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Rationale: Injurious agents often cause less severe injury in neonates as compared with adults.Objective: We hypothesized that maturational differences in lung inflammation induced by lipopolysaccharide (LPS) may be related to the nature of the nuclear factor (NF)-kappaB complex activated, and the profile of target genes expressed.Methods: Neonatal and adult mice were injected with intraperitoneal LPS. Lung inflammation was assessed by histology, and apoptosis was determined by TUNEL (terminal deoxynucleotidyl transferase UTP nick-end labeling). The expression of candidate inflammatory and apoptotic mediators was evaluated by quantitative real-time polymerase chain reaction and Western immunoblot.Results: Neonates demonstrated reduced inflammation and apoptosis, 24 hours after LPS exposure, as compared with adults. This difference was associated with persistent activation of NF-kappaB p65p50 heterodimers in the neonates in contrast to early, transient activation of p65p50 followed by sustained activation of p50p50 in the adults. Adults had increased expression of a panel of inflammatory and proapoptotic genes, and repression of antiapoptotic targets, whereas no significant changes in these mediators were observed in the neonates. Inhibition of NF-kappaB activity in the neonates decreased apoptosis, but heightened inflammation, with increased expression of the same inflammatory genes elevated in the adults. In contrast, inhibition of NF-kappaB in the adults resulted in partial suppression of the inflammatory response.Conclusions: NF-kappaB activation in the neonatal lung is antiinflammatory, protecting against LPS-mediated lung inflammation by repressing similar inflammatory genes induced in the adult. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to examine the association of cardiorespiratory events, including apnea, periodic breathing, intermittent hypoxemia (IH), and bradycardia, with late-onset sepsis for extremely preterm infants (<29 weeks of gestational age) on vs off invasive mechanical ventilation., Study Design: This is a retrospective analysis of data from infants enrolled in Pre-Vent (ClinicalTrials.gov identifier NCT03174301), an observational study in 5 level IV neonatal intensive care units. Clinical data were analyzed for 737 infants (mean gestational age: 26.4 weeks, SD 1.71). Monitoring data were available and analyzed for 719 infants (47 512 patient-days); of whom, 109 had 123 sepsis events. Using continuous monitoring data, we quantified apnea, periodic breathing, bradycardia, and IH. We analyzed the relationships between these daily measures and late-onset sepsis (positive blood culture >72 hours after birth and ≥5-day antibiotics)., Results: For infants not on a ventilator, apnea, periodic breathing, and bradycardia increased before sepsis diagnosis. During times on a ventilator, increased sepsis risk was associated with longer events with oxygen saturation <80% (IH80) and more bradycardia events before sepsis. IH events were associated with higher sepsis risk but did not dynamically increase before sepsis, regardless of ventilator status. A multivariable model including postmenstrual age, cardiorespiratory variables (apnea, periodic breathing, IH80, and bradycardia), and ventilator status predicted sepsis with an area under the receiver operator characteristic curve of 0.783., Conclusion: We identified cardiorespiratory signatures of late-onset sepsis. Longer IH events were associated with increased sepsis risk but did not change temporally near diagnosis. Increases in bradycardia, apnea, and periodic breathing preceded the clinical diagnosis of sepsis., Competing Interests: Declaration of Competing Interest Some authors have financial conflicts of interest. JRM and DEL own stock in Medical Prediction Sciences Corporation. JRM is a consultant for Nihon Kohden Digital Health Solutions, proceeds donated to the University of Virginia. ZAV is a consultant for Medtronic. All other authors have no financial conflicts to disclose. No authors have any nonfinancial conflicts of interest to disclose. Funding Support: We acknowledge the following NIH grants for funding the work presented in this manuscript. University of Virginia (NCT03174301): U01 HL133708, K23 HD097254, HL133708-05S1. Case Western Reserve University: U01 HL133643. Northwestern University: U01 HL133704. University of Alabama at Birmingham: U01 HL133536, K23 HL157618. University of Miami: U01 HL133689. Washington University: U01 HL133700, F., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants characterized by alveolar dysplasia, vascular simplification and dysmorphic vascular development. Supplemental oxygen and mechanical ventilation commonly used as life-saving measures in premature infants may cause BPD. microRNAs (miRNAs), a class of small, non-coding RNAs, regulate target gene expression mainly through post-transcriptional repression. miRNAs play important roles in modulating oxidative stress, proliferation, apoptosis, senescence, inflammatory responses, and angiogenesis. These cellular processes play pivotal roles in the pathogenesis of BPD. Accumulating evidence demonstrates that miRNAs are dysregulated in the lung of premature infants with BPD, and in animal models of this disease, suggesting contributing roles of dysregulated miRNAs in the development of BPD. Therefore, miRNAs are considered promising biomarker candidates and therapeutic agents for this disease. In this review, we discuss how dysregulated miRNAs and their modulation alter cellular processes involved in BPD. We then focus on therapeutic approaches targeting miRNAs for BPD. This review provides an overview of miRNAs as biomarkers, and highlights potential pathogenic roles, and therapeutic strategies for BPD using miRNAs., Competing Interests: The authors declare no conflict of interest. Given the role as Editorial Board Member, Hongwei Yao had no involvement in the peer-review of this article and has no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Graham Pawelec., (© 2024 The Author(s). Published by IMR Press.)

Objective. Highly comparative time series analysis (HCTSA) is a novel approach involving massive feature extraction using publicly available code from many disciplines. The Prematurity-Related Ventilatory Control (Pre-Vent) observational multicenter prospective study collected bedside monitor data from>700extremely preterm infants to identify physiologic features that predict respiratory outcomes. Approach . We calculated a subset of 33 HCTSA features on>7 M 10 min windows of oxygen saturation (SPO2) and heart rate (HR) from the Pre-Vent cohort to quantify predictive performance. This subset included representatives previously identified using unsupervised clustering on>3500HCTSA algorithms. We hypothesized that the best HCTSA algorithms would compare favorably to optimal PreVent physiologic predictor IH90&#95;DPE (duration per event of intermittent hypoxemia events below 90%). Main Results. The top HCTSA features were from a cluster of algorithms associated with the autocorrelation of SPO2 time series and identified low frequency patterns of desaturation as high risk. These features had comparable performance to and were highly correlated with IH90&#95;DPE but perhaps measure the physiologic status of an infant in a more robust way that warrants further investigation. The top HR HCTSA features were symbolic transformation measures that had previously been identified as strong predictors of neonatal mortality. HR metrics were only important predictors at early days of life which was likely due to the larger proportion of infants whose outcome was death by any cause. A simple HCTSA model using 3 top features outperformed IH90&#95;DPE at day of life 7 (.778 versus .729) but was essentially equivalent at day of life 28 (.849 versus .850). Significance . These results validated the utility of a representative HCTSA approach but also provides additional evidence supporting IH90&#95;DPE as an optimal predictor of respiratory outcomes., (Creative Commons Attribution license.)

Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest.

