Pathogenesis and Physiologic Mechanisms of Neonatal Pulmonary Hypertension: Preclinical Studies.

Neonatal pulmonary hypertension (PH) is a devastating disorder of the pulmonary vasculature characterized by elevated pulmonary vascular resistance and mean pulmonary arterial pressure. Occurring predominantly because of maldevelopment or maladaptation of the pulmonary vasculature, PH in neonates is associated with suboptimal short-term and long-term outcomes because its pathobiology is unclear in most circumstances, and it responds poorly to conventional pulmonary vasodilators. Understanding the pathogenesis and pathophysiology of neonatal PH can lead to novel strategies and precise therapies. The review is designed to achieve this goal by summarizing pulmonary vascular development and the pathogenesis and pathophysiology of PH associated with maladaptation, bronchopulmonary dysplasia, and congenital diaphragmatic hernia based on evidence predominantly from preclinical studies. We also discuss the pros and cons of and provide future directions for preclinical studies in neonatal PH.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates.

BACKGROUND: Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. METHODS: Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. RESULTS: We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. CONCLUSION: These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

Protective role of CXCR7 activation in neonatal hyperoxia-induced systemic vascular remodeling and cardiovascular dysfunction in juvenile rats.

Neonatal hyperoxia induces long-term systemic vascular stiffness and cardiovascular remodeling, but the mechanisms are unclear. Chemokine receptor 7 (CXCR7) represents a key regulator of vascular homeostasis and repair by modulating TGF-ß1 signaling. This study investigated whether pharmacological CXCR7 agonism prevents neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction in juvenile rats. Newborn Sprague Dawley rat pups assigned to room air or hyperoxia (85% oxygen), received CXCR7 agonist, TC14012 or placebo for 3 weeks. These rat pups were maintained in room air until 6 weeks when aortic pulse wave velocity doppler, cardiac echocardiography, aortic and left ventricular (LV) fibrosis were assessed. Neonatal hyperoxia induced systemic vascular stiffness and cardiac dysfunction in 6-week-old rats. This was associated with decreased aortic and LV CXCR7 expression. Early treatment with TC14012, partially protected against neonatal hyperoxia-induced systemic vascular stiffness and improved LV dysfunction and fibrosis in juvenile rats by decreasing TGF-ß1 expression. In vitro, hyperoxia-exposed human umbilical arterial endothelial cells and coronary artery endothelial cells had increased TGF-ß1 levels. However, treatment with TC14012 significantly reduced the TGF-ß1 levels. These results suggest that dysregulation of endothelial CXCR7 signaling may contribute to neonatal hyperoxia-induced systemic vascular stiffness and cardiac dysfunction.

Extracellular vesicles: pathogenic messengers and potential therapy for neonatal lung diseases.

Extracellular vesicles (EVs) are a heterogeneous group of nano-sized membranous structures increasingly recognized as mediators of intercellular and inter-organ communication. EVs contain a cargo of proteins, lipids and nucleic acids, and their cargo composition is highly dependent on the biological function of the parental cells. Their cargo is protected from the extracellular environment by the phospholipid membrane, thus allowing for safe transport and delivery of their intact cargo to nearby or distant target cells, resulting in modification of the target cell's gene expression, signaling pathways and overall function. The highly selective, sophisticated network through which EVs facilitate cell signaling and modulate cellular processes make studying EVs a major focus of interest in understanding various biological functions and mechanisms of disease. Tracheal aspirate EV-miRNA profiling has been suggested as a potential biomarker for respiratory outcome in preterm infants and there is strong preclinical evidence showing that EVs released from stem cells protect the developing lung from the deleterious effects of hyperoxia and infection. This article will review the role of EVs as pathogenic messengers, biomarkers, and potential therapies for neonatal lung diseases.

Case report: Fatal lung hyperinflammation in a preterm newborn with SARS-CoV-2 infection.

