The role of cytochrome P450 (CYP) enzymes in hyperoxic lung injury.

INTRODUCTION: Hyperoxic lung injury is a condition that can occur in patients in need of supplemental oxygen, such as premature infants with bronchopulmonary dysplasia or adults with acute respiratory distress syndrome. Cytochrome P450 (CYP) enzymes play critical roles in the metabolism of endogenous and exogenous compounds. AREAS COVERED: Through their complex pathways, some subfamilies of these enzymes may contribute to or protect against hyperoxic lung injury. Oxidative stress from reactive oxygen species (ROS) production is most likely a major contributor of hyperoxic lung injury. CYP1A enzymes have been shown to protect against hyperoxic lung injury while CYP1B enzymes seem to contribute to it. CYP2J2 enzymes help protect against hyperoxic lung injury by triggering EET production, thereby, increasing antioxidant enzymes. The metabolism of arachidonic acid to ω-terminal hydroxyeicosatetraenoic acid (20-HETEs) by CYP4A and CYP4F enzymes could impact hyperoxic lung injury via the vasodilating effects of 20-HETE. CYP2E1 and CYP2A enzymes may contribute to the oxidative stress in the lungs caused by ethanol- and nicotine-metabolism, respectively. EXPERT OPINION: Overall, the CYP enzymes, depending upon the isoform, play a contributory or protective role in hyperoxic lung injury, and are, therefore, ideal candidates for developing drugs that can treat oxygen-mediated lung injury.

Dual-specificity phosphatase (DUSP) genetic variants predict pulmonary hypertension in patients with bronchopulmonary dysplasia.

BACKGROUND: Pulmonary hypertension (PH) in patients with bronchopulmonary dysplasia (BPD) results from vasoconstriction and/or vascular remodeling, which can be regulated by mitogen-activated protein kinases (MAPKs). MAPKs are deactivated by dual-specificity phosphatases (DUSPs). We hypothesized that single-nucleotide polymorphisms (SNPs) in DUSP genes could be used to predict PH in BPD. METHODS: Preterm infants diagnosed with BPD (n = 188) were studied. PH was defined by echocardiographic criteria. Genomic DNA isolated from patient blood samples was analyzed for 31 SNPs in DUSP genes. Clinical characteristics and minor allele frequencies were compared between BPD-PH (cases) and BPD-without PH (control) groups. Biomarker models to predict PH in BPD using clinical and SNP data were tested by calculations of area under the ROC curve. RESULTS: In our BPD cohort, 32% (n = 61) had PH. Of the DUSP SNPs evaluated, DUSP1 SNP rs322351 was less common, and DUSP5 SNPs rs1042606 and rs3793892 were more common in cases than in controls. The best fit biomarker model combines clinical and DUSP genetic data with an area under the ROC curve of 0.76. CONCLUSION: We identified three DUSP SNPs as potential BPD-PH biomarkers. Combining clinical and DUSP genetic data yields the most robust predictor for PH in BPD.

Thioredoxin Reductase-1 Inhibition Augments Endogenous Glutathione-Dependent Antioxidant Responses in Experimental Bronchopulmonary Dysplasia.

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

Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia.

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2-/- mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by Nε -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.

MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung.

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.

UGT1A1*28 Genotypes and Respiratory Disease in Very Preterm Infants: A Cohort Study.

BACKGROUND: Respiratory disease in the very preterm infant is frequent and often severe. Bilirubin is both a potent neurotoxin and antioxidant, and may have a clinical impact on preterm respiratory disease. The Gilbert genotype, the UGT1A1*28 allele, is the major known genetic cause of variation in bilirubin. OBJECTIVES: To study the association between respiratory disease in the very preterm infant and the UGT1A1*28 allele. METHODS: This is a cohort study of 1,354 very preterm infants (gestational age <32 weeks) born in Jutland, Denmark in 1997-2011. Genotypes were obtained from the Danish Neonatal Screening Biobank, and clinical information was obtained from the databases of two tertiary neonatal intensive care units. Outcomes were the need for surfactant therapy, any need for and duration of supplementary oxygen and bronchopulmonary dysplasia (BPD). RESULTS: Per UGT1A1*28 allele, odds were increased for any need of supplementary oxygen (odds ratio 1.26; 1.05-1.50) and for BPD (odds ratio 1.71; 1.23-2.39), the need of supplementary oxygen increased by 6.38 days (1.87-10.89), and chance per day of no longer needing supplementary oxygen was reduced (hazard rate 0.84; 0.76-0.93). No effect was observed for need of surfactant treatment (odds ratio 1.08; 0.91-1.28). Hardy-Weinberg equilibrium was unlikely for the cohort (p < 0.012). This could be explained by death prior to genotype sampling. In tests of robustness this failed to explain the primary results. CONCLUSIONS: Compared to the common genotype, UGT1A1*28 genotypes were associated with an increased need of oxygen supplementation and risk of BPD in very preterm newborns.