Background: In extremely preterm infants, persistence of cardioventilatory events is associated with long-term morbidity. Therefore, the objective was to characterize physiologic growth curves of apnea, periodic breathing, intermittent hypoxemia, and bradycardia in extremely preterm infants during the first few months of life., Methods: The Prematurity-Related Ventilatory Control study included 717 preterm infants <29 weeks gestation. Waveforms were downloaded from bedside monitors with a novel sharing analytics strategy utilized to run software locally, with summary data sent to the Data Coordinating Center for compilation., Results: Apnea, periodic breathing, and intermittent hypoxemia events rose from day 3 of life then fell to near-resolution by 8-12 weeks of age. Apnea/intermittent hypoxemia were inversely correlated with gestational age, peaking at 3-4 weeks of age. Periodic breathing was positively correlated with gestational age peaking at 31-33 weeks postmenstrual age. Females had more periodic breathing but less intermittent hypoxemia/bradycardia. White infants had more apnea/periodic breathing/intermittent hypoxemia. Infants never receiving mechanical ventilation followed similar postnatal trajectories but with less apnea and intermittent hypoxemia, and more periodic breathing., Conclusions: Cardioventilatory events peak during the first month of life but the actual postnatal trajectory is dependent on the type of event, race, sex and use of mechanical ventilation., Impact: Physiologic curves of cardiorespiratory events in extremely preterm-born infants offer (1) objective measures to assess individual patient courses and (2) guides for research into control of ventilation, biomarkers and outcomes. Presented are updated maturational trajectories of apnea, periodic breathing, intermittent hypoxemia, and bradycardia in 717 infants born <29 weeks gestation from the multi-site NHLBI-funded Pre-Vent study. Cardioventilatory events peak during the first month of life but the actual postnatal trajectory is dependent on the type of event, race, sex and use of mechanical ventilation. Different time courses for apnea and periodic breathing suggest different maturational mechanisms., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Cellular senescence is a status of irreversible growth arrest, which can be triggered by the p53/p21 cip1 and p16 INK4 /Rb pathways via intrinsic and external factors. Senescent cells are typically enlarged and flattened, and characterized by numerous molecular features. The latter consists of increased surfaceome, increased residual lysosomal activity at pH 6.0 (manifested by increased activity of senescence-associated beta-galactosidase [SA- β -gal]), senescence-associated mitochondrial dysfunction, cytoplasmic chromatin fragment, nuclear lamin b1 exclusion, telomere-associated foci, and the senescence-associated secretory phenotype. These features vary depending on the stressor leading to senescence and the type of senescence. Cellular senescence plays pivotal roles in organismal aging and in the pathogenesis of aging-related diseases. Interestingly, senescence can also both promote and inhibit wound healing processes. We recently report that senescence as a programmed process contributes to normal lung development. Lung senescence is also observed in Down Syndrome, as well as in premature infants with bronchopulmonary dysplasia and in a hyperoxia-induced rodent model of this disease. Furthermore, this senescence results in neonatal lung injury. In this review, we briefly discuss the molecular features of senescence. We then focus on the emerging role of senescence in normal lung development and in the pathogenesis of bronchopulmonary dysplasia as well as putative signaling pathways driving senescence. Finally, we discuss potential therapeutic approaches targeting senescent cells to prevent perinatal lung diseases., Competing Interests: Declaration of competing interest Authors have declared that no conflict of interest exists.

Objectives: Mitochondrial dysfunction is implicated in the pathophysiology of psychiatric disorders. Levels of circulating cell-free mitochondrial DNA (cf-mtDNA) are observed to be altered in depression. However, the few studies that have measured cf-mtDNA in depression have reported conflicting findings. This study examined cf-mtDNA and depressive symptoms in low-active adults who smoke., Methods: Participants were adults 18 to 65 years old ( N = 109; 76% female) with low baseline physical activity and depressive symptoms recruited for a smoking cessation study. Self-report measures assessed depression severity, positive and negative affect, and behavioral activation. Blood was collected and analyzed for cf-mtDNA. Relationships between depressive symptoms and cf-mtDNA were examined with correlations and linear regression., Results: Levels of cf-mtDNA were associated with categorically defined depression (Center for Epidemiologic Studies Depression Scale score >15), lower positive affect, and decreased behavioral activation ( p < .05). Relationships remained significant after adjustment for age, sex, and nicotine dependence. In a linear regression model including all depressive symptom measures as predictors, Center for Epidemiologic Studies Depression Scale group and lower positive affect remained significant., Conclusions: This work suggests that mitochondrial changes are associated with depressive symptoms in low-active adults who smoke. Higher levels of cf-mtDNA in association with depression and with lower positive affect and decreased behavioral activation are consistent with a possible role for mitochondrial function in depressive symptoms., (Copyright © 2023 by the American Psychosomatic Society.)

Rationale: Immature control of breathing is associated with apnea, periodic breathing, intermittent hypoxemia, and bradycardia in extremely preterm infants. However, it is not clear if such events independently predict worse respiratory outcome. Objectives: To determine if analysis of cardiorespiratory monitoring data can predict unfavorable respiratory outcomes at 40 weeks postmenstrual age (PMA) and other outcomes, such as bronchopulmonary dysplasia at 36 weeks PMA. Methods: The Prematurity-related Ventilatory Control (Pre-Vent) study was an observational multicenter prospective cohort study including infants born at <29 weeks of gestation with continuous cardiorespiratory monitoring. The primary outcome was either "favorable" (alive and previously discharged or inpatient and off respiratory medications/O 2 /support at 40 wk PMA) or "unfavorable" (either deceased or inpatient/previously discharged on respiratory medications/O 2 /support at 40 wk PMA). Measurements and Main Results: A total of 717 infants were evaluated (median birth weight, 850 g; gestation, 26.4 wk), 53.7% of whom had a favorable outcome and 46.3% of whom had an unfavorable outcome. Physiologic data predicted unfavorable outcome, with accuracy improving with advancing age (area under the curve, 0.79 at Day 7, 0.85 at Day 28 and 32 wk PMA). The physiologic variable that contributed most to prediction was intermittent hypoxemia with oxygen saturation as measured by pulse oximetry <90%. Models with clinical data alone or combining physiologic and clinical data also had good accuracy, with areas under the curve of 0.84-0.85 at Days 7 and 14 and 0.86-0.88 at Day 28 and 32 weeks PMA. Intermittent hypoxemia with oxygen saturation as measured by pulse oximetry <80% was the major physiologic predictor of severe bronchopulmonary dysplasia and death or mechanical ventilation at 40 weeks PMA. Conclusions: Physiologic data are independently associated with unfavorable respiratory outcome in extremely preterm infants.