Vertical transmission of SARS-CoV-2 from mother to fetus is widely accepted. Whereas most infected neonates present with mild symptoms or are asymptomatic, respiratory distress syndrome (RDS) and abnormal lung images are significantly more frequent in COVID-19 positive neonates than in non-infected newborns. Fatality is rare and discordant meta-analyses of case reports and series relating perinatal maternal COVID-19 status to neonatal disease severity complicate their extrapolation as prognostic indicators. A larger database of detailed case reports from more extreme cases will be required to establish therapeutic guidelines and allow informed decision making. Here we report an unusual case of a 28 weeks' gestation infant with perinatally acquired SARS-CoV-2, who developed severe protracted respiratory failure. Despite intensive care from birth with first line anti-viral and anti-inflammatory therapy, respiratory failure persisted, and death ensued at 5 months. Lung histopathology showed severe diffuse bronchopneumonia, and heart and lung immunohistochemistry confirmed macrophage infiltration, platelet activation and neutrophil extracellular trap formation consistent with late multisystem inflammation. To our knowledge, this is the first report of SARS CoV-2 pulmonary hyperinflammation in a preterm newborn with fatal outcome.

c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

Col4a3-/- Mice on Balb/C Background Have Less Severe Cardiorespiratory Phenotype and SGLT2 Over-Expression Compared to 129x1/SvJ and C57Bl/6 Backgrounds.

Alport syndrome (AS) is a hereditary renal disorder with no etiological therapy. In the preclinical Col4a3-/- model of AS, disease progression and severity vary depending on mouse strain. The sodium-glucose cotransporter 2 (SGLT2) is emerging as an attractive therapeutic target in cardiac/renal pathologies, but its application to AS remains untested. This study investigates cardiorespiratory function and SGLT2 renal expression in Col4a3-/- mice from three different genetic backgrounds, 129x1/SvJ, C57Bl/6 and Balb/C. male Col4a3-/- 129x1/SvJ mice displayed alterations consistent with heart failure with preserved ejection fraction (HFpEF). Female, but not male, C57Bl/6 and Balb/C Col4a3-/- mice exhibited mild changes in systolic and diastolic function of the heart by echocardiography. Male C57Bl/6 Col4a3-/- mice presented systolic dysfunction by invasive hemodynamic analysis. All strains except Balb/C males demonstrated alterations in respiratory function. SGLT2 expression was significantly increased in AS compared to WT mice from all strains. However, cardiorespiratory abnormalities and SGLT2 over-expression were significantly less in AS Balb/C mice compared to the other two strains. Systolic blood pressure was significantly elevated only in mutant 129x1/SvJ mice. The results provide further evidence for strain-dependent cardiorespiratory and hypertensive phenotype variations in mouse AS models, corroborated by renal SGLT2 expression, and support ongoing initiatives to develop SGLT2 inhibitors for the treatment of AS.

Comparative Effects of Bone Marrow-derived Versus Umbilical Cord Tissue Mesenchymal Stem Cells in an Experimental Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a life-threatening condition in preterm infants with few effective therapies. Mesenchymal stem or stromal cells (MSCs) are a promising therapeutic strategy for BPD. The ideal MSC source for BPD prevention is however unknown. The objective of this study was to compare the regenerative effects of MSC obtained from bone marrow (BM) and umbilical cord tissue (UCT) in an experimental BPD model. In vitro, UCT-MSC demonstrated greater proliferation and expression of anti-inflammatory cytokines as compared to BM-MSC. Lung epithelial cells incubated with UCT-MSC conditioned media (CM) had better-wound healing following scratch injury. UCT-MSC CM and BM-MSC CM had similar pro-angiogenic effects on hyperoxia-exposed pulmonary microvascular endothelial cells. In vivo, newborn rats exposed to normoxia or hyperoxia (85% O2) from postnatal day (P) 1 to 21 were given intra-tracheal (IT) BM or UCT-MSC (1 × 106 cells/50 µL), or placebo (PL) on P3. Hyperoxia PL-treated rats had marked alveolar simplification, reduced lung vascular density, pulmonary vascular remodeling, and lung inflammation. In contrast, administration of both BM-MSC and UCT-MSC significantly improved alveolar structure, lung angiogenesis, pulmonary vascular remodeling, and lung inflammation. UCT-MSC hyperoxia-exposed rats however had greater improvement in some morphometric measures of alveolarization and less lung macrophage infiltration as compared to the BM-MSC-treated group. Together, these findings suggest that BM-MSC and UCT-MSC have significant lung regenerative effects in experimental BPD but UCT-MSC suppresses lung macrophage infiltration and promotes lung epithelial cell healing to a greater degree.

