transforming-growth-factor-beta and Hyperoxia

The carotid body may undergo plasticity changes during development/ageing and in response to environmental (hypoxia and hyperoxia), metabolic, and inflammatory stimuli. The different cell types of the carotid body express a wide series of growth factors and corresponding receptors, which play a role in the modulation of carotid body function and plasticity. In particular, type I cells express nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, insulin-like-growth factor-I and -II, basic fibroblast growth factor, epidermal growth factor, transforming growth factor-α and -β, interleukin-1β and -6, tumor necrosis factor-α, vascular endothelial growth factor, and endothelin-1. Many specific growth factor receptors have been identified in type I cells, indicating autocrine/paracrine effects. Type II cells may also produce growth factors and express corresponding receptors. Future research will have to consider growth factors in further experimental models of cardiovascular, metabolic, and inflammatory diseases and in human (normal and pathologic) samples. From a methodological point of view, microarray and/or proteomic approaches would permit contemporary analyses of large groups of growth factors. The eventual identification of physical interactions between receptors of different growth factors and/or neuromodulators could also add insights regarding functional interactions between different trophic mechanisms.

Topics: Animals; Brain-Derived Neurotrophic Factor; Carotid Body; Ciliary Neurotrophic Factor; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression Regulation; Glial Cell Line-Derived Neurotrophic Factor; Humans; Hyperoxia; Hypoxia; Insulin-Like Growth Factor I; Nerve Growth Factor; Neurotrophin 3; Receptors, Growth Factor; Transforming Growth Factor alpha; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Various pre- and postnatal risk factors, which act additively or synergistically induce an injurious inflammatory response in the airways and the pulmonary interstitium of preterm infants with bronchopulmonary dysplasia. This inflammatory response is characterized by an accumulation of neutrophils and macrophages as well as an arsenal of proinflammatory mediators that affect the endothelium and alveolar-capillary integrity. Besides proinflammatory cytokines and toxic oxygen radicals, lipid mediators as well as potent proteases may be responsible for acute lung injury. There is increasing evidence that an imbalance between pro- and anti-inflammatory factors, which should protect the alveoli and lung tissue, are key features in the pathogenesis of bronchopulmonary dysplasia. In addition, a subnormal generation of growth factors may affect alveolarization and vascular development in preterm infants with bronchopulmonary dysplasia. In this condensed review article, the current concepts on the possible role of inflammation in the evolution of bronchopulmonary dysplasia will be summarized.

Topics: Blood-Air Barrier; Bronchopulmonary Dysplasia; Chemotaxis; Chorioamnionitis; Cytokines; Endothelium; Female; Humans; Hyperoxia; Infant, Newborn; Infant, Premature; Inflammation Mediators; Intercellular Signaling Peptides and Proteins; Macrophages; Neutrophils; Oxygen; Peptide Hydrolases; Pneumonia; Pregnancy; Pulmonary Alveoli; Respiration, Artificial; Transforming Growth Factor beta

Hyperoxia-induced lung injury is characterized by acute alveolar injury, disrupted epithelial-mesenchymal signaling, oxidative stress, and surfactant dysfunction, yet currently, there is no effective treatment. Although a combination of aerosolized pioglitazone (PGZ) and a synthetic lung surfactant (B-YL peptide, a surfactant protein B mimic) prevents hyperoxia-induced neonatal rat lung injury, whether it is also effective in preventing hyperoxia-induced adult lung injury is unknown.. Using adult mice lung explants, we characterize the effects of 24 and 72-h (h) exposure to hyperoxia on 1) perturbations in Wingless/Int (Wnt) and Transforming Growth Factor (TGF)-β signaling pathways, which are critical mediators of lung injury, 2) aberrations of lung homeostasis and injury repair pathways, and 3) whether these hyperoxia-induced aberrations can be blocked by concomitant treatment with PGZ and B-YL combination.. Our study reveals that hyperoxia exposure to adult mouse lung explants causes activation of Wnt (upregulation of key Wnt signaling intermediates β-catenin and LEF-1) and TGF-β (upregulation of key TGF-β signaling intermediates TGF-β type I receptor (ALK5) and SMAD 3) signaling pathways accompanied by an upregulation of myogenic proteins (calponin and fibronectin) and inflammatory cytokines (IL-6, IL-1β, and TNFα), and alterations in key endothelial (VEGF-A and its receptor FLT-1, and PECAM-1) markers. All of these changes were largely mitigated by the PGZ + B-YL combination.. The effectiveness of the PGZ + B-YL combination in blocking hyperoxia-induced adult mice lung injury ex-vivo is promising to be an effective therapeutic approach for adult lung injury in vivo.

