Search

Competing Interests: Competing interests The authors declare no competing interests.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (>21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

Acute kidney injury (AKI) occurs in nearly 30% of sick neonates. Chronic kidney disease (CKD) can be detected in certain populations of sick neonates as early as 2 years. AKI is often part of a multisystem syndrome that negatively impacts developing organs resulting in short- and long-term pulmonary, neurodevelopmental, and cardiovascular morbidities. It is critical to incorporate kidney-related data into neonatal clinical trials in a uniform manner to better understand how neonatal AKI or CKD could affect an outcome of interest. Here, we provide expert opinion recommendations and rationales to support the inclusion of short- and long-term neonatal kidney outcomes using a tiered approach based on study design: (1) observational studies (prospective or retrospective) limited to data available within a center's standard practice, (2) observational studies involving prospective data collection where prespecified kidney outcomes are included in the design, (3) interventional studies with non-nephrotoxic agents, and (4) interventional studies with known nephrotoxic agents. We also provide recommendations for biospecimen collection to facilitate ancillary kidney specific research initiatives. This approach balances the costs of AKI and CKD ascertainment with knowledge gained. We advocate that kidney outcomes be included routinely in neonatal clinical study design. Consistent incorporation of kidney outcomes across studies will increase our knowledge of neonatal morbidity., (© 2024. The Author(s).)

Extremely preterm infants are often treated with supraphysiological oxygen, which contributes to the development of bronchopulmonary dysplasia (BPD). These same infants exhibit compromised antioxidant capacities due in part to selenium (Se) deficiency. Se is essential for basal and inducible antioxidant responses. The present study utilized a perinatal Se deficiency (SeD) mouse model to identify the combined effects of newborn hyperoxia exposure and SeD on alveolarization and antioxidant responses, including the identification of affected developmental pathways. Se-sufficient (SeS) and SeD C3H/HeN breeding pairs were generated, and pups were exposed to room air or 85% O 2 from birth to 14 d. Survival, antioxidant protein expression, and RNA seq analyses were performed. Greater than 40% mortality was observed in hyperoxia-exposed SeD pups. Surviving SeD pups had greater lung growth deficits than hyperoxia-exposed SeS pups. Gpx2 and 4 protein and Gpx activity were significantly decreased in SeD pups. Nrf2-regulated proteins, Nqo1 and Gclc were increased in SeD pups exposed to hyperoxia. RNA seq revealed significant decreases in the Wnt/β-catenin and Notch pathways. Se is a biologically relevant modulator of perinatal lung development and antioxidant responses, especially in the context of hyperoxia exposure. The RNA seq analyses suggest pathways essential for normal lung development are dysregulated by Se deficiency.

DNA methylation is necessary for developmental gene regulation, but adverse environments result in aberrant methylation and gene silencing. The current pilot study tested the hypothesis that treatment with DNA methylation inhibitors (decitabine; RG108) would improve alveolarization in a newborn murine model of severe bronchopulmonary dysplasia. Newborn mice exposed to maternal inflammation (LPS) and neonatal hyperoxia (85% O 2 ) were treated with decitabine (p3, 0.1 mg/kg; p2, 4, 6, 0.1 mg/kg; or p2, 4, 6, 0.15 mg/kg) or RG108 (p3, 0.0013 mg/kg) delivered intranasally. Modest improvements in alveolarization were observed with decitabine, but no differences were observed with RG108. Attenuated phospho-SMAD2/3 levels and greater surfactant protein C protein levels compared to vehicle were observed with some tested doses. No detrimental side effects were observed with the doses used in this study. In summary, our pilot investigations identified a safe dose for intranasal administration of both methylation inhibitors and provides a foundation for further studies into methylation inhibitors in the context of neonatal lung injury., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bronchopulmonary dysplasia (BPD) is a common lung disease of premature infants. Hyperoxia exposure and microbial dysbiosis are contributors to BPD development. However, the mechanisms linking pulmonary microbial dysbiosis to worsening lung injury are unknown. Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that regulates oxidative stress responses and modulates hyperoxia-induced lung injury. We hypothesized that airway dysbiosis would attenuate Nrf2-dependent antioxidant function, resulting in a more severe phenotype of BPD. Here, we show that preterm infants with a Gammaproteobacteria-predominant dysbiosis have increased endotoxin in tracheal aspirates, and mice monocolonized with the representative Gammaproteobacteria Escherichia coli show increased tissue damage compared with germ-free (GF) control mice. Furthermore, we show Nrf2-deficient mice have worse lung structure and function after exposure to hyperoxia when the airway microbiome is augmented with E. coli . To confirm the disease-initiating potential of airway dysbiosis, we developed a novel humanized mouse model by colonizing GF mice with tracheal aspirates from human infants with or without severe BPD, producing gnotobiotic mice with BPD-associated and non-BPD-associated lung microbiomes. After hyperoxia exposure, BPD-associated mice demonstrated a more severe BPD phenotype and increased expression of Nrf2 -regulated genes, compared with GF and non-BPD-associated mice. Furthermore, augmenting Nrf2 -mediated antioxidant activity by supporting colonization with Lactobacillus species improved dysbiotic-augmented lung injury. Our results demonstrate that a lack of protective pulmonary microbiome signature attenuates an Nrf2 -mediated antioxidant response, which is augmented by a respiratory probiotic blend. We anticipate antioxidant pathways will be major targets of future microbiome-based therapeutics for respiratory disease.

Decreased selenium (Se) levels during childhood and infancy are associated with worse respiratory health. Se is biologically active after incorporation into Se-containing antioxidant enzymes (AOE) and proteins. It is unknown how decreased maternal Se during pregnancy and lactation impacts neonatal pulmonary selenoproteins, growth, and lung development. Using a model of neonatal Se deficiency that limits Se intake to the dam during pregnancy and lactation, we evaluated which neonatal pulmonary selenoproteins are decreased in both the saccular (postnatal day 0, P0) and early alveolar (postnatal day 7, P7) stages of lung development. We found that Se deficient (SeD) pups weigh less and exhibit impaired alveolar development compared to Se sufficient (SeS) pups at P7. The activity levels of glutathione peroxidase (GPx) and thioredoxin reductase (Txnrd) were decreased at P0 and P7 in SeD lungs compared to SeS lungs. Protein content of GPx1, GPx3 and Txnrd1 were decreased in SeD lungs at P0 and P7, whereas Txnrd2 content was unaltered compared to SeS controls. The expression of NRF-2 dependent genes and several non-Se containing AOE were similar between SeS and SeD lungs. SeD lungs exhibited a decrease in selenoprotein N, an endoplasmic reticulum protein implicated in alveolar development, at both time points. We conclude that exposure to Se deficiency during pregnancy and lactation impairs weight gain and lung growth in offspring. Our data identify multiple selenoproteins in the neonatal lung that are vulnerable to decreased Se intake, which may impact oxidative stress and cell signaling under physiologic conditions as well as after oxidative stressors.

Omics analyses are commonly used for identifying pathways and genes responsible for physiologic and pathologic processes. Though sex is considered a biological variable in rigorous assessments of pulmonary responses to oxidant exposures, the contribution of the murine strain is largely ignored. This study utilized an unbiased integrated assessment of high-resolution metabolomic profiling and RNA-sequencing to explore sex- and strain-dependent pathways in adult mouse lungs. The results indicated that strain exhibited a greater influence than sex on pathways associated with inflammatory and oxidant/antioxidant responses and that interaction metabolites more closely resembled those identified as differentially expressed by strain. Metabolite analyses revealed that the components of the glutathione antioxidant pathway were different between strains, specifically in the formation of mixed disulfides. Additionally, selenium metabolites such as selenohomocystiene and selenocystathionine were similarly differentially expressed. Transcriptomic analysis revealed similar findings, as evidenced by differences in glutathione peroxidase, peroxiredoxin, and the inflammatory transcription factors RelA and Jun. In summary, an multi-omics integrated approach identified that murine strain disproportionately impacts baseline expression of antioxidant systems in lung tissues. We speculate that strain-dependent differences contribute to discrepant pulmonary responses in preclincal mouse models, with deleterious effects on clinical translation.