Systemic delivery of large-scale manufactured Wharton's Jelly mesenchymal stem cell-derived extracellular vesicles improves cardiac function after myocardial infarction.

INTRODUCTION: Cardiovascular disease and myocardial infarction are leading causes of morbidity and mortality in aged populations. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are under evaluation as a therapeutic option for the treatment of myocardial infarction. AIM: This study aimed to develop a large-scale manufacturing procedure to harvest clinical-grade EVs required for the translation of EVs to the clinic. METHODS AND RESULTS: We compared the efficiency of large scale MSC-derived EV production and characterized EV miRNA cargo using the Quantum bioreactor with either fetal bovine serum or human platelet lysate (PLT)-containing expansion media. We tested the potency of the EV products in a murine model of acute myocardial infarction. Our results demonstrate an advantage of the Quantum bioreactor as a large-scale platform for EV production using PLT media; however, both media produced EVs with similar effects in vivo. The systemic delivery of EV products improved cardiac function following myocardial infarctions as indicated by a significant improvement in ejection fraction as well as parameters of cardiac performance, afterload, contractility and lusitropy. CONCLUSION: These findings have important implications for scale-up strategies of EVs and will facilitate clinical trials for their clinical evaluation.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury.

BACKGROUND: Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. METHODS: Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. RESULTS: Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1ß and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. CONCLUSIONS: These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

Amniotic fluid-derived extracellular vesicles: characterization and therapeutic efficacy in an experimental model of bronchopulmonary dysplasia.

BACKGROUND AIMS: Extracellular vesicles (EVs) are being tested for their use as novel therapeutics. However, the optimal source of EVs is currently under investigation. Amniotic fluid (AF) is a natural source of EVs that can be easily obtained for use in regenerative medicine, yet AF-EV characterization has not been fully explored. METHODS: Here the authors demonstrate AF as a rich source of EVs and identify the microRNA and proteomic cargo. Bioinformatics analysis of this cargo revealed multiple pathway targets, including immunomodulatory, anti-inflammatory and free radical scavenging networks. The authors further demonstrated the therapeutic potential of this EV product as a novel preventative agent for bronchopulmonary dysplasia (BPD). RESULTS: Intra-tracheal administration of AF-EVs preserved alveolar development, attenuated vascular remodeling and pulmonary hypertension, decreased lung pro-inflammatory cytokine expression and reduced macrophage infiltration in an experimental BPD model. CONCLUSIONS: The authors' results suggest that AF is a viable biological fluid for EV harvest and that AF-EVs have strong therapeutic potential for pulmonary diseases, such as BPD, warranting further development to transition this novel EV product into the clinic.

Soluble Klotho, a biomarker and therapeutic strategy to reduce bronchopulmonary dysplasia and pulmonary hypertension in preterm infants.

Preterm infants with bronchopulmonary dysplasia (BPD) and pulmonary hypertension (PH) have accelerated lung aging and poor long-term outcomes. Klotho is an antiaging protein that modulates oxidative stress, angiogenesis and fibrosis. Here we test the hypothesis that decreased cord Klotho levels in preterm infants predict increased BPD-PH risk and early Klotho supplementation prevents BPD-like phenotype and PH in rodents exposed to neonatal hyperoxia. In experiment 1, Klotho levels were measured in cord blood of preterm infants who were enrolled in a longitudinal cohort study. In experiment 2, using an experimental BPD-PH model, rat pups exposed to room air or hyperoxia (85% O2) were randomly assigned to receive every other day injections of recombinant Klotho or placebo. The effect of Klotho on lung structure, PH and cardiac function was assessed. As compared to controls, preterm infants with BPD or BPD-PH had decreased cord Klotho levels. Early Klotho supplementation in neonatal hyperoxia-exposed rodents preserved lung alveolar and vascular structure, attenuated PH, reduced pulmonary vascular remodeling and improved cardiac function. Together, these findings have important implications as they suggest that perinatal Klotho deficiency contributes to BPD-PH risk and strategies that preserve Klotho levels, may improve long-term cardiopulmonary outcomes in preterm infants.

Effects of Klotho supplementation on hyperoxia-induced renal injury in a rodent model of postnatal nephrogenesis.

