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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.

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 C12FDG 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 C12FDG 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., Hyperoxia‐induced senescence is p53‐dependent, but not pRB‐dependent in MLE‐12 cells. Glycolysis was upregulated in hyperoxia‐exposed MLE‐12 cells, this increased glycolysis is derived from highly glycolytic senescent cells.

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.

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.Here we investigated the role of a very short exposure to hyperoxia (95% OIn 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.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.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.

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.

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.Bach1After 10 d recovery from neonatal hyperoxia, Bach1Bach1

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.

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.

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 (

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.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.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.The findings of the present study demonstrate that neonatal hyperoxia differentially affects pulmonary outcome in female and male adult mice.

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

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.

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.

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. Antioxid. Redox Signal. 21, 1881–1892.

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.

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.

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. Antioxid. Redox Signal. 20, 1743–1753.

The scaffold protein ISCU facilitates the assembly of iron-sulfur clusters (ISCs), which are essential cofactors for many vital metabolic processes. The mTOR pathways are central to nutrient and energy-sensing networks. Here, we demonstrate that mTORC1 associates with ISCU and phosphorylates ISCU at serine 14. This phosphorylation stabilized ISCU protein. Insufficiency of ISCU triggered by mTORC1 inhibition prevented ISC assembly. Sustained ISCU protein levels enhanced by mTORC1 sensitized TSC2-null cells to iron deprivation due to constitutive ISC biogenesis-triggered iron demand, which outstrips supply. We conclude that the mTORC1 pathway serves to modulate iron metabolism and homeostasis, and we speculate that iron deprivation may be an adjunct in the treatment of cancers characterized by constitutive mTORC1 activation.

The purpose of this position paper is to present a critical analysis of the challenges and limitations of the most widely used fluorescent probes for detecting and measuring reactive oxygen and nitrogen species. Where feasible, we have made recommendations for the use of alternate probes and appropriate analytical techniques that measure the specific products formed from the reactions between fluorescent probes and reactive oxygen and nitrogen species. We have proposed guidelines that will help present and future researchers with regard to the optimal use of selected fluorescent probes and interpretation of results.

Premature neonates with immature lungs often require supplemental oxygen for survival. Hyperoxic exposure in neonates results in impaired formation of alveoli and lung vasculature that is characteristic of bronchopulmonary dysplasia (BPD), which can have long term consequences for the development of the respiratory and nervous systems. We have shown that hyperoxia reduces levels of Rev-erbα, a transcriptional repressor and circadian regulator of metabolism, in the mouse lung. We hypothesized that reduction of Rev-erbα expression would alter metabolism in a mouse embryonic fibroblast (MEF) model of hyperoxic exposure. In order to determine changes in metabolic function and utilization of key substrates resulting from loss of Rev-erbα, we exposed WT and Rev-erbα KO MEFs to either air or hyperoxia (95% O2/5% CO2) for 24 hours. Control cells were exposed to air for 24 hours. Mitochondrial respiration, glycolysis, and substrate utilization were then determined using a Seahorse XF24 Bioanalyzer. Additionally, a 4 day growth curve was performed in air to determine differences in cell proliferation. Rev-erbα KO MEFs exposed to hyperoxia exhibited increased oxidative respiration and glycolysis compared to WT cells. Substrate utilization experiments showed that immediately following hyperoxic exposure, Rev-erbα KO MEFs were less dependent on glutamine and fatty acids, but had a higher capacity to metabolize glucose. Rev-erbα KO MEFs exhibit a higher basal rate of growth in air compared to WT. In conclusion, disruption of Rev-erbα in MEFs resulted in increased glycolysis and higher levels of proliferation. We speculate that these changes may render the cells more vulnerable to oxidative stress and less viable. We will test this using protein carbonyls as a measurement of oxidative stress levels and clonogenic assays as a measurement of cell survival.

Inhaled NO (iNO) may be protective against hyperoxic injury in the premature lung, but the mechanism is unknown. We hypothesized that NO would prevent hyperoxia-induced nuclear factor kappa B (NF-κB) activation in neonatal pulmonary microvascular endothelial cells [human pulmonary microvascular endothelial cell (HPMEC)] and prevent the up-regulation of target genes. After hyperoxic exposure (O2 >95%), nuclear NF-κB consensus sequence binding increased and was associated with IκBα degradation. Both of these findings were prevented by exposure to NO. Furthermore, intracellular adhesion molecule (ICAM)-1 mRNA and protein levels increased in cells exposed to hyperoxia, an effect abrogated by NO. To evaluate the potentially toxic effect of NO plus hyperoxia, cell viability and proliferation were assessed. Cells exposed to NO plus hyperoxia demonstrated improved survival as measured by trypan blue exclusion when compared with cells exposed to hyperoxia alone. These differences in cell death could not be attributed to apoptosis measured by caspase-3 activity. Finally, cellular proliferation inhibited by hyperoxia was rescued by concurrent exposure to NO. These data demonstrate that NO prevents hyperoxia-induced NF-κB activation in HPMEC and results in decreased expression of adhesion molecules and decreased cellular toxicity. This may help to explain the protective effects of NO on hyperoxic injury in the developing lung vasculature.