Background: Coronavirus disease 2019 (COVID-19) disproportionally affects pregnant women and their newborn; however, little is known about variables that modulate maternal-fetal immune response to infection., Methods: We prospectively studied socioeconomic, biologic, and clinical factors affecting humoral immunity in 87 unvaccinated pregnant women hospitalized in Buenos Aires for symptoms consistent with COVID-19., Results: The number of days between symptom onset and childbirth predicted maternal and newborn virus spike protein receptor binding domain (RBD)-specific immunoglobulin G (IgG). These findings suggest newborns may benefit less when mothers deliver soon after COVID-19 infection. Similarly, a longer time between symptom onset and birth predicted higher in utero transfer of maternal IgG and its concentration in cord blood. Older gestational age at birth was associated with lower maternal to cord blood IgG ratio. Of women with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, 87% developed RBD-specific IgA responses in breast milk within 96 hours of childbirth. IgA was not significantly associated with time from infection but correlated with maternal serum IgG and placental transfer., Conclusions: These results demonstrate the combined role of biologic, clinical, and socioeconomic variables associated with maternal RBD-specific antibodies and supports early vaccination strategies for COVID-19 in socioeconomically vulnerable pregnant women., Clinical Trials Registration: NCT04362956., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

In this state-of-the-art review, we highlight the major advances over the last 5 years in neonatal acute kidney injury (AKI). Large multicenter studies reveal that neonatal AKI is common and independently associated with increased morbidity and mortality. The natural course of neonatal AKI, along with the risk factors, mitigation strategies, and the role of AKI on short- and long-term outcomes, is becoming clearer. Specific progress has been made in identifying potential preventive strategies for AKI, such as the use of caffeine in premature neonates, theophylline in neonates with hypoxic-ischemic encephalopathy, and nephrotoxic medication monitoring programs. New evidence highlights the importance of the kidney in "crosstalk" between other organs and how AKI likely plays a critical role in other organ development and injury, such as intraventricular hemorrhage and lung disease. New technology has resulted in advancement in prevention and improvements in the current management in neonates with severe AKI. With specific continuous renal replacement therapy machines designed for neonates, this therapy is now available and is being used with increasing frequency in NICUs. Moving forward, biomarkers, such as urinary neutrophil gelatinase-associated lipocalin, and other new technologies, such as monitoring of renal tissue oxygenation and nephron counting, will likely play an increased role in identification of AKI and those most vulnerable for chronic kidney disease. Future research needs to be focused on determining the optimal follow-up strategy for neonates with a history of AKI to detect chronic kidney disease., Competing Interests: POTENTIAL CONFLICT OF INTEREST: For full disclosure, we provide here an additional list of other authors’ commitments and funding sources that are not directly related to this study: Dr Askenazi is a consultant for Baxter, Nuwellis, Medtronic Bioporto, the Acute Kidney Injury Foundation, and Seastar; he receives grant funding for studies not related to this project from Baxter, Nuwellis, Medtronic, and the National Institutes of Health; the other authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2021 by the American Academy of Pediatrics.)

Background: Alveolar macrophages (AMs) are resident inflammatory cells in the lung that serve as early sentinels of infection or injury. We have identified thioredoxin reductase 1 inhibition by gold compounds increases activation of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent pathways to attenuate inflammatory responses. The present studies utilized murine alveolar macrophages (MH-S) to test the hypothesis that the gold compound, auranofin (AFN), decreases interleukin (IL)-1β expression through NRF2-mediated interactions with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway genes and/or increases in glutathione synthesis., Methods: MH-S cells were treated with AFN and lipopolysaccharide (LPS) and analyzed at 6 and 24 h. The Il1b promoter was analyzed by chromatin immunoprecipitation for direct interaction with NRF2., Results: Expression of IL-1β, p-IκBα, p-p65 NF-kB, and NOD-, LRR-, and pyrin domain-containing protein 3 were elevated by LPS exposure, but only IL-1β expression was suppressed by AFN treatment. Both AFN and LPS treatments increased cellular glutathione levels, but attenuation of glutathione synthesis by buthionine sulfoximine (BSO) did not alter expression of Il-1β. Analysis revealed direct NRF2 binding to the Il1b promoter which was enhanced by AFN and inhibited the transcriptional activity of DNA polymerase II., Conclusions: Our data demonstrate that AFN-induced NRF2 activation directly suppresses IL-1β synthesis independent of NFκB and glutathione-mediated antioxidant mechanisms. NRF2 binding to the promoter region of IL1β directly inhibits transcription of the IL1β gene. Collectively, our research suggests that gold compounds elicit NRF2-dependent pulmonary protection by suppressing macrophage-mediated inflammation.

Background: MicroRNA (miR) are small conserved RNA that regulate gene expression post-transcription. Previous genome-wide analysis studies in preterm infants indicate that pathways of miR 219-5p are important in infants with Bronchopulmonary Dysplasia (BPD)., Methods: Here we report a prospective cohort study of extremely preterm neonates wherein infants diagnosed with severe BPD expressed increased airway miR-219-5p and decreased platelet derived growth factor receptor alpha (PDGFR-α), a target of mir-219-5p and a key regulator of alveolarization, compared to post-conception age-matched term infants., Results: miR-219-5p was highly expressed in the pulmonary epithelial lining in lungs of infants with BPD by in situ hybridization of human infant lungs. In both in vitro and in vivo (mouse) models of BPD, miR-219-5p was increased on exposure to hyperoxia compared with the normoxia control, with a complementary decrease of PDGFR-α. To further confirm the target relationship between miR-219 and PDGFR-α, pulmonary epithelial cells (MLE12) and lung primary fibroblasts were treated with a mimic of miR-219-5p and a locked nucleic acid (LNA) based inhibitor of miR-219-5p. In comparison with the control group, the level of miR-219 increased significantly after miR-219 mimic treatment, while the level of PDGFR-α declined markedly. LNA exposure increased PDGFR-α. Moreover, in BPD mouse model, over-expression of miR-219-5p inhibited alveolar development, indicated by larger alveolar spaces accompanied by reduced septation., Conclusions: Taken together, our results demonstrate that increased miR-219-5p contributes to the pathogenesis of BPD by targeting and reducing PDGFR-α. The use of specific miRNA antagonists may be a therapeutic strategy for preventing the development of BPD.

Maternal selenium (Se) deficiency is associated with decreased neonatal Se levels, which increases the risk for neonatal morbidities. There is a hierarchy to selenoprotein expression after Se deficiency in adult rodents, depending on the particular protein and organ evaluated. However, it is unknown how limited Se supply during pregnancy impacts neonatal selenoprotein expression. We used an Se-deficient diet to induce perinatal Se deficiency (SeD), initiated 2-4 weeks before onset of breeding and continuing through gestation. Neonatal plasma, liver, heart, kidney, and lung were collected on the day of birth and assessed for selenoproteins, factors required for Se processing, and non-Se containing antioxidant enzymes (AOE). Maternal SeD reduced neonatal circulating and hepatic glutathione peroxidase (GPx) activity, as well as hepatic expression of Gpx1 and selenophosphate synthetase 2 (Sps2). In contrast, the impact of maternal SeD on hepatic thioredoxin reductase 1, hepatic non-Se containing AOEs, as well as cardiac, renal, and pulmonary GPx activity, varied based on duration of maternal exposure to SeD diet. We conclude that the neonatal liver and circulation demonstrate earlier depletion in selenoenzyme activity after maternal SeD. Our data indicate that prolonged maternal SeD may escalate risk to the neonate by progressively diminishing Se-containing AOE across multiple organs.