Topics: Animals; Hyperoxia; Lung; Lung Injury; Mice; Pioglitazone; PPAR gamma; PPAR-gamma Agonists; Surface-Active Agents; Transforming Growth Factor beta

It is well known that supplemental oxygen used to treat preterm infants in respiratory distress is associated with permanently disrupting lung development and the host response to influenza A virus (IAV). However, many infants who go home with normally functioning lungs are also at risk for hyperreactivity after a respiratory viral infection. We recently reported a new, low-dose hyperoxia mouse model (40% for 8 days; 40×8) that causes a transient change in lung function that resolves, rendering 40×8 adult animals functionally indistinguishable from room air controls. Here we report that when infected with IAV, 40×8 mice display an early transient activation of TGFβ signaling and later airway hyperreactivity associated with peribronchial inflammation (profibrotic macrophages) and fibrosis compared with infected room air controls, suggesting neonatal oxygen induced hidden molecular changes that prime the lung for hyperreactive airways disease. Although searching for potential activators of TGFβ signaling, we discovered that thrombospondin-1 (TSP-1) is elevated in naïve 40×8 mice compared with controls and localized to lung megakaryocytes and platelets before and during IAV infection. Elevated TSP-1 was also identified in human autopsy samples of former preterm infants with bronchopulmonary dysplasia. These findings reveal how low doses of oxygen that do not durably change lung function may prime it for hyperreactive airways disease by changing expression of genes, such as TSP-1, thus helping to explain why former preterm infants who have normal lung function are susceptible to airway obstruction and increased morbidity after viral infection.

Topics: Animals; Bronchial Hyperreactivity; Bronchopulmonary Dysplasia; Cell Line; Disease Models, Animal; Dogs; Female; Humans; Hyperoxia; Influenza A virus; Influenza, Human; Madin Darby Canine Kidney Cells; Male; Mice; Mice, Inbred C57BL; Orthomyxoviridae Infections; Pulmonary Fibrosis; Thrombospondin 1; Transforming Growth Factor beta

Bronchopulmonary dysplasia (BPD) is a severe chronic lung disease in preterm infants. Circular RNAs (circRNAs) are key regulators of various biological processes. The present study aimed to explore the biological roles of circRNAs in BPD pathogenesis.. A newborn BPD rat model was developed to construct a circRNA library; Illumina deep sequencing (Illumina, San Diego, CA, USA) was used to reveal differential expression of circRNAs in the hyperoxia-induced BPD rat models. Sanger sequencing and a reverse transcription-polymerase chain reaction were performed to confirm circRNAs that may be related to BPD. After miRNA binding-site prediction, we constructed a network diagram of circRNA-competing endogenous RNAs (ceRNAs) related to transforming growth factor (TGF)-β and p53 pathways using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.. In total, 256 differentially expressed circRNAs were detected between the hyperoxia group and the normoxia group. Of these circRNAs, 195 were up-regulated and 61 were down-regulated. The differences of circRNA distribution between the two groups were analyzed and six circRNAs were validated in the tissue samples. GO analysis indicated that 6519 target genes were enriched in cell location and biological processes. KEGG pathway enrichment analysis showed that circRNAs involved in 242 KEGG pathways. A network diagram of circRNA-ceRNA related to TGF-β and p53 pathways was constructed.. CircRNAs are differentially expressed between the BPD model and control group. Many target genes of circRNAs are involved in the developmental process, which suggests that BPD may be associated with pathways including extracellular matrix-receptor interaction, vascular endothelial growth factor signaling and vascular smooth muscle contraction.

Topics: Animals; Bronchopulmonary Dysplasia; Computational Biology; Disease Models, Animal; Down-Regulation; Gene Expression Profiling; Gene Expression Regulation; Gene Ontology; High-Throughput Nucleotide Sequencing; Humans; Hyperoxia; Immunohistochemistry; Infant, Newborn; Lung; Rats; Rats, Sprague-Dawley; RNA, Circular; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Protein p53; Up-Regulation

Topics: Acute Kidney Injury; Animals; Animals, Newborn; Antioxidants; Body Weight; Cell Proliferation; Disease Models, Animal; Female; Glomerular Filtration Rate; Hyperoxia; Inflammation; Interleukin-6; Kidney Cortex; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Organ Size; Oxygen; Regeneration; Smad2 Protein; STAT3 Transcription Factor; Transforming Growth Factor beta