Heme is an essential cofactor in several enzymes of the electron transport chain (ETC) where its primary function is the coordination of iron to facilitate redox reactions. Unbound, free heme is strongly oxidative such that the cell has evolved mechanisms to prevent it from causing oxidative damage. This includes the catalytic breakdown of heme by heme oxygenase (HO-1), generating antioxidants CO and biliverdin. However the catabolism of heme also releases highly toxic free iron which seems counter productive to the antioxidant effort. We sought to explore the role of HO-1 in iron homeostasis using HO-1 knockout mouse embryonic fibroblasts and HO-1 deficient human liver cells. By Western blot these cells showed dysregulated iron handling including increased expression of transferrin receptor and decreased expression of ferritin and ferroportin compared to wild type controls. Additionally cytosolic aconitase activity was decreased in HO-1 deficient cells while the mutually exclusive mRNA binding activity of aconitase was increased. This switch is indicative of reduced FeS cluster availability. Consistent with FeS cluster deficiency we observed decreased frataxin mRNA levels, the product of which is involved in FeS cluster assembly in the mitochondria. Because FeS clusters are important in the ETC we examined mitochondrial respiration with a Seahorse Bioanalyzer (Agilent) and found it to be markedly reduced in HO-1 knockout cells. Interestingly, this was partially rescued by exogenous iron (1nM transferrin). Our findings suggest that an additional and important role of HO-1 is the maintenance of cellular iron homeostasis via the release of iron upon breakdown of heme.

Due to the advances in perinatal and neonatal care, preterm infants can survive from 22 weeks of gestation. Following extreme prematurity, mechanical ventilation and supplemental oxygen are employed to sustain life. Though supplemental oxygen therapy is clinically necessary it can cause impaired lung growth and repair due to perturbed energy metabolism. To better understand this, we investigated the role of hyperoxia on bioenergetics in mouse lung epithelial cells (MLE-12) exposed to hyperoxia (95% O2, 5% CO2). As short as 4 hours of hyperoxic exposure caused a reduction in the basal respiration, ATP production, and pyruvate utilization compared to air exposed controls as measured by the seahorse bioanalyzer. In addition, the decreased pyruvate utilization was not associated with altered mRNA levels of glucose transporters and mitochondrial pyruvate carriers, nor changes in glycolysis as measured by the glycolytic rate assay. Interestingly, MLE-12 cells exposed to hyperoxia had significantly increased mRNA and protein levels of UCP-1, a transport and uncoupling protein found in the inner mitochondrial membrane which can export monocarboxylate anions such as pyruvate. Despite this increase in UCP-1 we did not see an expected increase in proton leak. These data suggest that hyperoxia may enhance UCP-1 mediated export of pyruvate outside of the mitochondrial matrix rendering it unavailable for oxidative phosphorylation.