Cellular antioxidants protect against hyperoxic lung injury. The role of the glutathione (GSH) system in lung development and bronchopulmonary dysplasia (BPD) pathogenesis has not been systematically investigated. The current study utilized GSH reductase-deficient (Gsr-KO) neonatal mice to test the hypothesis that early disruption of the GSH system negatively impacts lung development and hyperoxic responses. Lungs from wild-type (Gsr-WT) and Gsr-KO mice were analyzed for histopathology, developmental markers, redox indices, and transcriptome profiling at different developmental stages following exposure to room air or hyperoxia (85% O 2 ) for up to 14 d. Lungs from Gsr-KO mice exhibited alveolar epithelial dysplasia in the embryonic and neonatal periods with relatively normal lung architecture in adulthood. GSH and its oxidized form (GSSG) were 50-70% lower at E19-PND14 in Gsr-KO lungs than in age-matched Gsr-WT. Differential gene expression between Gsr-WT and Gsr-KO lungs was analyzed at discrete developmental stages. Gsr-KO lungs exhibited downregulated cell cycle and DNA damage checkpoint genes at E19, as well as lung lipid metabolism and surfactant genes at PND5. In addition to abnormal baseline lung morphometry, Gsr-KO mice displayed a blunted response to hyperoxia. Hyperoxia caused a more robust upregulation of the lung thioredoxin system in Gsr-KO compared to Gsr-WT. Gsr-dependent, hyperoxia-responsive genes were highly associated with abnormal cytoskeleton, skeletal-muscular function, and tissue morphology at PND5. Overall, our data in Gsr-KO mice implicate the GSH system as a key regulator of lung development, cellular differentiation, and hyperoxic responses in neonatal mice., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Selenium (Se) levels decrease in the circulation during acute inflammatory states and sepsis, and are inversely associated with morbidity and mortality. A more specific understanding of where selenoproteins and Se processing are compromised during insult is needed. We investigated the acute signaling response in selenoenzymes and Se processing machinery in multiple organs after innate immune activation in response to systemic lipopolysaccharide (LPS)., Methods: Wild type (WT) adult male C57/B6 mice were exposed to LPS (5 mg/kg, intraperitoneal). Blood, liver, lung, kidney and spleen were collected from control mice as well as 2, 4, 8, and 24 h after LPS. Plasma Se concentration was determined by ICP-MS. Liver, lung, kidney and spleen were evaluated for mRNA and protein content of selenoenzymes and proteins required to process Se., Results: After 8 h of endotoxemia, plasma levels of Se and the Se transporter protein, SELENOP were significantly decreased. Consistent with this timing, the transcription and protein content of several hepatic selenoenzymes, including SELENOP, glutathione peroxidase 1 and 4 were significantly decreased. Furthermore, hepatic transcription and protein content of factors required for the Se processing, including selenophosphate synthetase 2 (Sps2), phosphoseryl tRNA kinase (Pstk), selenocysteine synthase (SepsecS), and selenocysteine lyase (Scly) were significantly decreased. Significant LPS-induced downregulation of these key selenium processing enzymes was observed in isolated hepatocytes. In contrast to the acute and dynamic changes observed in the liver, selenoenzymes did not decrease in the lung, kidney or spleen., Conclusion: Hepatic selenoenzyme production and Se processing factors decreased after endotoxemia. This was temporally associated with decreased circulating Se. In contrast to these active changes in the regulation of Se processing in the liver, selenoenzymes did not decrease in the lung, kidney or spleen. These findings highlight the need to further study the impact of innate immune challenges on Se processing in the liver and the impact of targeted therapeutic Se replacement strategies during innate immune challenge., (Copyright © 2020 Sherlock, Sjostrom, Sian, Delaney, Tipple, Krebs, Nozik-Grayck and Wright.)

Aims: Interleukin-1β (IL-1β) contributes to the development of bronchopulmonary dysplasia (BPD). Thioredoxin reductase-1 (Txnrd1) inhibition activates nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent responses. Txnrd1 activity is selenium (Se) dependent and Se deficiency is common in prematurity. Auranofin (AFN), a Txnrd1 inhibitor, decreases IL-1β levels and increases Nrf2 activation in lipopolysaccharide (LPS) treated alveolar macrophages. In lung epithelia, AFN-induced Nrf2 activation is Se dependent. We tested the hypothesis that the effects of Txnrd1 inhibition in alveolar macrophages are Se dependent., Main Methods: To establish Se sufficient (Se+) and deficient (Se-) conditions, alveolar (MH-S) macrophages were cultured in 2.5% fetal bovine serum (FBS) ± 25 nM Na 2 SeO 3 . Se- (2.5% FBS) and Se+ (2.5% FBS + 25 nM Na 2 SeO 3 ) cells were cultured in the presence or absence of 0.05 μg/mL LPS and/or 0.5 μM AFN. Nrf2 activation was determined by measuring NADPH quinone oxidoreductase-1 (Nqo1) and glutathione levels. IL-1β mRNA (Il1b) and protein levels were measured using qRT-PCR and ELISA. Data were analyzed by ANOVA followed by Tukey's post-hoc., Key Findings: We detected an independent effect of AFN, but not LPS, on Nqo1 expression and GSH levels in Se+ and Se- cells. LPS significantly increased Il1b and IL-1β levels in both groups. AFN-mediated attenuation of this effect was not impacted by Se status., Significance: The beneficial effects of Txnrd1 inhibition in alveolar macrophages are Se-independent and therefore unlikely to be diminished by clinical Se deficiency., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

As a result of preterm birth, immature kidneys are exposed to interventions in the NICU that promote survival, but are nephrotoxic. Furthermore, the duration of renal development may be truncated in these vulnerable neonates. Immaturity and nephrotoxic exposures predispose preterm newborns to acute kidney injury (AKI), particularly in the low birth weight and extremely preterm gestational age groups. Several studies have associated preterm birth as a risk factor for future chronic kidney disease (CKD). However, only a few publications have investigated the impact of neonatal AKI on CKD development. Here, we will review the evidence linking preterm birth and AKI in the NICU to CKD and highlight the knowledge gaps and opportunities for future research. For neonatal intensive care studies, we propose the inclusion of AKI as an important short-term morbidity outcome and CKD findings such as a reduced glomerular filtration rate in the assessment of long-term outcomes.

Electron Paramagnetic Resonance (EPR) spectroscopy coupled with spin traps/probes enables quantitative determination of reactive nitrogen and oxygen species (RNOS). Even with numerous studies using spin probes, the methodology has not been rigorously investigated. The autoxidation of spin probes has been commonly overlooked. Using the spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH), the present study has tested the effects of metal chelators, temperature, and oxygen content on the autoxidation of spin probes, where an optimized condition is refined for cell studies. The apparent rate of CMH autoxidation under this condition is 7.01 ± 1.60 nM/min, indicating low sensitivity and great variation of the CMH method and that CMH autoxidation rate should be subtracted from the generation rate of CMH-detectable oxidants (simplified as oxidants below) in samples. Oxidants in RAW264.7 cells are detected at an initial rate of 4.0 ± 0.7 pmol/min/10 6  cells, which is not considered as the rate of basal oxidants generation because the same method has failed to detect oxidant generation from the stimulation of phorbol-12-mysirate-13-acetate (PMA, 0.1 nmol/10 6  cells) in cells (2.5 ± 0.9 for PMA vs. 2.1 ± 1.5 pmol/min/10 6  cells for dimethyl sulfoxide (DMSO)-treated cells). In contrast, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which exhibits minimal autoxidation, reveals differences between PMA and DMSO treatment (0.26 ± 0.09 vs. -0.06 ± 0.12 pmol/min/10 6  cells), which challenges previous claims that spin probes are more sensitive than spin traps. We have also found that low temperature EPR measurements of frozen samples of CMH autoxidation provide lower signal intensity and greater variation compared to RT measurements of fresh samples. The current study establishes an example for method development of RNOS detection, where experimental details are rigorously considered and tested, and raises questions on the applications of spin probes and spin traps., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thioredoxin reductase-1 (TXNRD1) inhibition activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and prevents acute lung injury (ALI). Heme oxygenase-1 (HO-1) induction following TXNRD1 inhibition is Nrf2-dependent in airway epithelial (club) cells in vitro . The influence of club cell HO-1 on lung development and lung injury responses is poorly understood. The present studies characterized the effects of hyperoxia on club cell-specific HO-1 knockout (KO) mice. These mice were generated by crossing Hmox1 flox mice with transgenic mice expressing cre recombinase under control of the club cell-specific Scgb1a1 promoter. Baseline analyses of lung architecture and function performed in age-matched adult wild-type and KO mice indicated an increased alveolar size and airway resistance in HO-1 KO mice. In subsequent experiments, adult wild-type and HO-1 KO mice were either continuously exposed to >95% hyperoxia or room air for 72 h or exposed to >95 hyperoxia for 48 h followed by recovery in room air for 48 h. Injury was quantitatively assessed by calculating right lung/body weight ratios (g/kg). Analyses indicated an independent effect of hyperoxia but not genotype on right lung/body weight ratios in both wild-type and HO-1 KO mice. The magnitude of increases in right lung/body weight ratios was similar in mice of both genotypes. In the recovery model, an independent effect of hyperoxia but not genotype was also detected. In contrast to the continuous exposure model, right lung/body weight ratio mice were significantly elevated in HO-1 KO but not wild-type mice. Though club cell HO-1 does not alter hyperoxic sensitivity in adult mice, it significantly influences lung development and resolution of lung injury following acute hyperoxic exposure., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Katelyn Dunigan-Russell et al.)