Senescence causes age-related diseases and stress-related injury. Paradoxically, it is also essential for organismal development. Whether senescence contributes to lung development or injury in early life remains unclear. Here, we show that lung senescence occurred at birth and decreased throughout the saccular stage in mice. Reducing senescent cells at this stage disrupted lung development. In mice (<12 h old) exposed to hyperoxia during the saccular stage followed by air recovery until adulthood, lung senescence increased particularly in type II cells and secondary crest myofibroblasts. This peaked during the alveolar stage and was mediated by the p53/p21 pathway. Decreasing senescent cells during the alveolar stage attenuated hyperoxia-induced alveolar and vascular simplification. Conclusively, early programmed senescence orchestrates postnatal lung development whereas later hyperoxia-induced senescence causes lung injury through different mechanisms. This defines the ontogeny of lung senescence and provides an optimal therapeutic window for mitigating neonatal hyperoxic lung injury by inhibiting senescence., (© 2023. The Author(s).)

Background: Premature infants, subjected to supplemental oxygen and mechanical ventilation, may develop bronchopulmonary dysplasia, a chronic lung disease characterized by alveolar dysplasia and impaired vascularization. We and others have shown that hyperoxia causes senescence in cultured lung epithelial cells and fibroblasts. Although miR-34a modulates senescence, it is unclear whether it contributes to hyperoxia-induced senescence. We hypothesized that hyperoxia increases miR-34a levels, leading to cellular senescence., Methods: We exposed mouse lung epithelial (MLE-12) cells and primary human small airway epithelial cells to hyperoxia (95% O 2 /5% CO 2 ) or air (21% O 2 /5% CO 2 ) for 24 h. Newborn mice (< 12 h old) were exposed to hyperoxia (> 95% O 2 ) for 3 days and allowed to recover in room air until postnatal day 7. Lung samples from premature human infants requiring mechanical ventilation and control subjects who were not mechanically ventilated were employed., Results: Hyperoxia caused senescence as indicated by loss of nuclear lamin B1, increased p21 gene expression, and senescence-associated secretory phenotype factors. Expression of miR-34a-5p was increased in epithelial cells and newborn mice exposed to hyperoxia, and in premature infants requiring mechanical ventilation. Transfection with a miR-34a-5p inhibitor reduced hyperoxia-induced senescence in MLE-12 cells. Additionally, hyperoxia increased protein levels of the oncogene and tumor-suppressor Krüppel-like factor 4 (KLF4), which were inhibited by a miR-34a-5p inhibitor. Furthermore, KLF4 knockdown by siRNA transfection reduced hyperoxia-induced senescence., Conclusion: Hyperoxia increases miR-34a-5p, leading to senescence in lung epithelial cells. This is dictated in part by upregulation of KLF4 signaling. Therefore, inhibiting hyperoxia-induced senescence via miR-34a-5p or KLF4 suppression may provide a novel therapeutic strategy to mitigate the detrimental consequences of hyperoxia in the neonatal lung., (© 2022. The Author(s).)

Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in degrading heme into biliverdin and iron. HO-1 can also enter the nucleus and regulate gene transcription independent of its enzymatic activity. Whether HO-1 can alter gene expression through direct binding to target DNA remains unclear. Here, we performed HO-1 CHIP-seq and then employed 3D structural modeling to reveal putative HO-1 DNA binding domains. We identified three probable DNA binding domains on HO-1. Using the Proteinarium, we identified several genes as the most highly connected nodes in the interactome among the HO-1 gene binding targets. We further demonstrated that HO-1 modulates the expression of these key genes using Hmox1 deficient cells. Finally, mutation of four conserved amino acids (E215, I211, E201, and Q27) within HO-1 DNA binding domain 1 significantly increased expression of Gtpbp3 and Eif1 genes that were identified within the top 10 binding hits normalized by gene length predicted to bind this domain. Based on these data, we conclude that HO-1 protein is a putative DNA binding protein, and regulates targeted gene expression. This provides the foundation for developing specific inhibitors or activators targeting HO-1 DNA binding domains to modulate targeted gene expression and corresponding cellular function.

Background and Aims: Cell-free DNA (cfDNA) is elevated in several disease states. Metabolic syndrome is a constellation of factors associated with poor cardiometabolic outcomes. This study examined associations of cfDNA from the nucleus (cf-nDNA) and mitochondria (cf-mtDNA), C-reactive protein (CRP), and metabolic syndrome risk, in low-active smokers with depressive symptoms., Methods: Participants ( N  = 109; mean age 47) self-reported medical history. Physical activity was determined by accelerometry and anthropometrics were measured. Blood was collected and analyzed for cf-nDNA, cf-mtDNA, CRP, triglycerides, high-density lipoprotein, hemoglobin A1c. A continuous metabolic syndrome composite risk score was calculated. Relationships of cf-nDNA, cf-mtDNA, CRP, and cardiometabolic risk were examined with correlations and linear regression., Results: CRP and cf-nDNA were significantly associated with metabolic syndrome risk ( r  = .39 and r =  .31, respectively), cf-mtDNA was not ( r  = .01). In a linear regression, CRP and cf-nDNA significantly predicted the metabolic syndrome risk score, findings that remained significant controlling for age, gender, nicotine dependence, and physical activity., Conclusions: Associations of cf-nDNA with both CRP and metabolic risk suggest a role for cf-nDNA in inflammatory processes associated with metabolic syndrome. The negative findings for cf-mtDNA suggest distinct roles for cf-nDNA and cf-mtDNA in these processes., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Background: Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants that may cause long-term lung dysfunction. Accumulating evidence supports the vascular hypothesis of BPD, in which lung endothelial cell dysfunction drives this disease. We recently reported that endothelial carnitine palmitoyltransferase 1a (Cpt1a) is reduced by hyperoxia, and that endothelial cell-specific Cpt1a knockout mice are more susceptible to developing hyperoxia-induced injury than wild type mice. Whether Cpt1a upregulation attenuates hyperoxia-induced endothelial cell dysfunction and lung injury remains unknown. We hypothesized that upregulation of Cpt1a by baicalin or L-carnitine ameliorates hyperoxia-induced endothelial cell dysfunction and persistent lung injury., Methods: Lung endothelial cells or newborn mice (< 12 h old) were treated with baicalin or L-carnitine after hyperoxia (50% and 95% O 2 ) followed by air recovery., Results: We found that incubation with L-carnitine (40 and 80 mg/L) and baicalin (22.5 and 45 mg/L) reduced hyperoxia-induced apoptosis, impaired cell migration and angiogenesis in cultured lung endothelial cells. This was associated with increased Cpt1a gene expression. In mice, neonatal hyperoxia caused persistent alveolar and vascular simplification in a concentration-dependent manner. Treatment with L-carnitine (150 and 300 mg/kg) and baicalin (50 and 100 mg/kg) attenuated neonatal hyperoxia-induced alveolar and vascular simplification in adult mice. These effects were diminished in endothelial cell-specific Cpt1a knockout mice., Conclusions: Upregulating Cpt1a by baicalin or L-carnitine ameliorates hyperoxia-induced lung endothelial cell dysfunction, and persistent alveolar and vascular simplification. These findings provide potential therapeutic avenues for using L-carnitine and baicalin as Cpt1a upregulators to prevent persistent lung injury in premature infants with BPD., (© 2022. The Author(s).)