Lack of EC-SOD worsens alveolar and vascular development in a neonatal mouse model of bleomycin-induced bronchopulmonary dysplasia and pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) worsens clinical outcomes in former preterm infants with bronchopulmonary dysplasia (BPD). Oxidant stress disrupts alveolar and vascular development in models of BPD. Bleomycin causes oxidative stress and induces BPD and PAH in neonatal rats. Disruption in the vascular endothelial growth factor (VEGF) and nitric oxide signaling pathways contributes to BPD. We hypothesized that loss of EC-SOD would worsen PAH associated with BPD in a neonatal mouse model of bleomycin-induced BPD by disrupting the VEGF/NO signaling pathway. METHODS: Neonatal wild-type mice (WT), and mice lacking EC-SOD (EC-SOD KO) received intraperitoneal bleomycin (2 units/kg) or phosphate-buffered saline (PBS) three times weekly and were evaluated at weeks 3 or 4. RESULTS: Lack of EC-SOD impaired alveolar development and resulted in PH (elevated right ventricular systolic pressures, right ventricular hypertrophy (RVH)), decreased vessel density, and increased small vessel muscularization. Exposure to bleomycin further impaired alveolar development, worsened RVH and vascular remodeling. Lack of EC-SOD and bleomycin treatment decreased lung total and phosphorylated VEGFR2 and eNOS protein expression. CONCLUSION: EC-SOD is critical in preserving normal lung development and loss of EC-SOD results in disrupted alveolar development, PAH and vascular remodeling at baseline, which is further worsened with bleomycin and associated with decreased activation of VEGFR2.

Mouse lung development and NOX1 induction during hyperoxia are developmentally regulated and mitochondrial ROS dependent.

Animal models demonstrate that exposure to supraphysiological oxygen during the neonatal period compromises both lung and pulmonary vascular development, resulting in a phenotype comparable to bronchopulmonary dysplasia (BPD). Our prior work in murine models identified postnatal maturation of antioxidant enzyme capacities as well as developmental regulation of mitochondrial oxidative stress in hyperoxia. We hypothesize that consequences of hyperoxia may also be developmentally regulated and mitochondrial reactive oxygen species (ROS) dependent. To determine whether age of exposure impacts the effect of hyperoxia, neonatal mice were placed in 75% oxygen for 72 h at either postnatal day 0 (early postnatal) or day 4 (late postnatal). Mice exposed to early, but not late, postnatal hyperoxia demonstrated decreased alveolarization and septation, increased muscularization of resistance pulmonary arteries, and right ventricular hypertrophy (RVH) compared with normoxic controls. Treatment with a mitochondria-specific antioxidant, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), during early postnatal hyperoxia protected against compromised alveolarization and RVH. In addition, early, but not late, postnatal hyperoxia resulted in induction of NOX1 expression that was mitochondrial ROS dependent. Because early, but not late, exposure resulted in compromised lung and cardiovascular development, we conclude that the consequences of hyperoxia are developmentally regulated and decrease with age. Attenuated disease in mitoTEMPO-treated mice implicates mitochondrial ROS in the pathophysiology of neonatal hyperoxic lung injury, with potential for amplification of ROS signaling through NOX1 induction. Furthermore, it suggests a potential role for targeted antioxidant therapy in the prevention or treatment of BPD.

Arginase inhibition prevents bleomycin-induced pulmonary hypertension, vascular remodeling, and collagen deposition in neonatal rat lungs.

