial cells (PAECs). Comparisons were made by unpaired t-test and ANOVA. Results:There were no differences in age, hemoglobin, or oxygen saturation between groups. Mean PA pressure and left PA flow were higher, and right lung blood flow was lower in Glenn lambs compared to controls (p<0.05). All other baseline hemodynamics were similar. Glenn PAs had impaired relaxation to Ach. Glenn lung NO metabolite levels (NOx) and eNOS protein were lower, and ET-1 levels and prepro-ET-1 protein were higher than controls (p<0.05). Glenn PAECs had higher rates of proliferation and angiogenesis, and decreased apoptosis (p < 0.05). Conclusions:The initiation of non-pulsatile PBF following the Glenn induces early EC dysfunction independent of hypoxemia and polycythemia.

Co-spray dried inhalable powder formulations of fasudil monohydrochloride salt (FMCS) and inhalable lung surfactant-based nanocarriers composed of synthetic phospholipids, zwitterionic DPPC (1,2-palmitoyl-sn-glycero-3-phosphocholine) and anionic DPPG (1,2-dipalmitoyl-sn-glycero-3-[phosphor-rac-1-glycerol]) sodium salt, were designed and optimized using organic solution advanced spray drying. FMCS can potentially be used for the treatment of various complex pulmonary diseases with this current work focusing on pulmonary arterial hypertension. Comprehensive physicochemical characterization, electron and optical microscopy imaging, thermal analysis, molecular fingerprinting spectroscopy, in vitro aerosol dispersion performance with human dry powder inhaler (DPI) devices, in vitro membrane permeation and drug release, and in vitro human cellular studies were conducted. Well-defined, small, and smooth nanoparticles/microparticles in the solid state were engineered at different molar ratios of FMCS/DPPC/DPPG (25:75, 50:50, and 75:25) and successfully produced as inhalable powders having the properties necessary for targeted pulmonary delivery as dry powder inhalers. In vitro aerosol performance demonstrated excellent aerosol dispersion with different DPI devices. The phospholipid bilayer biophysical properties were confirmed and retained following cospray drying. Sustained release of fasudil drug and in vitro biocompatibility were demonstrated on human lung cells from different airway regions.