The identification and development of novel therapeutic strategies for acute lung injury is urgently required. It has been previously demonstrated that microRNA (miR)‑16 suppresses the level of transforming growth factor (TGF)‑β in acute lung injury (ALI). Therefore, the present study investigated the role of miR‑16 in the phenotype, cell proliferation and apoptosis, and the involvement of TGF‑β/Smad family member 2 (Smad2) and JAK/signal transducer and activator of transcription (STAT)3 signaling, of primary human alveolar type II epithelial cells (AECII). Following transfection with miR‑16 mimics, AECII cells were exposed to hyperoxia for 24 h. Subsequently, immunofluorescence staining of surfactant protein‑A (SP‑A) was performed, and cell proliferation and apoptosis were investigated by Cell Counting Kit‑8 assays and annexin V‑fluorescein isothiocyanate/propidium iodide staining, respectively. Furthermore, the expression levels of miR‑16, TGF‑β, Smad2, phosphorylated‑Smad2, JAK and STAT3 were detected by western blotting and/or reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that miR‑16 levels and SP‑A fluorescence were markedly inhibited by hyperoxia. Furthermore, transfection of AECII cells with miR‑16 mimics increased SP‑A fluorescence in hyperoxia‑treated AECII cells, significantly reversed hyperoxia‑induced reductions in cell proliferation and inhibited hyperoxia‑induced apoptosis. Finally, miR‑16 mimics modulated the mRNA and protein expression of components of the TGF‑β/Smad2 and JAK/STAT3 pathways in AECII cells following hyperoxia. In conclusion, the results of the present study indicate that overexpression of miR‑16 may exert a protective effect in AECII cells against cell apoptosis and ALI, which may be associated with TGF‑β/Smad2 and JAK/STAT3 signaling pathways. This may also represent a promising target for novel therapeutic strategies for acute lung injury.

Topics: Alveolar Epithelial Cells; Apoptosis; Cell Proliferation; Cells, Cultured; Gene Expression; Humans; Hyperoxia; Janus Kinases; MicroRNAs; Signal Transduction; Smad2 Protein; STAT3 Transcription Factor; Transfection; Transforming Growth Factor beta

Caffeine is widely used to manage apnea of prematurity, and reduces the incidence of bronchopulmonary dysplasia (BPD). Deregulated transforming growth factor (TGF)-β signaling underlies arrested postnatal lung maturation in BPD. It is unclear whether caffeine impacts TGF-β signaling or postnatal lung development in affected lungs.. Caffeine downregulated expression of type I and type III TGF-β receptors, and Smad2; and potentiated TGF-β signaling in vitro. In vivo, caffeine administration normalized body mass under hyperoxic conditions, and normalized Smad2 phosphorylation detected in lung homogenates; however, caffeine administration neither improved nor worsened lung structure in hyperoxia-exposed mice, in which postnatal lung maturation was blunted.. Caffeine modulated TGF-β signaling in vitro and in vivo. Caffeine administration was well-tolerated by newborn mice, but did not influence the course of blunted postnatal lung maturation in a hyperoxia-based experimental mouse model of BPD.

Topics: Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Caffeine; Cells, Cultured; Disease Models, Animal; Fibroblasts; Hyperoxia; Mice, Inbred C57BL; Phosphorylation; Protein Serine-Threonine Kinases; Proteoglycans; Pulmonary Alveoli; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad2 Protein; Time Factors; Transforming Growth Factor beta