Arginase is an enzyme that limits substrate L-arginine bioavailability for the production of nitric oxide by the nitric oxide synthases and produces L-ornithine, which is a precursor for collagen formation and tissue remodeling. We studied the pulmonary vascular effects of arginase inhibition in an established model of repeated systemic bleomycin sulfate administration in neonatal rats that results in pulmonary hypertension and lung injury mimicking the characteristics typical of bronchopulmonary dysplasia. We report that arginase expression is increased in the lungs of bleomycin-exposed neonatal rats and that treatment with the arginase inhibitor amino-2-borono-6-hexanoic acid prevented the bleomycin-induced development of pulmonary hypertension and deposition of collagen. Arginase inhibition resulted in increased L-arginine and L-arginine bioavailability and increased pulmonary nitric oxide production. Arginase inhibition also normalized the expression of inducible nitric oxide synthase, and reduced bleomycin-induced nitrative stress while having no effect on bleomycin-induced inflammation. Our data suggest that arginase is a promising target for therapeutic interventions in neonates aimed at preventing lung vascular remodeling and pulmonary hypertension.

Substance P protects against hyperoxic-induced lung injury in neonatal rats.

The aim of the study was to investigate the effects of substance P (SP) in hyperoxia-induced lung injury in newborn rats. Thirty-two rat pups were randomly divided into four groups: normoxia/saline, normoxia/SP, hyperoxia/saline and hyperoxia/SP. In a separate set of experiments, the neonatal rat pups were exposed to 21% or >95% O2 for 14 days with or without intraperitoneal administration of SP. On day 14, the animals were sacrificed and the lungs were processed for histology and biochemical analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used for the detection of apoptosis. Antioxidant capacity was assessed by glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), oxidative stress was assessed by determining the extent of formation of malondialdehyde (MDA), activities of NADPH oxidase activity, and formation of reactive oxygen species (ROS). The activity of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2) proteins and expression of NF-E2-related factor 2 (NRF2) were detected by Western blot, and the expression of p-p38 was detected by immunofluorescence analysis. Compared with the hyperoxia treatment, the lung damage was significantly ameliorated following the SP treatment. Furthermore, the lungs from the pups exposed to hyperoxia TUNEL-positive nuclei increased markedly and decreased significantly after SP treatment. The levels of MDA decreased and that of GSH-Px and SOD increased following the SP treatment. The SP treatment significantly suppressed the activity of NADPH oxidase and reduced ROS production. SP stimulation may result in blocking p38 MAPK and ERK signaling pathways, and the activities of p-p38 and p-ERK, and expression of NRF2 decreased following the SP treatment. These findings indicate that SP can ameliorate hyperoxic lung injury through decreasing cell apoptosis, elevating antioxidant activities, and attenuating oxidative stress.

Role of serine proteases in the regulation of interleukin-877 during the development of bronchopulmonary dysplasia in preterm ventilated infants.

RATIONALE: The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-877) is a less potent chemoattractant than other shorter isoforms. Although interleukin-877 is abundant in the preterm circulation, its regulation in the preterm lung is unknown. OBJECTIVES: To study expression and processing of pulmonary interleukin-877 in preterm infants who did and did not develop bronchopulmonary dysplasia. METHODS: Total interleukin-8 and interleukin-877 were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function. MAIN RESULTS: Peak total interleukin-8 and interleukin-877 concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-877 to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-877 to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity in vitro (p<0.01). CONCLUSIONS: Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-877 by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia.

Superoxide dismutase 3 dysregulation in a murine model of neonatal lung injury.