BACKGROUND: Hyperoxia (HO) causes kidney injury in preterm infants; however, whether these effects are modifiable is unknown. We hypothesized that administration of exogenous soluble Klotho, a kidney-derived antioxidant, would attenuate HO-induced kidney injury during postnatal nephrogenesis in rats. METHODS: Sprague Dawley neonatal rats assigned to normoxia (21% O2) or HO (85% O2) groups from postnatal day (P) 1 to 21 were randomly assigned to receive alternate day intraperitoneal injections of recombinant Klotho or placebo for 3 weeks. They were recovered in normoxia for an additional 3 weeks and sacrificed at 6 weeks. Renal artery resistance and pulsatility indices, tubular injury scores, glomerular area, and renal antioxidant capacity were assessed. RESULTS: Rodents exposed to HO during postnatal nephrogenesis had reduced kidney Klotho expression, glomerulomegaly, and higher tubular injury scores. Exogenous Klotho administration improved renal perfusion as indicated by decreases in both resistance and pulsatility indices and increased antioxidant enzyme expression. CONCLUSIONS: HO exposure during postnatal nephrogenesis in rodents results in a decline in kidney Klotho expression, decreased renal perfusion, enlarged glomerular size, and tubular injury. The exogenous administration of Klotho attenuated HO-induced kidney injury and augmented antioxidant capacity.

Neonatal hyperoxia exposure induces aortic biomechanical alterations and cardiac dysfunction in juvenile rats.

Supplemental oxygen (O2 ) therapy in preterm infants impairs lung development, but the impact of O2 on long-term systemic vascular structure and function has not been well-explored. The present study tested the hypothesis that neonatal O2 therapy induces long-term structural and functional alterations in the systemic vasculature, resulting in vascular stiffness observed in children and young adults born preterm. Newborn Sprague-Dawley rats were exposed to normoxia (21% O2 ) or hyperoxia (85% O2 ) for 1 and 3 weeks. A subgroup exposed to 3 weeks hyperoxia was recovered in normoxia for an additional 3 weeks. Aortic stiffness was assessed by pulse wave velocity (PWV) using Doppler ultrasound and pressure myography. Aorta remodeling was assessed by collagen deposition and expression. Left ventricular (LV) function was assessed by echocardiography. We found that neonatal hyperoxia exposure increased vascular stiffness at 3 weeks, which persisted after normoxic recovery at 6 weeks of age. These findings were accompanied by increased PWV, aortic remodeling, and altered LV function as evidenced by decreased ejection fraction, cardiac output, and stroke volume. Importantly, these functional changes were associated with increased collagen deposition in the aorta. Together, these findings demonstrate that neonatal hyperoxia induces early and sustained biomechanical alterations in the systemic vasculature and impairs LV function. Early identification of preterm infants who are at risk of developing systemic vascular dysfunction will be crucial in developing targeted prevention strategies that may improve the long-term cardiovascular outcomes in this vulnerable population.

Intra-tracheal administration of a naked plasmid expressing stromal derived factor-1 improves lung structure in rodents with experimental bronchopulmonary dysplasia.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is characterized by alveolar simplification and disordered angiogenesis. Stromal derived factor-1 (SDF-1) is a chemokine which modulates cell migration, proliferation, and angiogenesis. Here we tested the hypothesis that intra-tracheal (IT) administration of a naked plasmid DNA expressing SDF-1 would attenuate neonatal hyperoxia-induced lung injury in an experimental model of BPD, by promoting angiogenesis. DESIGN/METHODS: Newborn Sprague-Dawley rat pups (n = 18-20/group) exposed to room air (RA) or hyperoxia (85% O2) from postnatal day (P) 1 to 14 were randomly assigned to receive IT a naked plasmid expressing SDF-1, JVS-100 (Juventas Therapeutics, Cleveland, Ohio) or placebo (PL) on P3. Lung alveolarization, angiogenesis, inflammation, vascular remodeling and pulmonary hypertension (PH) were assessed on P14. PH was determined by measuring right ventricular systolic pressure (RVSP) and the weight ratio of the right to left ventricle + septum (RV/LV + S). Capillary tube formation in SDF-1 treated hyperoxia-exposed human pulmonary microvascular endothelial cells (HPMEC) was determined by matrigel assay. Data is expressed as mean ± SD and analyzed by two-way ANOVA. RESULTS: Exposure of neonatal pups to 14 days of hyperoxia decreased lung SDF-1 gene expression. Moreover, whilst hyperoxia exposure inhibited capillary tube formation in HPMEC, SDF-1 treatment increased tube length and branching in HPMEC. PL-treated hyperoxia-exposed pups had decreased alveolarization and lung vascular density. This was accompanied by an increase in RVSP, RV/LV + S, pulmonary vascular remodeling and inflammation. In contrast, IT JVS-100 improved lung structure, reduced inflammation, PH and vascular remodeling. CONCLUSIONS: Intratracheal administration of a naked plasmid expressing SDF-1 improves alveolar and vascular structure in an experimental model of BPD. These findings suggest that therapies which modulate lung SDF-1 expression may have beneficial effects in preterm infants with BPD.