Bronchopulmonary dysplasia (BPD), a long-term respiratory morbidity of prematurity, is characterized by attenuated alveolar and vascular development. Supplemental oxygen and immature antioxidant defenses contribute to BPD development. Our group identified thioredoxin reductase-1 (TXNRD1) as a therapeutic target to prevent BPD. The present studies evaluated the impact of the TXNRD1 inhibitor aurothioglucose (ATG) on pulmonary responses and gene expression in newborn C57BL/6 pups treated with saline or ATG (25 mg/kg ip) within 12 h of birth and exposed to room air (21% O 2 ) or hyperoxia (>95% O 2 ) for 72 h. Purified RNA from lung tissues was sequenced, and differential expression was evaluated. Hyperoxic exposure altered ~2,000 genes, including pathways involved in glutathione metabolism, intrinsic apoptosis signaling, and cell cycle regulation. The isolated effect of ATG treatment was limited primarily to genes that regulate angiogenesis and vascularization. In separate studies, pups were treated as described above and returned to room air until 14 days. Vascular density analyses were performed, and ANOVA indicated an independent effect of hyperoxia on vascular density and alveolar architecture at 14 days. Consistent with RNA-seq analyses, ATG significantly increased vascular density in room air, but not in hyperoxia-exposed pups. These findings provide insights into the mechanisms by which TXNRD1 inhibitors may enhance lung development.

Hydrogen sulfide, nitric oxide, and carbon monoxide are endogenously produced gases that regulate various signaling pathways. The role of these transmitters is complex as constitutive production of these molecules may have anti-inflammatory, anti-microbial, and/or vasodilatory effects whereas induced production or formation of secondary metabolites may lead to cellular death. Given this fine line between friend and foe, therapeutic attenuation of these molecules' production has involved both inhibition of endogenous formation and therapeutic supplementation. All three gases have been implicated as regulators of critical aspects of neonatal physiology, and in turn, comorbidities including necrotizing enterocolitis, hypoxic ischemic encephalopathy, and pulmonary hypertension. In this review, we present current perspectives on these associations, highlight areas where insights remain sparse, and identify areas for potential for future investigations., Competing Interests: Declaration of competing interest The authors have indicated they have no potential conflicts of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Significance: Redox homeostasis is finely tuned and governed by distinct intracellular mechanisms. The dysregulation of this either by external or internal events is a fundamental pathophysiologic base for many pulmonary diseases. Recent Advances: Based on recent discoveries, it is increasingly clear that cellular redox state and oxidation of signaling molecules are critical modulators of lung disease and represent a final common pathway that leads to poor respiratory outcomes. Critical Issues: Based on the wide variety of stimuli that alter specific redox signaling pathways, improved understanding of the disease and patient-specific alterations are needed for the development of therapeutic targets. Further Directions: For the full comprehension of redox signaling in pulmonary disease, it is essential to recognize the role of reactive oxygen intermediates in modulating biological responses. This review summarizes current knowledge of redox signaling in pulmonary development and pulmonary vascular disease.

Preterm birth is a primary cause of worldwide childhood mortality. Bronchopulmonary dysplasia, characterized by impaired alveolar and lung vascular development, affects 25-50% of extremely low birth weight (BW; <1 kg) infants. Abnormalities in lung function persist into childhood in affected infants and are second only to asthma in terms of childhood respiratory disease healthcare costs. While advances in the medical care of preterm infants have reduced mortality, the incidence of BPD has not decreased in the past 10 years. Reactive oxygen intermediates play a key role in the development of lung disease but, despite promising preclinical therapies, antioxidants have failed to translate into meaningful clinical interventions to decrease the incidence of lung disease in premature infants. In this review we will summarize the state of the art research developments in regards to antioxidants and premature lung disease and discuss the limitations of antioxidant therapies in order to more fully comprehend the reasons why therapeutic antioxidant administration failed to prevent BPD. Finally we will review promising therapeutic strategies and targets., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The etiology of late-onset Alzheimer's disease is unknown. Recent epidemiological studies suggest that exposure to high levels of ozone (O 3 ) may be a risk factor for late-onset Alzheimer's disease. Nonetheless, whether and how O 3 exposure contributes to AD development remains to be determined. In this study, we tested the hypothesis that O 3 exposure synergizes with the genetic risk factor APOE ε4 and aging leading to AD, using male apolipoprotein E (apoE)4 and apoE3 targeted replacement mice as men have increased risk exposure to high levels of O 3 via working environments and few studies have addressed APOE ε4 effects on males. Surprisingly, our results show that O 3 exposure impairs memory in old apoE3, but not old apoE4 or young apoE3 and apoE4, male mice. Further studies show that old apoE4 mice have increased hippocampal activities or expression of some enzymes involved in antioxidant defense, diminished protein oxidative modification, and neuroinflammation following O 3 exposure compared with old apoE3 mice. These novel findings highlight the complexity of interactions between APOE genotype, age, and environmental exposure in AD development., (Published by Elsevier Inc.)

Fetal development occurs in a relatively hypoxemic environment, and birth represents significant oxidative stress. Premature infants are disadvantaged by a lack of maternal antioxidant transfer and impaired endogenous antioxidant responses. O 2 metabolism is essential for life and its biochemical reactions are dynamic, compartmentalized, and difficult to characterize in vivo. There is a growing appreciation for the role of reactive oxygen species in nonpathologic processes, including regulation of cell signaling and mitochondrial function. There are several gaps in the knowledge about the role of reactive oxygen species in normal development and how oxidative stress alters normal signaling and subsequent development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

L-N G -Nitro arginine methyl ester (L-NAME) has been widely applied for several decades in both basic and clinical research as an antagonist of nitric oxide synthase (NOS). Herein, we show that L-NAME slowly releases NO from its guanidino nitro group. Daily pretreatment of rats with L-NAME potentiated mesenteric vasodilation induced by nitrodilators such as nitroglycerin, but not by NO. Release of NO also occurred with the NOS-inactive enantiomer D-NAME, but not with L-arginine or another NOS inhibitor L-NMMA, consistent with the presence or absence of a nitro group in their structure and their nitrodilator-potentiating effects. Metabolic conversion of the nitro group to NO-related breakdown products was confirmed using isotopically-labeled L-NAME. Consistent with Fenton chemistry, transition metals and reactive oxygen species accelerated the release of NO from L-NAME. Both NO production from L-NAME and its nitrodilator-potentiating effects were augmented under inflammation. NO release by L-NAME can confound its intended NOS-inhibiting effects, possibly by contributing to a putative intracellular NO store in the vasculature., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Premature birth is a growing and serious public health problem affecting more than one of every ten infants worldwide. Bronchopulmonary dysplasia (BPD) is the most common neonatal morbidity associated with prematurity and infants with BPD suffer from increased incidence of respiratory infections, asthma, other forms of chronic lung illness, and death (Day and Ryan, Pediatr Res 81: 210-213, 2017; Isayama et la., JAMA Pediatr 171:271-279, 2017). BPD is now understood as a longitudinal disease process influenced by the intrauterine environment during gestation and modulated by gene-environment interactions throughout the neonatal and early childhood periods. Despite of this concept, there remains a paucity of multidisciplinary team-based approaches dedicated to the comprehensive study of this complex disease., Methods: The Discovery BPD (D-BPD) Program involves a cohort of infants < 1,250 g at birth prospectively followed until 6 years of age. The program integrates analysis of detailed clinical data by machine learning, genetic susceptibility and molecular translation studies., Discussion: The current gap in understanding BPD as a complex multi-trait spectrum of different disease endotypes will be addressed by a bedside-to-bench and bench-to-bedside approach in the D-BPD program. The D-BPD will provide enhanced understanding of mechanisms, evolution and consequences of lung diseases in preterm infants. The D-BPD program represents a unique opportunity to combine the expertise of biologists, neonatologists, pulmonologists, geneticists and biostatisticians to examine the disease process from multiple perspectives with a singular goal of improving outcomes of premature infants., Trial Registration: Does not apply for this study.