Ventilatory support, such as supplemental oxygen, used to save premature infants impairs the growth of the pulmonary microvasculature and distal alveoli, leading to bronchopulmonary dysplasia (BPD). Although lung cellular composition changes with exposure to hyperoxia in neonatal mice, most human BPD survivors are weaned off oxygen within the first weeks to months of life, yet they may have persistent lung injury and pulmonary dysfunction as adults. We hypothesized that early-life hyperoxia alters the cellular landscape in later life and predicts long-term lung injury. Using single-cell RNA sequencing, we mapped lung cell subpopulations at postnatal day (pnd)7 and pnd60 in mice exposed to hyperoxia (95% O 2 ) for 3 days as neonates. We interrogated over 10,000 cells and identified a total of 45 clusters within 32 cell states. Neonatal hyperoxia caused persistent compositional changes in later life (pnd60) in all five type II cell states with unique signatures and function. Premature infants requiring mechanical ventilation with different durations also showed similar alterations in these unique signatures of type II cell states. Pathologically, neonatal hyperoxic exposure caused alveolar simplification in adult mice. We conclude that neonatal hyperoxia alters the lung cellular landscape in later life, uncovering neonatal programing of adult lung dysfunction., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in premature infants. Accumulating evidence shows that dysregulated metabolism of glucose, lipids and amino acids are observed in premature infants. Animal and cell studies demonstrate that abnormal metabolism of these substrates results in apoptosis, inflammation, reduced migration, abnormal proliferation or senescence in response to hyperoxic exposure, and that rectifying metabolic dysfunction attenuates neonatal hyperoxia-induced alveolar simplification and vascular dysgenesis in the lung. BPD is often associated with several comorbidities, including pulmonary hypertension and neurodevelopmental abnormalities, which significantly increase the morbidity and mortality of this disease. Here, we discuss recent progress on dysregulated metabolism of glucose, lipids and amino acids in premature infants with BPD and in related in vivo and in vitro models. These findings suggest that metabolic dysregulation may serve as a biomarker of BPD and plays important roles in the pathogenesis of this disease. We also highlight that targeting metabolic pathways could be employed in the prevention and treatment of BPD., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Prolonged exposure to high oxygen concentrations in premature infants, although lifesaving, can induce lung oxidative stress and increase the risk of developing BPD, a form of chronic lung disease. The lung alveolar epithelium is damaged by sustained hyperoxia, causing oxidative stress and alveolar simplification; however, it is unclear what duration of exposure to hyperoxia negatively impacts cellular function., Methods: Here we investigated the role of a very short exposure to hyperoxia (95% O 2 , 5% CO 2 ) on mitochondrial function in cultured mouse lung epithelial cells and neonatal mice., Results: In epithelial cells, 4 h of hyperoxia reduced oxidative phosphorylation, respiratory complex I and IV activity, utilization of mitochondrial metabolites, and caused mitochondria to form elongated tubular networks. Cells allowed to recover in air for 24 h exhibited a persistent global reduction in fuel utilization. In addition, neonatal mice exposed to hyperoxia for only 12 h demonstrated alveolar simplification at postnatal day 14., Conclusion: A short exposure to hyperoxia leads to changes in lung cell mitochondrial metabolism and dynamics and has a long-term impact on alveolarization. These findings may help inform our understanding and treatment of chronic lung disease., Impact: Many studies use long exposures (up to 14 days) to hyperoxia to mimic neonatal chronic lung disease. We show that even a very short exposure to hyperoxia leads to long-term cellular injury in type II-like epithelial cells. This study demonstrates that a short (4 h) period of hyperoxia has long-term residual effects on cellular metabolism. We show that neonatal mice exposed to hyperoxia for a short time (12 h) demonstrate later alveolar simplification. This work suggests that any exposure to clinical hyperoxia leads to persistent lung dysfunction., (© 2020. International Pediatric Research Foundation, Inc.)

The transcriptional repressor Rev-erbα is known to down-regulate fatty acid metabolism and gluconeogenesis gene expression. In animal models, disruption of Rev-erbα results in global changes in exercise performance, oxidative capacity, and blood glucose levels. However, the complete extent to which Rev-erbα-mediated transcriptional repression of metabolism impacts cell function remains unknown. We hypothesized that loss of Rev-erbα in a mouse embryonic fibroblast (MEF) model would result in global changes in metabolism. MEFs lacking Rev-erbα exhibited a hypermetabolic phenotype, demonstrating increased levels of glycolysis and oxidative phosphorylation. Rev-erbα deletion increased expression of hexokinase II, transketolase, and ribose-5-phosphate isomerase genes involved in glycolysis and the pentose phosphate pathway (PPP), and these effects were not mediated by the transcriptional activator BMAL1. Upregulation of oxidative phosphorylation was not accompanied by an increase in mitochondrial biogenesis or numbers. Rev-erbα repressed proliferation via glycolysis, but not the PPP. When treated with H 2 O 2 , cell viability was reduced in Rev-erbα knockout MEFs, accompanied by increased ratio of oxidized/reduced NADPH, suggesting that perturbation of the PPP reduces capacity to mount an antioxidant defense. These findings uncover novel mechanisms by which glycolysis and the PPP are modulated through Rev-erbα, and provide new insights into how Rev-erbα impacts proliferation.

Supplemental oxygen and mechanical ventilation commonly used in premature infants may lead to chronic lung disease of prematurity, which is characterized by arrested alveolar development and dysmorphic vascular development. Hyperoxia is also known to dysregulate p53, senescence, and metabolism. However, whether these changes in p53, senescence, and metabolism are intertwined in response to hyperoxia is still unknown. Given that the lung epithelium is the first cell to encounter ambient oxygen during a hyperoxic exposure, we used mouse lung epithelial cells (MLE-12), surfactant protein expressing type II cells, to explore whether hyperoxic exposure alters senescence and glycolysis. We measured glycolytic rate using a Seahorse Bioanalyzer assay and senescence using a senescence-associated β galactosidase activity assay with X-gal and C 12 FDG as substrates. We found that hyperoxic exposure caused senescence and increased glycolysis as well as reduced proliferation. This was associated with increased double stranded DNA damage, p53 phosphorylation and nuclear localization. Furthermore, hyperoxia-induced senescence was p53-dependent, but not pRB-dependent, as shown in p53KO and pRBKO cell lines. Despite the inhibitory effects of p53 on glycolysis, we observed that glycolysis was upregulated in hyperoxia-exposed MLE-12 cells. This was attributable to a subpopulation of highly glycolytic senescent cells detected by C 12 FDG sorting. Nevertheless, inhibition of glycolysis did not prevent hyperoxia-induced senescence. Therapeutic strategies modulating p53 and glycolysis may be useful to mitigate the detrimental consequences of hyperoxia in the neonatal lung., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Dysmorphic pulmonary vascular growth and abnormal endothelial cell (EC) proliferation are paradoxically observed in premature infants with bronchopulmonary dysplasia (BPD), despite vascular pruning. The pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis, generates NADPH as a reducing equivalent and ribose 5-phosphate for nucleotide synthesis. It is unknown whether hyperoxia, a known mediator of BPD in rodent models, alters glycolysis and the PPP in lung ECs. We hypothesized that hyperoxia increases glycolysis and the PPP, resulting in abnormal EC proliferation and dysmorphic angiogenesis in neonatal mice. To test this hypothesis, lung ECs and newborn mice were exposed to hyperoxia and allowed to recover in air. Hyperoxia increased glycolysis and the PPP. Increased PPP, but not glycolysis, caused hyperoxia-induced abnormal EC proliferation. Blocking the PPP reduced hyperoxia-induced glucose-derived deoxynucleotide synthesis in cultured ECs. In neonatal mice, hyperoxia-induced abnormal EC proliferation, dysmorphic angiogenesis, and alveolar simplification were augmented by nanoparticle-mediated endothelial overexpression of phosphogluconate dehydrogenase, the second enzyme in the PPP. These effects were attenuated by inhibitors of the PPP. Neonatal hyperoxia augments the PPP, causing abnormal lung EC proliferation, dysmorphic vascular development, and alveolar simplification. These observations provide mechanisms and potential metabolic targets to prevent BPD-associated vascular dysgenesis.

Bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants, results from mechanical ventilation and hyperoxia, amongst other factors. Although most BPD survivors can be weaned from supplemental oxygen, many show evidence of cardiovascular sequelae in adulthood, including pulmonary hypertension and pulmonary vascular remodeling. Endothelial-mesenchymal transition (EndoMT) plays an important role in mediating vascular remodeling in idiopathic pulmonary arterial hypertension. Whether hyperoxic exposure, a known mediator of BPD in rodent models, causes EndoMT resulting in vascular remodeling and pulmonary hypertension remains unclear. We hypothesized that neonatal hyperoxic exposure causes EndoMT, leading to the development of pulmonary hypertension in adulthood. To test this hypothesis, newborn mice were exposed to hyperoxia and then allowed to recover in room air until adulthood. Neonatal hyperoxic exposure gradually caused pulmonary vascular and right ventricle remodeling as well as pulmonary hypertension. Male mice were more susceptible to developing pulmonary hypertension compared to female mice, when exposed to hyperoxia as newborns. Hyperoxic exposure induced EndoMT in mouse lungs as well as in cultured lung microvascular endothelial cells (LMVECs) isolated from neonatal mice and human fetal donors. This was augmented in cultured LMVECs from male donors compared to those from female donors. Using primary mouse LMVECs, hyperoxic exposure increased phosphorylation of both Smad2 and Smad3, but reduced Smad7 protein levels. Treatment with a selective TGF-β inhibitor SB431542 blocked hyperoxia-induced EndoMT in vitro. Altogether, we show that neonatal hyperoxic exposure caused vascular remodeling and pulmonary hypertension in adulthood. This was associated with increased EndoMT. These novel observations provide mechanisms underlying hyperoxia-induced vascular remodeling and potential approaches to prevent BPD-associated pulmonary hypertension by targeting EndoMT. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Heme oxygenase (HO) consists of inducible (HO-1) and constitutive (HO-2) isoforms that are encoded by Hmox1 and Hmox2 genes, respectively. As an anti-inflammatory and antioxidant molecule, HO participates in the development of metabolic diseases. Whether Hmox deficiency causes metabolic abnormalities under basal conditions remains unclear. We hypothesized that HO-1 and HO-2 differentially affect global and adipose tissue metabolism. To test this hypothesis, we determined insulin sensitivity, glucose tolerance, energy expenditure, and respiratory exchange ratio in global Hmox1 -/- and Hmox2 -/- mice. Body weight was reduced in female but not male Hmox1 -/- and Hmox2 -/- mice. Reduced insulin sensitivity and physical activity were observed in Hmox1 -/- but not Hmox2 -/- mice. Deletion of either Hmox1 or Hmox2 had no effects on glucose tolerance, energy expenditure or respiratory exchange ratio. Mitochondrial respiration was unchanged in gonadal fat pads (white adipose tissue, WAT) of Hmox1 -/- mice. Hmox2 deletion increased proton leak and glycolysis in gonadal, but not interscapular fat tissues (brown adipose tissue, BAT). Uncoupling protein and Hmox1 genes were unchanged in gonadal fat pads of Hmox2 -/- mice. Conclusively, HO-1 maintains insulin sensitivity, while HO-2 represses glycolysis and proton leak in the WAT under basal condition. This suggests that HO-1 and HO-2 differentially modulate metabolism, which may impact the metabolic syndrome.

Heme oxygenase-1 is induced by many cellular stressors and catalyzes the breakdown of heme to generate carbon monoxide and bilirubin, which confer cytoprotection. The role of HO-1 likely extends beyond the simple production of antioxidants, for example HO-1 activity has also been implicated in metabolism, but this function remains unclear. Here we used an HO-1 knockout lung cell line to further define the contribution of HO-1 to cellular metabolism. We found that knockout cells exhibit reduced growth and mitochondrial respiration, measured by oxygen consumption rate. Specifically, we found that HO-1 contributed to electron transport chain activity and utilization of certain mitochondrial fuels. Loss of HO-1 had no effect on intracellular non-heme iron concentration or on proteins whose levels and activities depend on available iron. We show that HO-1 supports essential functions of mitochondria, which highlights the protective effects of HO-1 in diverse pathologies and tissue types. Our results suggest that regulation of heme may be an equally significant role of HO-1.

Treatments with supplemental oxygen in premature infants can impair lung development, leading to bronchopulmonary dysplasia (BPD). Although a stage-specific alteration of lung lipidome occurs during postnatal lung development, whether neonatal hyperoxia, a known mediator of BPD in rodent models, changes lipid profiles in mouse lungs is still to be elucidated. To answer this question, newborn mice were exposed to hyperoxia for 3 days and allowed to recover in normoxia until postnatal day (pnd) 7 and pnd14, time-points spanning the peak stage of alveologenesis. A total of 2263 lung lipid species were detected by liquid chromatography-mass spectrometry, covering 5 lipid categories and 18 lipid subclasses. The most commonly identified lipid species were glycerophospholipids, followed by sphingolipids and glycerolipids. In normoxic conditions, certain glycerophospholipid and glycerolipid species augmented at pnd14 compared to pnd7. At pnd7, hyperoxia generally increased glycerophospholipid, sphingolipid, and glycerolipid species. Hyperoxia increased NADPH, acetyl CoA, and citrate acid but reduced carnitine and acyl carnitine. Hyperoxia increased oxidized glutathione but reduced catalase. These changes were not apparent at pnd14. Hyperoxia reduced docosahexaenoic acid and arachidonic acid at pnd14 but not at pnd7. Altogether, the lung lipidome changes throughout alveolarization. Neonatal hyperoxia alters the lung lipidome, which may contribute to alveolar simplification and dysregulated vascular development.