Bronchopulmonary dysplasia (BPD), a common chronic respiratory disease that occurs after premature birth, is believed to be secondary to oxidative damage from hyperoxia and inflammation, which leads to impaired alveolar formation and chronic lung dysfunction. We hypothesized that extracellular superoxide dismutase (SOD)3, an antioxidant uniquely targeted to the extracellular matrix (ECM) and alveolar fluid, might have a different response (down-regulation) to hyperoxic injury and recovery in room air (RA), thereby contributing to the persistent airspace injury and inflammation. We used a murine BPD model using postnatal hyperoxia (O2) (4 or 5 d) followed by short-term recovery (14 d) in RA, which mimics the durable effects after injury during alveolar development. This was associated with significantly increased mRNA expression for antioxidant genes mediated by nuclear factor erythroid 2-related factor (Nrf2) in the O2 (n = 4) versus RA group (n = 5). SOD3, an Nrf2-independent antioxidant, was significantly reduced in the O2-exposed mice compared with RA. Immunohistochemistry revealed decreased and disrupted SOD3 deposition in the alveolar ECM of O2-exposed mice. Furthermore, this distinct hyperoxic antioxidant and injury profile was reproducible in murine lung epithelial 12 cells exposed to O2. Overexpression of SOD3 rescued the injury measures in the O2-exposed cells. We establish that reduced SOD3 expression correlates with alveolar injury measures in the recovered neonatal hyperoxic lung, and SOD3 overexpression attenuates hyperoxic injury in an alveolar epithelial cell line. Such findings suggest a candidate mechanism for the pathogenesis of BPD that may lead to targeted interventions.

GSTM1 and GSTT1 gene polymorphisms as major risk factors for bronchopulmonary dysplasia in a Chinese Han population.

Bronchopulmonary dysphasia (BPD) is a complex multifactorial disease with an obvious genetic predisposition. Oxidative stress plays an important role in its pathogenesis. Glutathione S-transferases (GSTs) detoxify metabolites produced by oxidative stress within the cell and protect the cells against injury. In the present study, the hypothesis that polymorphisms in the GSTM1 and GSTT1 genes are associated with BPD in Chinese Han infants was examined. Sixty infants with BPD and 100 gestational age and birth weight-matched preterm infants without BPD were recruited. Genotyping for GSTM1 and GSTT1 was performed by multiplex polymerase chain reaction (PCR). The GSTM1 null genotype was more prevalent in BPD infants (65.0%) than in the control subjects (48.0%), which yielded higher risk towards BPD (odds ratio (OR): 2.012, 95% confidence interval (CI)=1.040-3.892, p=0.037). There was no statistically significant association of GSTT1 genotype with BPD (OR: 1.691, 95% CI=0.884-3.236, p=0.111), although the frequency of GSTT1 null genotype was higher among the BPD subjects (60.0%) than in the control patients (47.0%). GSTM1 and GSTT1 double null genotype was also higher in BPD group (38.3%) than in controls (21.0%) with a higher risk towards BPD (OR: 2.338, 95%CI=1.151-4.751, p=0.017). The results suggest that null genotypes of GSTM1 and GSTT1 genes may contribute to the development of BPD in our Chinese Han population.

Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a common and serious complication of premature birth, characterized by a pronounced arrest of alveolar development. The underlying pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix (ECM) are emerging as candidate disease pathomechanisms. In this study, the expression and regulation of three members of the lysyl hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and Plod3) in clinical BPD, as well as in an experimental animal model of BPD, were addressed. All three enzymes were localized to the septal walls in developing mouse lungs, with Plod1 also expressed in the vessel walls of the developing lung and Plod3 expressed uniquely at the base of developing septa. The expression of plod1, plod2, and plod3 was upregulated in the lungs of mouse pups exposed to 85% O2, an experimental animal model of BPD. Transforming growth factor (TGF)-ß increased plod2 mRNA levels and activated the plod2 promoter in vitro in lung epithelial cells and in lung fibroblasts. Using in vivo neutralization of TGF-ß signaling in the experimental animal model of BPD, TGF-ß was identified as the regulator of aberrant plod2 expression. PLOD2 mRNA expression was also elevated in human neonates who died with BPD or at risk for BPD, compared with neonates matched for gestational age at birth or chronological age at death. These data point to potential roles for lysyl hydroxylases in normal lung development, as well as in perturbed late lung development associated with BPD.

Sphingosine kinase 1 deficiency confers protection against hyperoxia-induced bronchopulmonary dysplasia in a murine model: role of S1P signaling and Nox proteins.

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

Targeting glycogen synthase kinase-3ß to prevent hyperoxia-induced lung injury in neonatal rats.