Background: Aurothioglucose- (ATG-) mediated inhibition of thioredoxin reductase-1 (TXNRD1) improves alveolarization in experimental murine bronchopulmonary dysplasia (BPD). Glutathione (GSH) mediates susceptibility to neonatal and adult oxidative lung injury. We have previously shown that ATG attenuates hyperoxic lung injury and enhances glutathione- (GSH-) dependent antioxidant defenses in adult mice., Hypothesis: The present studies evaluated the effects of TXNRD1 inhibition on GSH-dependent antioxidant defenses in newborn mice in vivo and lung epithelia in vitro ., Methods: Newborn mice received intraperitoneal ATG or saline prior to room air or 85% hyperoxia exposure. Glutamate-cysteine ligase (GCL) catalytic (Gclc) and modifier (Gclm) mRNA levels, total GSH levels, total GSH peroxidase (GPx) activity, and Gpx2 expression were determined in lung homogenates. In vitro , murine transformed club cells (mtCCs) were treated with the TXNRD1 inhibitor auranofin (AFN) or vehicle in the presence or absence of the GCL inhibitor buthionine sulfoximine (BSO)., Results: In vivo , ATG enhanced hyperoxia-induced increases in Gclc mRNA levels, total GSH contents, and GPx activity. In vitro , AFN increased Gclm mRNA levels, intracellular and extracellular GSH levels, and GPx activity. BSO prevented AFN-induced increases in GSH levels., Conclusions: Our data are consistent with a model in which TXNRD1 inhibition augments hyperoxia-induced GSH-dependent antioxidant responses in neonatal mice. Discrepancies between in vivo and in vitro results highlight the need for methodologies that permit accurate assessments of the GSH system at the single-cell level.

Background: Hyperoxia is a frequently employed therapy for prematurely born infants, induces lung injury and contributes to development of bronchopulmonary dysplasia (BPD). BPD is characterized by decreased cellular proliferation, cellular migration, and failure of injury repair systems. Actin binding proteins (ABPs) such as VASP, cofilin1, and profilin1 regulate cell proliferation and migration via modulation of actin dynamics. Lung mesenchymal stem cells (L-MSCs) initiate repair processes by proliferating, migrating, and localizing to sites of injury. These processes have not been extensively explored in hyperoxia induced lung injury and repair., Methods: ABPs and CD146 + L-MSCs were analyzed by immunofluorescence in human lung autopsy tissues from infants with and without BPD and by western blot in lung tissue homogenates obtained from our murine model of newborn hyperoxic lung injury., Results: Decreased F-actin content, ratio of VASP pS157/VASPpS239 , and profilin 1 expression were observed in human lung tissues but this same pattern was not observed in lungs from hyperoxia-exposed newborn mice. Increases in cofilin1 expression were observed in both human and mouse tissues at 7d indicating a dysregulation in actin dynamics which may be related to altered growth. CD146 levels were elevated in human and newborn mice tissues (7d)., Conclusion: Altered phosphorylation of VASP and expression of profilin 1 and cofilin 1 in human tissues indicate that the pathophysiology of BPD involves dysregulation of actin binding proteins. Lack of similar changes in a mouse model of hyperoxia exposure imply that disruption in actin binding protein expression may be linked to interventions or morbidities other than hyperoxia alone.

The trace element selenium (Se) contributes to redox signaling, antioxidant defense, and immune responses in critically ill neonatal and adult patients. Se is required for the synthesis and function of selenoenzymes including thioredoxin (Trx) reductase-1 (TXNRD1) and glutathione peroxidases (GPx). We have previously identified TXNRD1, primarily expressed by airway epithelia, as a promising therapeutic target to prevent lung injury, likely via nuclear factor E2-related factor 2 (Nrf2)-dependent mechanisms. The present studies utilized the TXNRD1 inhibitor auranofin (AFN) to test the hypothesis that Se positively influences Nrf2 activation and selenoenzyme responses in lung epithelial cells. Murine transformed Club cells (mtCCs) were supplemented with 0, 10, 25, or 100 nM Na 2 SeO 3 to create a range of Se conditions and were cultured in the presence or absence of 0.5 μM AFN. TXNRD1 and GPX2 protein expression and enzymatic activity were significantly greater upon Se supplementation (p < 0.05). AFN treatment (0.5 μM AFN for 1 h) significantly inhibited TXNRD1 but not GPx activity (p < 0.001). Recovery of TXNRD1 activity following AFN treatment was significantly enhanced by Se supplementation (p < 0.041). Finally, AFN-induced Nrf2 transcriptional activation was significantly greater in mtCCs supplemented in 25 or 100 nM Na 2 SeO 3 when compared to non-supplemented controls (p < 0.05). Our novel studies indicate that Se levels positively influence Nrf2 activation and selenoenzyme responses following TXNRD1 inhibition. These data suggest that Se status significantly influences physiologic responses to TXNRD1 inhibitors. In conclusion, correction of clinical Se deficiency, if present, will be necessary for optimal therapeutic effectiveness of TXNRD1 inhibitors in the prevention of lung disease., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Thioredoxin reductase-1 (TXNRD1) inhibition effectively activates nuclear factor (erythroid-derived 2)-like 2 (Nrf2) responses and attenuates lung injury in acute respiratory distress syndrome (ARDS) and bronchopulmonary dysplasia (BPD) models. Upon TXNRD1 inhibition, heme oxygenase-1 (HO-1) is disproportionally increased compared with Nrf2 target NADPH quinone oxidoreductase-1 (Nqo1). HO-1 has been investigated as a potential therapeutic target in both ARDS and BPD. TXNRD1 is predominantly expressed in airway epithelial cells; however, the mechanism of HO-1 induction by TXNRD1 inhibitors is unknown. We tested the hypothesis that TXNRD1 inhibition induces HO-1 via Nrf2-dependent mechanisms. Wild-type (WT), Nrf2 KO1.3 , and Nrf2 KO2.2 cells were morphologically indistinguishable, indicating that Nrf2 can be deleted from murine-transformed club cells (mtCCs) using CRISPR/Cas9 gene editing. Hemin, a Nrf2-independent HO-1-inducing agent, significantly increased HO-1 expression in WT, Nrf2 KO1.3 , and Nrf2 KO2.2 . Auranofin (AFN) (0.5 µM) inhibited TXNRD1 activity by 50% and increased Nqo1 and Hmox1 mRNA levels by 6- and 24-fold, respectively, in WT cells. Despite similar levels of TXNRD1 inhibition, Nqo1 mRNA levels were not different between control and AFN-treated Nrf2 KO1.3 and Nrf2 KO2.2 . AFN slightly increased Hmox1 mRNA levels in Nrf2 KO1.3 and Nrf2 KO2.2 cells compared with controls. AFN failed to increase HO-1 protein in Nrf2 KO1.3 and Nrf2 KO2.2 compared with a 36-fold increase in WT mtCCs. Our data indicate that Nrf2 is the primary mechanism by which TXNRD1 inhibitors increase HO-1 in lung epithelia. Future studies will use ARDS and BPD models to define the role of HO-1 in attenuation of lung injury by TXNRD1 inhibitors.