Preterm newborns with immature lungs receive life-saving supplemental oxygen. This treatment has improved survival rates but can also be toxic. Hyperoxia impairs lung development, causing long term respiratory and neurodevelopmental consequences. There are no therapeutic targets for supplemental oxygen toxicity. We have previously shown that exposure to hyperoxia (95% O2/5 % CO2) for 24 hours reduced levels of the transcriptional repressor and metabolic regulator, Rev-erbα, in mouse lung fibroblasts. We hypothesized that loss of Rev-erbα alters metabolism and substrate utilization in a mouse embryonic fibroblast (MEF) model. To test this, knockout (KO) and wild type (WT) MEF were grown to 80% confluency. Oxidative phosphorylation (oxphos), glycolysis, and substrate utilization were measured using a Seahorse XF24 Bioanalyzer. Rev-erbα KO MEF had increased oxphos compared with WT. Levels of glycolytic extracellular acidification were unchanged. Mitochondrial to nuclear DNA copy number ratios, measured by determining the levels of mitochondrial NADH dehydrogenase 1 over nuclear hexokinase 2, by PCR, were also unchanged. This demonstrated that increased oxphos was not caused by mitochondrial biogenesis. Rev-erbα KO MEF exhibited increased expression of ribose-5-phosphate isomerase and transketolase mRNA. These enzymes provide metabolic intermediates to glycolysis as part of the oxidative pentose phosphate pathway and may increase the amount of pyruvate available for oxphos. Rev-erbα KO MEF additionally exhibited a reduced dependency on fatty acids and glutamine. Reduced dependency on fatty acids may be related to a four-fold increase in expression of acyl-CoA synthetase 6 compared with WT. This enzyme is known to provide acyl-CoA for triacylgylceride synthesis in neurons, but whether it directs acyl-CoA to or away from beta oxidation in MEFs is unclear. In conclusion, loss of Rev-erbα increases oxphos and reduces fatty acid dependency in MEFs. We speculate that loss of Rev-erbα transcriptional repression causes upregulation of enzymes governing oxphos and acyl-CoA utilization for energy.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease, which is characterized by alveolar dysplasia and impaired vascularization in premature infants. BPD can develop pulmonary hypertension (PH) in adolescence and adulthood. This is corroborated by previous reports showing that hyperoxic exposure in newborn mice caused PH in adult mice. Endothelial-to-mesenchymal cells (EndoMT), a biological process where endothelial cells (ECs) progressively lose their specific markers and gain mesenchymal phenotype, is one of the contributing factors for development of PH. It has been shown that inhibition of fatty acid oxidation (FAO) augments the magnitude of EndoMT in ECs. This was attenuated by enhancing FAO via overexpression of carnitine palmitoyltransferase 1a (Cpt1a), a rate-limiting step of the carnitine shuttle. However, no information is available on whether hyperoxic exposure reduces FAO, resulting in EndoMT in lung ECs. We hypothesized that hyperoxic exposure causes EndoMT by modulating FAO in lung ECs. To test this hypothesis, mouse fetal lung EC line (MFLM-91U) and primary lung microvascular endothelial cells (LMVECs) were exposed to hyperoxia (95% O2/5% CO2) for 24 h. Some cultures were placed in air for 1, 3, or 7 days post exposure (air recovery). We found that hyperoxic exposure followed by air recovery reduced FAO as determined by the Seahorse XF24 Analyzer. The levels of markers for mesenchymal cells (i.e., vimentin, CD44, Snail1, Snug, Twist1, and α-SMA) were increased, while the EC markers (i.e., vWF, PECAM-1/CD31 and CD34) were reduced in lung ECs exposed to hyperoxia followed by air recovery. Surprisingly, gene deletion of Cpt1a attenuated hyperoxia-induced reduction of CD34 and CD31 gene expression. In conclusion, hyperoxic exposure followed by air recovery causes EndoMT in lung ECs, and inhibition of FAO rescues this phenotype. These findings may provide therapeutic approach targeting Cpt1a to preventing PH in BPD patients.

Zinc protoporphyrin IX (ZnPP), an endogenous heme analogue that inhibits heme oxygenase (HO) activity, represses tumor growth. It can also translocate into the nucleus and up-regulate heme oxygenase 1 (HMOX1) gene expression. Here, we demonstrate that tumor cell proliferation was inhibited by ZnPP, whereas tin protoporphyrin (SnPP), another equally potent HO-1 inhibitor, had no effect. Microarray analysis on 128 tumorigenesis related genes showed that ZnPP suppressed genes involved in cell proliferation and angiogenesis. Among these genes, CYCLIN D1 (CCND1) was specifically inhibited as were its mRNA and protein levels. Additionally, ZnPP inhibited CCND1 promoter activity through an Sp1 and Egr1 overlapping binding site (S/E). We confirmed that ZnPP modulated the S/E site, at least partially by associating with Sp1 and Egr1 proteins rather than direct binding to DNA targets. Furthermore, administration of ZnPP significantly inhibited cyclin D1 expression and progression of a B-cell leukemia/lymphoma 1 tumor in mice by preferentially targeting tumor cells. These observations show HO independent effects of ZnPP on cyclin D1 expression and tumorigenesis.

For nearly 100 y, pediatricians have regularly used oxygen to treat neonatal and childhood diseases. During this time, it has become clear that oxygen is toxic and that overzealous use can lead to significant morbidity. As we have learned more about the appropriate clinical indications for oxygen therapy, studies at the bench have begun to elucidate the molecular mechanisms by which cells respond to hyperoxia. In this review, we discuss transcription factors whose activity is regulated by oxygen, including nuclear factor, erythroid 2-related factor 2 (Nrf2), activator protein 1 (AP-1), p53, nuclear factor kappaB (NF-kappaB), signal transducers and activators of transcription protein (STAT), and ccat/enhancer binding protein (CEBP). Special attention is paid to the mechanisms by which hyperoxia affects these transcription factors in the lung. Finally, we identify downstream targets of these transcription factors, with a focus on heme oxygenase-1. A better understanding of how oxygen affects various signaling pathways could lead to interventions aimed at preventing hyperoxic injury.