Stromal derived factor-1 mediates the lung regenerative effects of mesenchymal stem cells in a rodent model of bronchopulmonary dysplasia.

BACKGROUND: Mesenchymal stem cells (MSCs) attenuate lung injury in experimental models of bronchopulmonary dysplasia (BPD). Stromal derived factor-1 (SDF-1), a chemokine secreted by MSCs, modulates angiogenesis and stem cell recruitment. Here we tested the hypothesis that SDF-1 mediates MSC protective effects in experimental BPD by modulating angiogenesis. METHODS: SDF-1 was knocked down in MSCs using lentiviral vectors carrying anti-SDF-1 short hairpin RNA (MSC-SDF KD). Non-silencing short hairpin RNA was used as control (MSC-NS control). Newborn rats exposed to normoxia or hyperoxia (FiO2 = 0.85) for 3 weeks, were randomly assigned to receive a single intra-tracheal injection (IT) of MSC-NS control or MSC-SDF KD (1 × 106 cells/50 µl) or placebo on postnatal day 7. The degree of alveolarization, lung angiogenesis, inflammation, and pulmonary hypertension (PH) were assessed at postnatal day 21. RESULTS: Administration of IT MSC-NS control improved lung alveolarization, angiogenesis and inflammation, and attenuated PH in newborn rats with hyperoxia-induced lung injury (HILI). In contrast, knockdown of SDF-1 in MSCs significantly reduced their beneficial effects on alveolarization, angiogenesis, inflammation and PH. CONCLUSIONS: The therapeutic benefits of MSCs in neonatal HILI are in part mediated by SDF-1, through anti-inflammatory and angiogenesis promoting mechanisms. Therapies directly targeting this chemokine may provide a novel strategy for the treatment of BPD.

Intra-Amniotic Soluble Endoglin Impairs Lung Development in Neonatal Rats.

Soluble endoglin (sENG) is increased in the amniotic fluid of women with preeclampsia and chorioamnionitis. Preterm infants born to women with these disorders have an increased risk of aberrant lung development. Whether this increased risk is secondary to elevated sENG levels is unclear. The objective of this study was to determine whether intrauterine exposure to an adenovirus overexpressing sENG impairs neonatal lung angiogenesis by modulating lung endothelial nitric oxide synthase (eNOS) signaling. Pregnant Sprague-Dawley rats were randomly assigned to receive ultrasound-guided intra-amniotic injections of adenovirus overexpressing sENG (Ad-sENG) or control adenovirus (Ad-control) on embryonic day 17. After this exposure, rat pups were maintained in normoxia and evaluated on postnatal day 14. Intra-amniotic Ad-sENG decreased lung vascular endothelial growth factor receptor 2 and eNOS expression in rat pups. This was accompanied by a marked decrease in lung angiogenesis and alveolarization. Ad-sENG-exposed pups also had an increase in right ventricular systolic pressure, weight ratio of right ventricle to left ventricle plus septum, and pulmonary vascular remodeling. In addition, exposure of human pulmonary artery endothelial cells to recombinant sENG reduced endothelial tube formation and protein levels of eNOS, phosphorylated eNOS, and phosphorylated Smad1/5. Together, our findings demonstrate that intrauterine exposure to an adenovirus overexpressing sENG disrupts lung development by impairing Smad1/5-eNOS signaling. We speculate that sENG-mediated dysregulation of Smad1/5-eNOS signaling contributes to impaired lung development and potentially primes the developing lung for further postnatal insults. Further studies exploring the relationship between amniotic fluid sENG levels and preterm respiratory outcomes will be necessary.