Dinitrosyl iron complexes (DNICs) are important intermediates in the metabolism of nitric oxide (NO). They have been considered to be NO storage adducts able to release NO, scavengers of excess NO during inflammatory hypotensive shock, and mediators of apoptosis in cancer cells, among many other functions. Currently, all studies of DNICs in biological matrices use electron paramagnetic resonance (EPR) for both detection and quantification. EPR is limited, however, by its ability to detect only paramagnetic mononuclear DNICs even though EPR-silent binuclear are likely to be prevalent. Furthermore, physiological concentrations of mononuclear DNICs are usually lower than the EPR detection limit (1 μM). We have thus developed a chemiluminescence-based method for the selective detection of both DNIC forms at physiological, pathophysiological, and pharmacologic conditions. We have also demonstrated the use of the new method in detecting DNIC formation in the presence of nitrite and nitrosothiols within biological fluids and tissue. This new method, which can be used alone or in tandem with EPR, has the potential to offer insight about the involvement of DNICs in many NO-dependent pathways., (Copyright © 2018. Published by Elsevier Inc.)

Glutathione-liganded binuclear dinitrosyl iron complex (glut-BDNIC) has been proposed to be a donor of nitric oxide (NO). This study was undertaken to investigate the mechanisms of vasoactivity, systemic hemodynamic effects, and pharmacokinetics of glut-BDNIC. To test the hypothesis that glut-BDNICs vasodilate by releasing NO in its reduced [nitroxyl (HNO)] state, a bioassay method of isolated, preconstricted ovine mesenteric arterial rings was used in the presence of selective scavengers of HNO or NO free radical (NO • ); the vasodilatory effects of glut-BDNIC were found to have characteristics similar to those of an HNO donor and markedly different than an NO • donor. In addition, products of the reaction of glut-BDNIC with CPTIO [2-(4-carboxyphenyl)-4,4,5-tetramethyl imidazoline-1-oxyl-3-oxide] were found to have electron paramagnetic characteristics similar to those of an HNO donor compared with an NO • donor. In contrast to S -nitroso-glutathione, which was vasodilative both in vitro and in vivo, the potency of glut-BDNIC-mediated vasodilation was markedly diminished in both rats and sheep. Wire myography showed that plasma albumin contributed to this loss of hypotensive effects, an effect abolished by modification of the cysteine-thiol residue of albumin. High doses of glut-BDNIC caused long-lasting hypotension in rats that can be at least partially attributed to its long circulating half-life of ∼44 minutes. This study suggests that glut-BDNIC is an HNO donor, and that its vasoactive effects are modulated by binding to the cysteine residue of plasma proteins, such as albumin., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

We previously showed that the thioredoxin reductase-1 (TrxR1) inhibitor aurothioglucose (ATG) improves alveolarization in hyperoxia-exposed newborn C3H/HeN mice. Our data supported a mechanism by which the protective effects of ATG are mediated via sustained nuclear factor E2-related factor 2 (Nrf2) activation in hyperoxia-exposed C3H/HeN mice 72 h after ATG administration. Given that inbred mouse strains have differential sensitivity and endogenous Nrf2 activation by hyperoxia, the present studies utilized two C57BL/6 exposure models to evaluate the effects of ATG on lung development and Nrf2 activation. The first model (0-14 days) was used in our C3H/HeN studies and the 2nd model (4-14 days) is well characterized in C57BL/6 mice. ATG significantly inhibited lung TrxR1 activity in both models; however, there was no effect on parameters of alveolarization in C57BL/6 mice. In sharp contrast to C3H/HeN mice, there was no effect of ATG on pulmonary NADPH quinone oxidoreductase-1 ( Nqo1) and heme oxygenase-1 ( Hmox1) at 72 h in either C57BL/6 model. In conclusion, although ATG inhibited TrxR1 activity in the lungs of newborn C57BL/6 mice, effects on lung development and sustained Nrf2-dependent pulmonary responses were blunted. These findings also highlight the importance of strain-dependent hyperoxic sensitivity in evaluation of potential novel therapies.

Nitrite and S-nitrosothiols (SNOs) are both byproducts of nitric oxide (NO) metabolism and are proposed to cause vasodilation via activation of soluble guanylate cyclase (sGC). We have previously reported that while SNOs are potent vasodilators at physiological concentrations, nitrite itself only produces vasodilation at supraphysiological concentrations. Here, we tested the hypothesis that sub-vasoactive concentrations of nitrite potentiate the vasodilatory effects of SNOs. Multiple exposures of isolated sheep arteries to S-nitroso-glutathione (GSNO) resulted in a tachyphylactic decreased vasodilatory response to GSNO but not to NO, suggesting attenuation of signaling steps upstream from sGC. Exposure of arteries to 1 μM nitrite potentiated the vasodilatory effects of GSNO in naive arteries and abrogated the tachyphylactic response to GSNO in pre-exposed arteries, suggesting that nitrite facilitates GSNO-mediated activation of sGC. In intact anesthetized sheep and rats, inhibition of NO synthases to decrease plasma nitrite levels attenuated vasodilatory responses to exogenous infusions of GSNO, an effect that was reversed by exogenous infusion of nitrite at sub-vasodilating levels. This study suggests nitrite potentiates SNO-mediated vasodilation via a mechanism that lies upstream from activation of sGC., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Even with advances in the care of preterm infants, chronic lung disease or bronchopulmonary dysplasia (BPD) continues to be a significant pulmonary complication. Among those diagnosed with BPD, a subset of infants develop severe BPD with disproportionate pulmonary morbidities. In addition to decreased alveolarization, these infants develop obstructive and/or restrictive lung function due to increases in or dysregulation of extracellular matrix proteins. Analyses of plasma obtained from preterm infants during the first week of life indicate that circulating miR-29b is suppressed in infants that subsequently develop BPD and that decreased circulating miR-29b is inversely correlated with BPD severity. Our mouse model mimics the pathophysiology observed in infants with severe BPD, and we have previously reported decreased pulmonary miR-29b expression in this model. The current studies tested the hypothesis that adeno-associated 9 (AAV9)-mediated restoration of miR-29b in the developing lung will improve lung alveolarization and minimize the deleterious changes in matrix deposition. Pregnant C3H/HeN mice received an intraperitoneal LPS injection on embryonic day 16 and newborn pups were exposed to 85% oxygen from birth to 14 days of life. On postnatal day 3 , AAV9-miR-29b or AAV9-control was administered intranasally. Mouse lung tissues were then analyzed for changes in miR-29 expression, alveolarization, and matrix protein levels and localization. Although only modest improvements in alveolarization were detected in the AAV9-miR29b-treated mice at postnatal day 28 , treatment completely attenuated defects in matrix protein expression and localization. Our data suggest that miR-29b restoration may be one component of a novel therapeutic strategy to treat or prevent severe BPD in prematurely born infants., (Copyright © 2017 the American Physiological Society.)