To determine whether prophylaxis with etanercept, an anti-inflammatory drug, would decrease the severity of lung injury in a neonatal rat model of bronchopulmonary dysplasia (BPD);. Rat pups were divided into three groups: pups exposed to room air (group 1; n = 10), to hyperoxia + placebo (group 2; n = 9), and to hyperoxia + etanercept (group 3; n = 8). Lung morphology was assessed by alveolar surface area percentage, which is a measure of alveolar size. The severities of lung inflammation and antioxidant capacity were assessed by quantifying tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), malondialdehyde (MDA), and superoxide dismutase (SOD) from lung homogenate;. The percentage of alveolar surface areas were significantly higher in group 3 compared to group 2 (p = .004) and similar in both group 1 and group 3 (p = .21). The mean level of lung MDA was significantly higher in group 2 compared to group 1 and group 3 (p < .05 for both). Lung homogenate SOD activities in group 3 was significantly higher than group 2 (p < .001). Furthermore, group 3 pups had lower levels of TNF-α and TGF-β in lung homogenate than that in group 2 (p < .05 for both) but similar in both group 1 and group 3;. Etanercept has favorable effects on alveolarization as well as inflammation and oxidative stress markers in a neonatal rat model of BPD.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchopulmonary Dysplasia; Disease Models, Animal; Etanercept; Female; Humans; Hyperoxia; Infant, Newborn; Infant, Premature; Lung; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Wistar; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Septation of the gas-exchange saccules of the morphologically immature mouse lung requires regulated timing, spatial direction, and dosage of transforming growth factor (TGF)-β signaling. We found that neonatal hyperoxia acutely initially diminished saccular TGF-β signaling coincident with alveolar simplification. However, sustained hyperoxia resulted in a biphasic response and subsequent up-regulation of TGF-β signaling, ultimately resulting in bronchopulmonary dysplasia. Significantly, we found that the TGF-β-induced matricellular protein (TGFBI) was similarly biphasically altered in response to hyperoxia. Moreover, genetic ablation revealed that TGFBI was required for normal alveolar structure and function. Although the phenotype was not neonatal lethal, Tgfbi-deficient lungs were morphologically abnormal. Mutant septal tips were stunted, lacked elastin-positive tips, exhibited reduced proliferation, and contained abnormally persistent alveolar α-smooth muscle actin myofibroblasts. In addition, Tgfbi-deficient lungs misexpressed TGF-β-responsive follistatin and serpine 1, and transiently suppressed myofibroblast platelet-derived growth factor α differentiation marker. Finally, despite normal lung volume, Tgfbi-null lungs displayed diminished elastic recoil and gas exchange efficiency. Combined, these data demonstrate that initial suppression of the TGF-β signaling apparatus, as well as loss of key TGF-β effectors (like TGFBI), underlies early alveolar structural defects, as well as long-lasting functional deficits routinely observed in chronic lung disease of infancy patients. These studies underline the complex (and often contradictory) role of TGF-β and indicate a need to design studies to associate alterations with initial appearance of phenotypical changes suggestive of bronchopulmonary dysplasia.

Topics: Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Extracellular Matrix Proteins; Hyperoxia; Lung; Mice; Myofibroblasts; Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Bronchopulmonary dysplasia (BPD) is a main chronic lung disease commonly occurs in preterm infants. BPD is characterized by impaired alveolarization and vascularization of the developing lung. Transforming growth factor-β (TGF-β) signaling pathway is known to play an important role during lung vascular development. In the present study, we examined whether the regulation of TGF-β-ALK-Smad signaling pathway influence on the disruption of pulmonary vascular development in newborn rats as hyperoxia-induced BPD model.. Newborn rats were continuously exposed to 21% or 85% O2 for 7 days, and subsequently kept in normoxic condition for another 14 days. Lung tissues harvested at each time point were evaluated for the expression of TGF-β1, ALK1, ALK5, phosphorylated Smad1/5, phosphorylated Smad2/3, VEGF, and endoglin, as accessed by both biochemical and immunohistological analyses.. Double-fluorescence immunohistochemical staining indicated these molecules were mainly expressed in pulmonary endothelial cells. The expression of TGF-β1 and ALK5 mRNA and protein were significantly increased in D5 hyperoxia group, while that of ALK1 mRNA and protein were significantly decreased. The level of phosphorylated Smad1/5 was significantly decreased in D7 hyperoxia group, whereas that of phosphorylated Smad2/3 was oppositely increased. In addition, the expression of vascular endothelial growth factor (VEGF) mRNA was increased at D1 with subsequent decrease in D7 hyperoxia group. There was no significantly difference in endoglin expression in entire experimental period.. These results indicate that exposure to hyperoxia altered the balance between TGF-β-ALK1-Smad1/5 and TGF-β-ALK5-Smad2/3 pathways in pulmonary endothelial cells, which may ultimately lead to the development of BPD.

Topics: Anaplastic Lymphoma Kinase; Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Hyperoxia; Immunohistochemistry; Lung; Rats; Receptor Protein-Tyrosine Kinases; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