Premature infants are often exposed to positive pressure ventilation and supplemental oxygen, which leads to the development of chronic lung disease (CLD). There are currently no standard serum biomarkers used for prediction or early detection of patients who go on to develop CLD. MicroRNAs (miRNAs) are a novel class of naturally occurring, short, noncoding substances that regulate gene expression at the posttranscriptional level and cause translational inhibition and/or mRNA degradation and present in body fluids packaged in extracellular vesicles (EVs), rendering them remarkably stable. Our aim was to evaluate miRNAs identified in serum EVs of premature infants as potential biomarkers for CLD. Serum EVs were extracted from premature infants at birth and on the 28th day of life (DOL). Using a human miRNA array, we identified 62 miRNAs that were universally expressed in CLD patients and non-CLD patients. Of the 62 miRNAs, 59 miRNAs and 44 miRNAs were differentially expressed on DOL0 and DOL28 in CLD and non-CLD patients, respectively. Of these miRNAs, serum EV miR-21 was upregulated in CLD patients on DOL28 compared with levels at birth and downregulated in non-CLD patients on DOL28 compared with levels at birth. In neonatal mice exposed to hyperoxia for 7days, as a model of CLD, five miRNAs (miR-34a, miR-21, miR-712, miR-682, and miR-221) were upregulated, and 7 miRNAs (miR-542-5p, miR-449a, miR-322, miR-190b, miR-153, miR-335-3p, miR-377) were downregulated. MiR-21 was detected as a common miRNA that changed in CLD patients and in the hyperoxia exposed mice. We conclude that EV miR-21 may be a biomarker of CLD.

Endothelial-to-mesenchymal transition (EndoMT) is a process of transdifferentiation where endothelial cells gradually adopt the phenotypic characteristics of mesenchymal cells. This phenomenon was first discovered in embryonic heart development. The mechanisms underlying EndoMT are due to the activation of transforming growth factor-β, bone morphogenetic protein, Wingless/Integrated, or Notch signaling pathways. The EndoMT can be modulated by pathological processes, including inflammation, disturbed shear stress, vascular stiffness, and metabolic dysregulation. Recent studies have shown that EndoMT is implicated in the pathogenesis of chronic lung diseases, including pulmonary hypertension and lung fibrosis. Lung pathology of bronchopulmonary dysplasia can be mimicked in rodents exposed to hyperoxia as neonates. Although hyperoxic exposure reduces an endothelial cell marker platelet and endothelial cell adhesion molecule but increases a mesenchymal cell biomarker α-smooth muscle actin in vitro in human pulmonary endothelial cells, there is no direct evidence showing EndoMT in the development of bronchopulmonary dysplasia. Both pulmonary hypertension and lung fibrosis occur in long-term survivors with bronchopulmonary dysplasia. In this review, we discuss the EndoMT and its modulation by pathological processes. We then focus on the role of EndoMT in the pathogenesis of these chronic lung diseases, and discuss therapeutic approaches targeting the EndoMT using its negative regulators., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In neonates, hyperoxia or positive pressure ventilation causes continued lung injury characterized by simplified vascularization and alveolarization, which are the hallmarks of bronchopulmonary dysplasia. Although endothelial cells (ECs) have metabolic flexibility to maintain cell function under stress, it is unknown whether hyperoxia causes metabolic dysregulation in ECs, leading to lung injury. We hypothesized that hyperoxia alters EC metabolism, which causes EC dysfunction and lung injury. To test this hypothesis, we exposed lung ECs to hyperoxia (95% O 2 /5% CO 2 ) followed by air recovery (O 2 /rec). We found that O 2 /rec reduced mitochondrial oxidative phosphorylation without affecting mitochondrial DNA copy number or mitochondrial mass and that it specifically decreased fatty acid oxidation (FAO) in ECs. This was associated with increased ceramide synthesis and apoptosis. Genetic deletion of carnitine palmitoyltransferase 1a (Cpt1a), a rate-limiting enzyme for carnitine shuttle, further augmented O 2 /rec-induced apoptosis. O 2 /rec-induced ceramide synthesis and apoptosis were attenuated when the FAO was enhanced by l-carnitine. Newborn mice were exposed to hyperoxia (>95% O 2 ) between Postnatal Days 1 and 4 and were administered l-carnitine (150 and 300 mg/kg, i.p.) or etomoxir, a specific Cpt1 inhibitor (30 mg/kg, i.p.), daily between Postnatal Days 10 and 14. Etomoxir aggravated O 2 /rec-induced apoptosis and simplified alveolarization and vascularization in mouse lungs. Similarly, arrested alveolarization and reduced vessel numbers were further augmented in EC-specific Cpt1a -knockout mice compared with wild-type littermates in response to O 2 /rec. Treatment with l-carnitine (300 mg/kg) attenuated O 2 /rec-induced lung injury, including simplified alveolarization and decreased vessel numbers. Altogether, enhancing FAO protects against hyperoxia-induced EC apoptosis and lung injury in neonates.

Background: The increasing incidence of bronchopulmonary dysplasia in premature babies may be due in part to immature ventilatory control, contributing to hypoxemia. The latter responds to ventilation and/or oxygen therapy, treatments associated with adverse sequelae. This is an overview of the Prematurity-Related Ventilatory Control Study which aims to analyze the under-utilized cardiorespiratory continuous waveform monitoring data to delineate mechanisms of immature ventilatory control in preterm infants and identify predictive markers., Methods: Continuous ECG, heart rate, respiratory, and oxygen saturation data will be collected throughout the NICU stay in 500 infants < 29 wks gestation across 5 centers. Mild permissive hypercapnia, and hyperoxia and/or hypoxia assessments will be conducted in a subcohort of infants along with inpatient questionnaires, urine, serum, and DNA samples., Results: Primary outcomes will be respiratory status at 40 wks and quantitative measures of immature breathing plotted on a standard curve for infants matched at 36-37 wks. Physiologic and/or biologic determinants will be collected to enhance the predictive model linking ventilatory control to outcomes., Conclusions: By incorporating bedside monitoring variables along with biomarkers that predict respiratory outcomes we aim to elucidate individualized cardiopulmonary phenotypes and mechanisms of ventilatory control contributing to adverse respiratory outcomes in premature infants.

The metabolism of nutrient substrates, including glucose, glutamine, and fatty acids, provides acetyl-CoA for the tricarboxylic acid cycle to generate energy, as well as metabolites for the biosynthesis of biomolecules, including nucleotides, proteins, and lipids. It has been shown that metabolism of glucose, fatty acid, and glutamine plays important roles in modulating cellular proliferation, differentiation, apoptosis, autophagy, senescence, and inflammatory responses. All of these cellular processes contribute to the pathogenesis of chronic lung diseases, including bronchopulmonary dysplasia, chronic obstructive pulmonary disease, and pulmonary fibrosis. Recent studies demonstrate that metabolic reprogramming occurs in patients with and animal models of chronic lung diseases, suggesting that metabolic dysregulation may participate in the pathogenesis and progression of these diseases. In this review, we briefly discuss the catabolic pathways for glucose, glutamine, and fatty acids, and focus on how metabolic reprogramming of these pathways impacts cellular functions and leads to the development of these chronic lung diseases. We also highlight how targeting metabolic pathways can be utilized in the prevention and treatment of these diseases.