Maternal obesity induces chronic inflammatory responses that impact the fetus/neonate during the perinatal period. Inflammation, iron regulation, and myelination are closely interconnected and disruptions in these processes may have deleterious effects on neurodevelopment. Hepcidin levels are increased in response to inflammation causing subsequent decreases in ferroportin and available iron needed for myelination. Our current studies were designed to test the hypotheses that: 1) maternal high fat diet (HFD) prior to and during pregnancy is sufficient to induce inflammation and alter iron regulation in the brain of the offspring, and 2) HFD exposure is associated with altered myelination and neurobehavioral deficits in the offspring. Our data revealed modest increases in inflammatory cytokines in the serum of dams fed HFD prior to pregnancy compared to dams fed a control diet (CD). Early increases in IL-5 and decreases in IL-10 were observed in serum at PN7 while IL-5 remained elevated at PN21 in the HFD-exposed pups. At PN0, most cytokine levels in whole brain homogenates were higher in the pups born to HFD-fed dams but were not different or were lower than in pups born to CD-fed dams at PN21. Conversely, the inflammation mediated transcription factor Nurr77 remained elevated at PN21. At birth, brain hepcidin, ferroportin, and l-ferritin levels were elevated in pups born to HFD-fed dams compared to pups born to CD-fed dams. Hepcidin levels remained elevated at PN7 and PN21 while ferroportin and l-ferritin levels were lower at PN7 and were not different at PN21. Decreases in myelination in the medial cortex were observed in male but not in female pups born to maternal HFD-fed dams at PN21. These structural changes correlated with changes in behavior (novel object recognition) in at 4months in males only. Our data indicate that maternal obesity (HFD) results in disruption of iron regulation in the brains of the offspring with structural and neurobehavioral deficits in males., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We previously demonstrated that decreased miR-17∼92 cluster expression was 1) present in lungs from human infants who died with bronchopulmonary dysplasia (BPD); 2) inversely correlated with DNA methyltransferase (DNMT) expression and promoter methylation; and 3) correlated with a subsequent diagnosis of BPD at 36 wk gestational age. We tested the hypothesis that plasma miR-17 levels would be lowest in infants who ultimately develop severe BPD. Secondly, we utilized our well-characterized murine model of severe BPD that combines perinatal inflammation with postnatal hyperoxia to test the hypothesis that alterations in lung miR-17∼92, DNMT, and promoter methylation in our model would mirror our findings in tissues from premature human infants. Plasma was obtained during the first 5 days of life from premature infants born ≤32 wk gestation. Lung tissues were harvested from mice exposed to maternal inflammation and neonatal hyperoxia for 14 days after birth. miR-17∼92 cluster expression and DNA methyltransferase expression were measured by qRT-PCR, and promoter methylation was assessed by Methyl-Profiler assay. Plasma miR-17 levels are significantly lower in the first week of life in human infants who develop severe BPD compared with mild or moderate BPD. Data from our severe BPD murine model reveal that lung miR-17∼92 cluster expression is significantly attenuated, and levels inversely correlated with DNMT expression and miR-17∼92 cluster promoter methylation. Collectively, our data support a plausible role for epigenetically altered miR-17∼92 cluster in the pathogenesis of severe BPD., (Copyright © 2016 the American Physiological Society.)

Oxygen toxicity and antioxidant deficiencies contribute to the development of bronchopulmonary dysplasia. Aurothioglucose (ATG) and auranofin potently inhibit thioredoxin reductase-1 (TrxR1), and TrxR1 disruption activates nuclear factor E2-related factor 2 (Nrf2), a regulator of endogenous antioxidant responses. We have shown previously that ATG safely and effectively prevents lung injury in adult murine models, likely via Nrf2-dependent mechanisms. The current studies tested the hypothesis that ATG would attenuate hyperoxia-induced lung developmental deficits in newborn mice. Newborn C3H/HeN mice were treated with a single dose of ATG or saline within 12 hours of birth and were exposed to either room air or hyperoxia (85% O2). In hyperoxia, ATG potently inhibited TrxR1 activity in newborn murine lungs, attenuated decreases in body weight, increased the transcription of Nrf2-regulated genes nicotinamide adenine dinucleotide phosphate reduced quinone oxidoreductase-1 (NQO1) and heme oxygenase 1, and attenuated alterations in alveolar development. To determine the impact of TrxR1 inhibition on Nrf2 activation in vitro, murine alveolar epithelial-12 cells were treated with auranofin, which inhibited TrxR1 activity, enhanced Nrf2 nuclear levels, and increased NQO1 and heme oxygenase 1 transcription. Our novel data indicate that a single injection of the TrxR1 inhibitor ATG attenuates hyperoxia-induced alterations in alveolar development in newborn mice. Furthermore, our data support a model in which the effects of ATG treatment likely involve Nrf2 activation, which is consistent with our findings in other lung injury models. We conclude that TrxR1 represents a novel therapeutic target to prevent oxygen-mediated neonatal lung injury.

Rationale: Bronchopulmonary dysplasia remains a significant cause of neonatal morbidity; however, the identification of novel targets to predict or prevent the development of bronchopulmonary dysplasia remains elusive. Proper microRNA (miR)-17∼92 cluster is necessary for normal lung development, and alterations in expression are reported in other pulmonary diseases. The overall hypothesis for our work is that altered miR-17∼92 cluster expression contributes to the molecular pathogenesis of bronchopulmonary dysplasia., Objectives: The current studies tested the hypothesis that alterations in miR-17∼92 cluster and DNA methyltransferase expression are present in bronchopulmonary dysplasia., Methods: miR-17∼92 cluster expression, promoter methylation, and DNA methyltransferase expression were determined in autopsy lung samples obtained from premature infants who died with bronchopulmonary dysplasia, or from term/near-term infants who died from nonrespiratory causes. Expression of miR-17∼92 cluster members miR-17 and -19b was measured in plasma samples collected in the first week of life from a separate cohort of preterm infants at a second institution in whom bronchopulmonary dysplasia was diagnosed subsequently., Measurements and Main Results: Autopsy tissue data indicated that miR-17∼92 expression is significantly lower in bronchopulmonary dysplasia lungs and is inversely correlated with promoter methylation and DNA methyltransferase expression when compared with that of control subjects without bronchopulmonary dysplasia. Plasma sample analyses indicated that miR-17 and -19b expression was decreased in infants who subsequently developed bronchopulmonary dysplasia., Conclusions: Our data are the first to demonstrate altered expression of the miR-17∼92 cluster in bronchopulmonary dysplasia. The consistency between our autopsy and plasma findings further support our working hypothesis that the miR-17∼92 cluster contributes to the molecular pathogenesis of bronchopulmonary dysplasia.

We have previously shown that an adverse perinatal environment significantly alters lung growth and development and results in persistently altered cardiopulmonary physiology in adulthood. Our model of maternal LPS treatment followed by 14 days of neonatal hyperoxia exposure causes severe pulmonary disease characterized by permanent decreases in alveolarization and diffuse interstitial fibrosis. The current investigations tested the hypothesis that dysregulation of Notch signaling pathways contributes to the permanently altered lung phenotype in our model and that the improvements we have observed previously with maternal docosahexaenoic acid (DHA) supplementation are mediated through normalization of Notch-related protein expression. Results indicated that inflammation (IL-6 levels) and oxidation (F2a-isoprostanes) persisted through 8 wk of life in mice exposed to LPS/O2 perinatally. These changes were attenuated by maternal DHA supplementation. Modest but inconsistent differences were observed in Notch-pathway proteins Jagged 1, DLL 1, PEN2, and presenilin-2. We detected substantial increases in markers of apoptosis including PARP-1, APAF-1, caspase-9, BCL2, and HMGB1, and these increases were attenuated in mice that were nursed by DHA-supplemented dams during the perinatal period. Although Notch signaling is not significantly altered at 8 wk of age in mice with perinatal exposure to LPS/O2, our findings indicate that persistent apoptosis continues to occur at 8 wk of age. We speculate that ongoing apoptosis may contribute to persistently altered lung development and may further enhance susceptibility to additional pulmonary disease. Finally, we found that maternal DHA supplementation prevented sustained inflammation, oxidation, and apoptosis in our model., (Copyright © 2015 the American Physiological Society.)

Significance: Fetal lung development takes place in hypoxia meaning that premature birth is hyperoxia for the prematurely born infant. The most common respiratory morbidity afflicting premature infants is bronchopulmonary dysplasia (BPD). Pathophysiologically, BPD represents the impact of injury, including O2 toxicity, to the immature developing lung that causes arrested lung development., Recent Advances: The thioredoxin (Trx) system, which is predominantly expressed in pulmonary epithelia in the newborn lung, acts as an antioxidant system; however, it is increasingly recognized as a key redox regulator of signal transduction and gene expression via thiol-disulfide exchange reactions., Critical Issues: This review focuses on the contribution of Trx family proteins toward normal and aberrant lung development, in particular, the roles of the Trx system in hyperoxic responses of alveolar epithelial cells, aberrant lung development in animal models of BPD, O2-dependent signaling processes, and possible therapeutic efficacy in preventing O2-mediated lung injury., Future Directions: The significant contribution of the Trx system toward redox regulation of key developmental pathways necessary for proper lung development suggests that therapeutic strategies focused on preserving pulmonary Trx function could significantly improve the outcomes of prematurely born human infants.