The pathological hallmarks of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, include inflammation, arrested alveolarization, and dysregulated angiogenesis. Severe BPD is often complicated by pulmonary hypertension (PH) that significantly increases morbidity and mortality. Glycogen synthase kinase (GSK)-3ß plays a pivotal role in embryonic development, cell proliferation and survival, and inflammation by modulating multiple signaling pathways, particularly the nuclear transcription factor, NF-κB, and Wnt/ß-catenin pathways. Aberrant GSK-3ß signaling is linked to BPD. We tested the hypothesis that inhibition of GSK-3ß is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia or hyperoxia (90% oxygen), and received daily intraperitoneal injections of placebo (DMSO) or SB216763, a specific pharmacological inhibitor of GSK-3ß, for 14 days. Hyperoxia exposure in the presence of the placebo increased GSK-3ß phosphorylation, which was correlated with increased inflammation, decreased alveolarization and angiogenesis, and increased pulmonary vascular remodeling and PH. However, treatment with SB216763 decreased phosphorylation of NF-κB p65, expression of monocyte chemotactic protein-1, and lung inflammation during hyperoxia. Furthermore, treatment with the GSK-3ß inhibitor also improved alveolarization and angiogenesis, and decreased pulmonary vascular remodeling and PH. These data indicate that GSK-3ß signaling plays an important role in the pathogenesis of hyperoxia-induced neonatal lung injury, and that inhibition of GSK-3ß is beneficial in preventing inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting GSK-3ß signaling may offer a novel strategy to prevent and treat preterm infants with BPD.

Glycogen synthase kinase-3ß/ß-catenin signaling regulates neonatal lung mesenchymal stromal cell myofibroblastic differentiation.

In bronchopulmonary dysplasia (BPD), alveolar septa are thickened with collagen and α-smooth muscle actin-, transforming growth factor (TGF)-ß-positive myofibroblasts. We examined the biochemical mechanisms underlying myofibroblastic differentiation, focusing on the role of glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin signaling pathway. In the cytoplasm, ß-catenin is phosphorylated on the NH(2) terminus by constitutively active GSK-3ß, favoring its degradation. Upon TGF-ß stimulation, GSK-3ß is phosphorylated and inactivated, allowing ß-catenin to translocate to the nucleus, where it activates transcription of genes involved in myofibroblastic differentiation. We examined the role of ß-catenin in TGF-ß1-induced myofibroblastic differentiation of neonatal lung mesenchymal stromal cells (MSCs) isolated from tracheal aspirates of premature infants with respiratory distress. TGF-ß1 increased ß-catenin expression and nuclear translocation. Transduction of cells with GSK-3ß S9A, a nonphosphorylatable, constitutively active mutant that favors ß-catenin degradation, blocked TGF-ß1-induced myofibroblastic differentiation. Furthermore, transduction of MSCs with ΔN-catenin, a truncation mutant that cannot be phosphorylated on the NH(2) terminus by GSK-3ß and is not degraded, was sufficient for myofibroblastic differentiation. In vivo, hyperoxic exposure of neonatal mice increases expression of ß-catenin in α-smooth muscle actin-positive myofibroblasts. Similar changes were found in lungs of infants with BPD. Finally, low-passage unstimulated MSCs from infants developing BPD showed higher phospho-GSK-3ß, ß-catenin, and α-actin content compared with MSCs from infants not developing this disease, and phospho-GSK-3ß and ß-catenin each correlated with α-actin content. We conclude that phospho-GSK-3ß/ß-catenin signaling regulates α-smooth muscle actin expression, a marker of myofibroblast differentiation, in vitro and in vivo. This pathway appears to be activated in lung mesenchymal cells from patients with BPD.