In premature neonates, hyperoxic exposure contributes to lung injury characterized by simplified alveolarization and arrested vascularization. These are the hallmarks of bronchopulmonary dysplasia, a disease with long-term consequences on pulmonary and neurodevelopmental function. Lung vascular development and endothelial cell signals are synergistically important for normal alveolarization. It has been shown that metabolism of nutrients such as glucose, fatty acid, and glutamine is key in controlling proliferation, differentiation, apoptosis, autophagy, senescence, and inflammatory responses, which contribute to the pathogenesis of chronic lung diseases, including bronchopulmonary dysplasia. Recent studies show that metabolic reprogramming occurs in vitro in cells and in vivo in animal models and more importantly in patients with bronchopulmonary dysplasia, suggesting that metabolic dysregulation may participate in the pathogenesis and progression of these diseases. Although endothelial cells rely mainly on glycolysis for bioenergetics, they have the metabolic flexibility to maintain cell function under stress or nutrient deprivation. Others have shown that hyperoxia decreases glycolysis and oxidative phosphorylation in epithelial cells. Nevertheless, endothelial cells show enhanced mitochondrial fatty acid use after exposure to hyperoxia. This may serve to preserve endothelial cell proliferation and alveolarization, and thereby mitigate neonatal hyperoxic lung injury., Competing Interests: Potential Conflicts of Interest: None disclosed.

Background: BTB and CNC homology 1 (Bach1) is a transcriptional repressor of heme oxygenase (HO)-1. The effects of Bach1 disruption on hyperoxic lung injury in newborn mice have not been determined. We aimed to investigate the role of Bach1 in the newborns exposed to hyperoxia., Methods: Bach1 -/- and WT newborn mice were exposed to 21% or 95% oxygen for 4 d and were then allowed to recover in room air. Lung histology was assessed and lung Bach1, HO-1, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 mRNA levels were evaluated using RT-PCR. Lung inflammatory cytokine levels were determined using cytometric bead arrays., Results: After 10 d recovery from neonatal hyperoxia, Bach1 -/- mice showed improved lung alveolarization compared with WT. HO-1, IL-6, and MCP-1 mRNA levels and IL-6 and MCP-1 protein levels were significantly increased in the Bach1 -/- lungs exposed to neonatal hyperoxia. Although an increase in apoptosis was observed in the Bach1 -/- and WT lungs after neonatal hyperoxia, there were no differences in apoptosis between these groups., Conclusion: Bach1 -/- newborn mice were well-recovered from hyperoxia-induced lung injury. This effect is likely achieved by the antioxidant/anti-inflammatory activity of HO-1 or by the transient overexpression of proinflammatory cytokines.

In the lung, heme oxygenase-1 (HO-1) is developmentally regulated, with its highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared with adults. However, few reports have addressed the functional effect of microRNAs (miRNAs) in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure, and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) and adult (2 mo old) mice were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low-density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-untranslated region of the transcriptional repressor BTB and CNC homology 1 (Bach1) and regulates its expression, and subsequently leads to higher levels of lung HO-1 mRNA compared with levels in adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its effect on Bach1. This report is the first to show developmental differences in lung miR-196a and its effect on Bach1 and HO-1 expression at baseline and in hyperoxia., (Copyright © 2016 the American Physiological Society.)

Diurnal oscillations in the expression of antioxidant genes imply that protection against oxidative stress is circadian-gated. We hypothesized that stabilization of the core circadian gene Rev-erbα (Nr1d1) improves cellular bioenergetics and protects against nutrient deprivation and oxidative stress. Compared to WT, mouse lung fibroblasts (MLG) stably transfected with a degradation resistant Rev-erbα (Ser(55/59) to Asp; hence referred to as SD) had 40% higher protein content, 1.5-fold higher mitochondrial area (confocal microscopy), doubled oxidative phosphorylation by high-resolution respirometry (Oroboros) and were resistant to glucose deprivation for 24h. This resulted from a 4-fold reduction in mitophagy (L3CB co-localized with MitoTracker Red) versus WT. Although PGC1α protein expression was comparable between SD and WT MLG cells, the role of mitochondrial biogenesis in explaining increased mitochondrial mass in SD cells was less clear. Embryonic fibroblasts (MEF) from C57Bl/6-SD transgenic mice, had a 9-fold induction of FoxO1 mRNA and increased mRNA of downstream antioxidant targets heme oxygenase-1 (HO-1), Mn superoxide dismutase and catalase (1.5, 2 fold and 2 fold respectively) versus WT. This allowed the SD cells to survive 1h incubation with 500 µM H2O2 as well as 24h of exposure to 95% O2 and remain attached whereas most WT cells did not. These observations establish a mechanistic link between the metabolic functions of Rev-erbα with mitochondrial homeostasis and protection against oxidative stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aim: Premature infants are often exposed to hyperoxia to maintain adequate oxygenation, which may lead to the development of bronchopulmonary dysplasia (BPD). Sex-specific differences exist in the development and severity of BPD. Only a few studies have examined the mechanisms underlying these sex-related differences. The aim of the present study is to examine the sex-related long-term effects of neonatal hyperoxia on the lungs of adult mice., Materials and Methods: Newborn mice were exposed to 95% oxygen (hyperoxia) for 96 hours and were allowed to recover in room air to adulthood (8 weeks of age). Lung tissues were excised at 4 days, 14 days, or 8 weeks of age. Short-term effects of neonatal hyperoxia on the mouse lung and sex-related differences in pulmonary function, airway hyper-responsiveness, and lung structure in adult mice were assessed., Results: Neonatal hyperoxia was found to have no differential effect on body weight, muscarinic acetylcholine receptor gene expression, or bronchiolar epithelial thickness in adult mice. Respiratory resistance was increased and sensitivity to methacholine was decreased in male adult mice following exposure to neonatal hyperoxia, whereas delayed alveolarization was observed in female adult mice following exposure to neonatal hyperoxia., Conclusions: The findings of the present study demonstrate that neonatal hyperoxia differentially affects pulmonary outcome in female and male adult mice.