Vascular remodeling and smooth muscle cell proliferation are hallmark pathogenic features of pulmonary artery hypertension. MicroRNAs, endogenously expressed small noncoding RNAs, regulate gene expression at the posttranscriptional level. It has previously been shown that miR-17 overexpression in cultured human pulmonary artery smooth muscle cell (hPASMC) resulted in increased viable cell number. Previously, we have found that arginase II promotes hypoxia-induced proliferation in hPASMC. Therefore, we hypothesized that miR-17 would be upregulated by hypoxia in hPASMC and would result in greater arginase II expression. We found that levels of miR-17-5p and arginase II were significantly greater in cultured hPASMC exposed to 1% O2 for 48 h than in hPASMC exposed to 21% O2 for 48 h. Furthermore, inhibiting miR-17-5p expression decreased hypoxia-induced arginase II protein levels in hPASMC. Conversely, overexpressing miR-17-5p resulted in greater arginase II protein levels. Somewhat surprisingly, arginase II inhibition was associated with lower miR-17-5p expression in both normoxic and hypoxic hPASMC, whereas overexpressing arginase II resulted in greater miR-17-5p expression in hPASMC. These findings suggest that hypoxia-induced arginase II expression is not only regulated by miR-17-5p but also that there is a feedback loop between arginase II and miR-17-5p in hPASMC. We also found that the arginase II-mediated regulation of miR-17-5p was independent of either p53 or c-myc. We also found that l-arginine, the substrate for arginase II, and l-ornithine, the amino acid product of arginase II, were not involved in the regulation of miR-17-5p expression., (Copyright © 2014 the American Physiological Society.)

Aims: Inflammation and oxygen toxicity increase free radical production and contribute to the development of acute respiratory distress syndrome (ARDS), which is a significant cause of morbidity and mortality in intensive care patients. We have previously reported increased glutathione (GSH) levels in lung epithelial cells in vitro and attenuated adult murine hyperoxic lung injury in vivo after pharmacological thioredoxin reductase-1 (TrxR1) inhibition. Using a murine ARDS model, we tested the hypothesis that aurothioglucose (ATG) treatment increases pulmonary GSH levels, attenuates lung injury, and decreases mortality in a GSH-dependent manner., Results: Adult mice received a single intratracheal dose of 0.375 μg/g lipopolysaccharide (LPS) 12 h before a single intraperitoneal injection of 25 mg/kg ATG. Control mice received intratracheal and/or intraperitoneal saline. Mice were then exposed to room air or hyperoxia (>95% O2). Lung injury was assessed by bronchoalveolar lavage protein concentrations. Expression of glutamate-cysteine ligase modifier subunit (GCLM), GSH, cytokines, and chemokines was determined. Exposure to LPS/hyperoxia induced inflammation and lung injury. ATG treatment significantly attenuated lung injury, increased lung GCLM expression and GSH levels, and decreased mortality. GSH depletion completely prevented the protective effects of ATG in LPS/hyperoxia-exposed mice., Innovation: ATG treatment significantly attenuates lung injury and enhances survival in a clinically relevant murine model of ARDS. The protective effects of ATG are GSH dependent., Conclusion: Augmentation of GSH systems by TrxR1 inhibition could represent a promising therapeutic approach to attenuate oxidant-mediated lung injury and improve patient outcomes.

The preterm infant is often exposed to maternal and neonatal inflammatory stimuli and is born with immature lungs, resulting in a need for oxygen therapy. Nutritional intervention with docosahexaenoic acid (DHA; 6.3 g/kg of diet) has been shown to attenuate inflammation in various human diseases. Previous studies demonstrated that maternal DHA supplementation during late gestation and lactation attenuated hyperoxic lung injury in newborn mouse pups. In the present studies, we tested the hypothesis that DHA supplementation to the dam would reduce hyperoxic lung injury and growth deficits in a more severe model of systemic maternal inflammation, including lipopolysaccharide (LPS) and neonatal hyperoxia exposure. On embryonic day 16, dams were placed on DHA (6.3 g DHA/kg diet) or control diets and injected with saline or LPS. Diets were maintained through weaning. At birth, pups were placed in room air or hyperoxia for 14 d. Improvements in birth weight (P < 0.01), alveolarization (P ≤ 0.01), and pulmonary function (P ≤ 0.03) at 2 and 8 wk of age were observed in pups exposed to perinatal inflammation and born to DHA-supplemented dams compared with control diet-exposed pups. These improvements were associated with decreases in tissue macrophage numbers (P < 0.01), monocyte chemoattractant protein-1 expression (P ≤ 0.05), and decreases in soluble receptor for advanced glycation end products concentrations (P < 0.01) at 2 and 8 wk. Furthermore, DHA supplementation attenuated pulmonary fibrosis, which was associated with the reduction of matrix metalloproteinases 2, 3, and 8 (P ≤ 0.03) and collagen mRNA (P ≤ 0.05), and decreased collagen (P < 0.01) and vimentin (P ≤ 0.03) protein concentrations. In a model of severe inflammation, maternal DHA supplementation lessened inflammation and improved lung growth in the offspring. Maternal supplementation with DHA may be a therapeutic strategy to reduce neonatal inflammation.

Pathological pulmonary artery smooth muscle cell (PASMC) proliferation contributes to pulmonary vascular remodeling in pulmonary hypertensive diseases associated with hypoxia. Both the hypoxia-inducible factor (HIF) and phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt) pathways have been implicated in hypoxia-induced PASMC proliferation. Thioredoxin-1 (Trx1) is a ubiquitously expressed protein that is involved in redox-dependent signaling via HIF and PI3K-Akt in cancer. The role of Trx1 in PASMC proliferation has not been elucidated. The present studies tested the hypothesis that Trx1 regulates hypoxia-induced PASMC proliferation via HIF and/or PI3K- and Akt-dependent mechanisms. Following exposure to chronic hypoxia, our data indicate that Trx1 activity is increased in adult murine lungs. Furthermore, hypoxia-induced increases in cellular proliferation are correlated with increased Trx1 expression, HIF activation, and Akt activation in cultured human PASMC. Both small-interfering RNA-mediated knockdown and pharmacological Trx1 inhibition attenuated hypoxia-induced PASMC proliferation, HIF activation, and Akt activation. While Trx1 knockdown suppressed hypoxia-induced PI3K-Akt activation in PASMC, PI3K-Akt inhibition prevented hypoxia-induced proliferation but had no effect on hypoxia-induced increases in Trx1 or HIF activation. Thus, our findings indicate that Trx1 contributes to hypoxia-induced PASMC proliferation by modulating HIF activation and subsequent PI3K-Akt activation. These novel data suggest that Trx1 might represent a novel therapeutic target to prevent hypoxic PASMC proliferation.

Supraphysiological O2 concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2(+/+)), heterozygous (COX-2(+/-)), and homozygous (COX-2(-/-)) transgenic mice.Micewere exposed to room air (21% O2) or hyperoxia (85% O2) for 14 days.Aspirin-injected and COX-2(-/-) pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D2 metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Altered functions of the lung epithelial surface likely contribute to the respiratory morbidities in infants with bronchopulmonary dysplasia (BPD). Infants with BPD exhibit decreased expressions of secretoglobins (SCGBs), including Clara cell secretory protein (CCSP). Expression of lung SCGB and annexin A1 (ANXA1) is persistently altered in CCSP knockout mice suggesting that CCSP indirectly influences innate immune responses. The present studies tested the hypothesis that neonatal hyperoxic exposure induces deficits in CCSP expression that are associated with persistent alterations in lung SCGB and ANXA1 expression. Newborn C3H/HeN mice were exposed to room air (RA) or 85% O2 from birth and were sacrificed at 14 d or returned to RA for 14 d. Neonatal hyperoxia followed by RA recovery was associated with decreased lung CCSP and SCGB3A1 protein but not mRNA expression. Hyperoxia-induced alterations in the charge characteristics of ANXA1 were unchanged by RA recovery and were associated with elevated lung macrophage numbers. These findings support a model in which hyperoxia-induced alterations in Clara cell function influence lung innate immune function through effects on immunomodulatory proteins. Studies to determine the mechanism(s) by which CCSP alterations affect SCGBs, ANXA1, and innate immune responses in BPD are warranted.