Vasculoprotective effects of heme oxygenase-1 in a murine model of hyperoxia-induced bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is characterized by simplified alveolarization and arrested vascular development of the lung with associated evidence of endothelial dysfunction, inflammation, increased oxidative damage, and iron deposition. Heme oxygenase-1 (HO-1) has been reported to be protective in the pathogenesis of diseases of inflammatory and oxidative etiology. Because HO-1 is involved in the response to oxidative stress produced by hyperoxia and is critical for cellular heme and iron homeostasis, it could play a protective role in BPD. Therefore, we investigated the effect of HO-1 in hyperoxia-induced lung injury using a neonatal transgenic mouse model with constitutive lung-specific HO-1 overexpression. Hyperoxia triggered an increase in pulmonary inflammation, arterial remodeling, and right ventricular hypertrophy that was attenuated by HO-1 overexpression. In addition, hyperoxia led to pulmonary edema, hemosiderosis, and a decrease in blood vessel number, all of which were markedly improved in HO-1 overexpressing mice. The protective vascular response may be mediated at least in part by carbon monoxide, due to its anti-inflammatory, antiproliferative, and antiapoptotic properties. HO-1 overexpression, however, did not prevent alveolar simplification nor altered the levels of ferritin and lactoferrin, proteins involved in iron binding and transport. Thus the protective mechanisms elicited by HO-1 overexpression primarily preserve vascular growth and barrier function through iron-independent, antioxidant, and anti-inflammatory pathways.

Hyperoxia impairs alveolar formation and induces senescence through decreased histone deacetylase activity and up-regulation of p21 in neonatal mouse lung.

Alveolar development comprises the transition of lung architecture from saccules to gas-exchange units during late gestation and early postnatal development. Exposure to hyperoxia disrupts developmental signaling pathways and causes alveolar hypoplasia as seen in bronchopulmonary dysplasia affecting preterm human newborns. Expanding literature suggests that epigenetic changes caused by environmental triggers during development may lead to heritable changes in gene expression. Given recent data on altered histone deacetylase (HDAC) activity in lungs of humans and animal models with airspace enlargement/emphysema, we hypothesized that alveolar hypoplasia from hyperoxia exposure in neonatal mice is a consequence of cell cycle arrest and reduced HDAC activity and up-regulation of the cyclin-dependent kinase inhibitor, p21. We exposed newborn mice to hyperoxia and compared lung morphologic and epigenetic changes to room air controls. Furthermore, we pretreated a subgroup of animals with the macrolide antibiotic azithromycin (AZM), known to possess antiinflammatory properties. Our results showed that hyperoxia exposure resulted in alveolar hypoplasia and was associated with decreased HDAC1 and HDAC2 and increased p53 and p21 expression. Furthermore, AZM did not confer protection against hyperoxia-induced alveolar changes. These findings suggest that alveolar hypoplasia caused by hyperoxia is mediated by epigenetic changes affecting cell cycle regulation/senescence during lung development.

A role for matrix metalloproteinase 9 in IFNγ-mediated injury in developing lungs: relevance to bronchopulmonary dysplasia.

We noted a marked increase in IFNγ mRNA in newborn (NB) murine lungs after exposure to hyperoxia. We sought to evaluate the role of IFNγ in lung injury in newborns. Using a unique triple-transgenic (TTG), IFNγ-overexpressing, lung-targeted, externally regulatable NB murine model, we describe a lung phenotype of impaired alveolarization, resembling human bronchopulmonary dysplasia (BPD). IFNγ-mediated abnormal lung architecture was associated with increased cell death and the upregulation of cell death pathway mediators caspases 3, 6, 8, and 9, and angiopoietin 2. Moreover, an increase was evident in cathepsins B, H, K, L, and S, and in matrix metalloproteinases (MMPs) 2, 9, 12, and 14. The IFNγ-mediated abnormal lung architecture was found to be MMP9-dependent, as indicated by the rescue of the IFNγ-induced pulmonary phenotype and survival during hyperoxia with a concomitant partial deficiency of MMP9. This result was concomitant with a decrease in caspases 3, 6, 8, and 9 and angiopoietin 2, but an increase in the expression of angiopoietin 1. In addition, NB IFNγ TTG mice exhibited significantly decreased survival during hyperoxia, compared with littermate controls. Furthermore, as evidence of clinical relevance, we show increased concentrations of the downstream targets of IFNγ chemokine (C-X-C motif) ligands (CXCL10 and CXCL11) in baboon and human lungs with BPD. IFNγ and its downstream targets may contribute significantly to the final common pathway of hyperoxia-induced injury in the developing lung and in human BPD.