Free radicals and oxidants are now implicated in physiological responses and in several diseases. Given the wide range of expertise of free radical researchers, application of the greater understanding of chemistry has not been uniformly applied to biological studies. We suggest that some widely used methodologies and terminologies hamper progress and need to be addressed. We make the case for abandonment and judicious use of several methods and terms and suggest practical and viable alternatives. These changes are suggested in four areas: use of fluorescent dyes to identify and quantify reactive species, methods for measurement of lipid peroxidation in complex biological systems, claims of antioxidants as radical scavengers, and use of the terms for reactive species., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Significance: Premature and sick neonates are often exposed to high concentrations of oxygen, which results in lung injury and long-term adverse consequences. Nevertheless, neonates are more tolerant to hyperoxia than are adults. This may be, in part, explained by the high lung content of heme oxygenase-1 (HO-1), the rate-limiting enzyme in the degradation of heme and an important stress protein. The abundance of HO-1 dictates its cytoprotective and deleterious effects. Interestingly, in response to hyperoxia, lung HO-1 mRNA is not further up-regulated in neonates, suggesting that lung HO-1 gene expression is tightly regulated so as to optimize cytoprotection when faced with an oxidative stress such as hyperoxia., Recent Advances: In addition to the lack of induction of HO-1 mRNA, neonatal lung HO-1 protein is observed in the nucleus in neonatal mice exposed to hyperoxia but not in adults, which is further evidence for the developmental regulation of HO-1. Nuclear HO-1 had unique properties independent of its enzymatic activity. In addition, there has been increasing evidence that nuclear HO-1 contributes to cellular proliferation and malignant transformation in several human cancers., Critical Issues: Since HO-1 has dual effects in cytoprotection and cellular proliferation, the titration of HO-1 effects is critical to ensure beneficial actions against oxidative stress., Future Directions: Much more has to be understood about the specific roles of HO-1 so as to manipulate its abundance and/or nuclear migration to maximize the therapeutic benefit of this pleiotropic protein in the neonatal lung.

With oxidative injury as well as in some solid tumors and myeloid leukemia cells, heme oxygenase-1 (HO-1), the anti-oxidant, anti-inflammatory, and anti-apoptotic microsomal stress protein, migrates to the nucleus in a truncated and enzymatically inactive form. However, the function of HO-1 in the nucleus is not completely clear. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor and master regulator of numerous antioxidants and anti-apoptotic proteins, including HO-1, also accumulates in the nucleus with oxidative injury and in various types of cancer. Here we demonstrate that in oxidative stress, nuclear HO-1 interacts with Nrf2 and stabilizes it from glycogen synthase kinase 3β (GSK3β)-mediated phosphorylation coupled with ubiquitin-proteasomal degradation, thereby prolonging its accumulation in the nucleus. This regulation of Nrf2 post-induction by nuclear HO-1 is important for the preferential transcription of phase II detoxification enzymes such as NQO1 as well as glucose-6-phosphate dehydrogenase (G6PDH), a regulator of the pentose phosphate pathway. Using Nrf2 knock-out cells, we further demonstrate that nuclear HO-1-associated cytoprotection against oxidative stress depends on an HO-1/Nrf2 interaction. Although it is well known that Nrf2 induces HO-1 leading to mitigation of oxidant stress, we propose a novel mechanism by which HO-1, by modulating the activation of Nrf2, sets an adaptive reprogramming that enhances antioxidant defenses., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: The response to oxidative stress and inflammation varies with diurnal rhythms. Nevertheless, it is not known whether circadian genes are regulated by these stimuli. We evaluated whether Rev-erbα, a key circadian gene, was regulated by oxidative stress and/or inflammation in vitro and in a mouse model., Results: A unique sequence consisting of overlapping AP-1 and nuclear factor kappa B (NFκB) consensus sequences was identified on the mouse Rev-erbα promoter. This sequence mediates Rev-erbα promoter activity and transcription in response to oxidative stress and inflammation. This region serves as an NrF2 platform both to receive oxidative stress signals and to activate Rev-erbα, as well as an NFκB-binding site to repress Rev-erbα with inflammatory stimuli. The amplitude of the rhythmicity of Rev-erbα was altered by pre-exposure to hyperoxia or disruption of NFκB in a cell culture model of circadian simulation. Oxidative stress overcame the inhibitory effect of NFκB binding on Rev-erbα transcription. This was confirmed in neonatal mice exposed to hyperoxia, where hyperoxia-induced lung Rev-erbα transcription was further increased with NFκB disruption. Interestingly, this effect was not observed in similarly exposed adult mice., Innovation: These data provide novel mechanistic insights into how key circadian genes are regulated by oxidative stress and inflammation in the neonatal lung., Conclusion: Rev-erbα transcription and circadian oscillation are susceptible to oxidative stress and inflammation in the neonate. Due to Rev-erbα's role in cellular metabolism, this could contribute to lung cellular function and injury from inflammation and oxidative stress.

Oxygen toxicity contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Neonatal mice exposed to hyperoxia develop a simplified lung structure that resembles BPD. Sustained activation of the transcription factor NF-κB and increased expression of protective target genes attenuate hyperoxia-induced mortality in adults. However, the effect of enhancing hyperoxia-induced NF-κB activity on lung injury and development in neonatal animals is unknown. We performed this study to determine whether sustained NF-κB activation, mediated through IκBβ overexpression, preserves lung development in neonatal animals exposed to hyperoxia. Newborn wild-type (WT) and IκBβ-overexpressing (AKBI) mice were exposed to hyperoxia (>95%) or room air from day of life (DOL) 0-14, after which all animals were kept in room air. Survival curves were generated through DOL 14. Lung development was assessed using radial alveolar count (RAC) and mean linear intercept (MLI) at DOL 3 and 28 and pulmonary vessel density at DOL 28. Lung tissue was collected, and NF-κB activity was assessed using Western blot for IκB degradation and NF-κB nuclear translocation. WT mice demonstrated 80% mortality through 14 days of exposure. In contrast, AKBI mice demonstrated 60% survival. Decreased RAC, increased MLI, and pulmonary vessel density caused by hyperoxia in WT mice were significantly attenuated in AKBI mice. These findings were associated with early and sustained NF-κB activation and expression of cytoprotective target genes, including vascular endothelial growth factor receptor 2. We conclude that sustained hyperoxia-induced NF-κB activation improves neonatal survival and preserves lung development. Potentiating early NF-κB activity after hyperoxic exposure may represent a therapeutic intervention to prevent BPD., (Copyright © 2014 the American Physiological Society.)

Significance: Many reports have underscored the importance of the heme degradation pathway that is regulated by heme oxygenase (HO). This reaction releases bile pigments and carbon monoxide (CO), which are important antioxidant and signaling molecules. Thus, the reaction of HO-1 would have significant cytoprotective effects. Nevertheless, the importance of this protein goes beyond its enzymatic action. New evidence outlines significant effects of inactive forms of the HO-1 protein., Recent Advances: In fact, the role of the HO protein in cellular signaling, including transcription factor activation, binding to proteins, phosphorylation, and modulation of protein function, among others, has started being elucidated. The mechanism by which the inducible form of HO-1, in particular, can migrate to various cellular compartments to mediate important signaling or how and why it binds to key transcription factors and other proteins that are important in DNA repair is also described in several physiologic systems., Critical Issues: The signaling functions of HO-1 may have particular relevance in clinical circumstances, including cancer, as redistribution of HO-1 into the nuclear compartment is observed with cancer progression and metastasis. In addition, along with oxidative stress, the pleiotropic functions of HO-1 modulate antioxidant defense. In organ transplantation, HO and its byproducts suppress rejection at multiple levels and in sepsis-induced pulmonary dysfunction, inhaled CO or modulation of HO activity can change the course of the disease in animals., Future Directions: It is hoped that a more detailed understanding of the various signaling functions of HO will guide therapeutic approaches for complex diseases.