IL-1β creates persistent pulmonary inflammation accompanied by elevated transforming growth factor β (TGF-β levels and is associated with abnormal elastogenesis, which is observed in bronchopulmonary dysplasia (BPD). Although progress has been made in this field, the mechanisms underlying this process remain only partially understood.. We assessed aberrant elastin localization-associated signaling in mouse pups exposed to 85% O2 treated with either IL-1Ra or 1D11, using morphometric analyses, quantitative RT-PCR, immunostaining, and ELISA. We also evaluated the derivation of elastin-producing cells using dual marker tracking. The regulatory mechanisms of IL-1β were investigated in vitro in lung epithelial and mesenchymal cells.. Elevated levels of IL-1β, αvβ6 and TGF-β1 were each associated with aberrant elastin production in O2-exposed lungs. IL-1Ra abolished TGF-β1 activation and αvβ6 upregulation, which occurred as a result of exposure to hyperoxia, whereas 1D11 had no discernible effect on the expression of either αvβ6 or IL-1β even following O2-exposure, suggesting that IL-1β was initially induced. Additionally, double staining revealed the presence of epithelium-derived elastin-producing cells, which was confirmed via in vitro IL-1β stress-induced epithelial-mesenchymal transformation (EMT) morphological and molecular marker changes, which may explain the altered lung elastin deposition and defective septation observed in BPD.. These data support the hypothesis that IL-1β was initially induced by hyperoxia; αvβ6 subsequently interacted with and activated TGF-β1, acting as an epithelial/mesenchymal signaling molecule that contributed to excessive alveolar elastogenesis, the primary pathological feature of BPD.

Topics: Animals; Antigens, Neoplasm; Cell Line; Down-Regulation; Elastin; Epithelial Cells; Epithelial-Mesenchymal Transition; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Hyperoxia; Integrins; Interleukin 1 Receptor Antagonist Protein; Interleukin-1beta; Mice, Inbred C57BL; Morphogenesis; Phenotype; Pulmonary Alveoli; Rats; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

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)-β 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-β signaling in the experimental animal model of BPD, TGF-β 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.

Topics: Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Cell Line; Epithelial Cells; Female; Humans; Hyperoxia; Infant, Newborn; Lung; Male; Mice; Mice, Inbred C57BL; Pregnancy; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; Transforming Growth Factor beta; Up-Regulation

Aberrant remodelling of the extracellular matrix in the developing lung may underlie arrested alveolarisation associated with bronchopulmonary dysplasia (BPD). Transglutaminases are regulators of extracellular matrix remodelling. Therefore, the expression and activity of transglutaminases were assessed in lungs from human neonates with BPD and in a rodent model of BPD. Transglutaminase expression and localisation were assessed by RT-PCR, immunoblotting, activity assay and immunohistochemical analyses of human and mouse lung tissues. Transglutaminase regulation by transforming growth factor (TGF)-β was investigated in lung cells by luciferase-based reporter assay and RT-PCR. TGF-β signalling was neutralised in vivo in an animal model of BPD, to determine whether TGF-β mediated the hyperoxia-induced changes in transglutaminase expression. Transglutaminase 2 expression was upregulated in the lungs of preterm infants with BPD and in the lungs of hyperoxia-exposed mouse pups, where lung development was arrested. Transglutaminase 2 localised to the developing alveolar septa. TGF-β was identified as a regulator of transglutaminase 2 expression in human and mouse lung epithelial cells. In vivo neutralisation of TGF-β signalling partially restored normal lung structure and normalised lung transglutaminase 2 mRNA expression. Our data point to a role for perturbed transglutaminase 2 activity in the arrested alveolarisation associated with BPD.

Topics: Animals; Bronchopulmonary Dysplasia; Epithelial Cells; Extracellular Matrix; Female; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; GTP-Binding Proteins; Humans; Hyperoxia; Infant; Infant, Newborn; Infant, Premature; Lung; Male; Mice; Protein Glutamine gamma Glutamyltransferase 2; Pulmonary Alveoli; Signal Transduction; Transforming Growth Factor beta; Transglutaminases

To investigate the effect of hypoxia or hyperoxia on the progression of hepatic fibrosis and to examine the role of transforming growth factor-β (TGF-β) in the livers of rats exposed to hypoxic or hyperoxic conditions.. Male Sprague-Dawley rats were injected intraperitoneally with thioacetamide to induce hepatic fibrosis and were randomly divided into a hypoxia group, a hyperoxia group and an untreated control group. Ten rats in the hypoxia group were exposed to an altitude of 20000 ft for 1 h/d during 7 wk. Ten rats in the hyperoxia group were exposed to a water depth of 20 m with 100% oxygen supply for 1 h/d during 7 wk. We evaluated the degree of hepatic fibrosis using Masson trichrome stain and examined the expression level of hepatic TGF-β mRNA using quantitative real-time reverse transcriptase-polymerase chain reaction analysis.. Eight of 10 rats exposed to hypoxia showed diffuse and confluent fibrosis with the formation of structurally abnormal parenchymal nodules involving the entire liver, consistent with hepatic cirrhosis. Nine of 10 rats exposed to hyperoxia also demonstrated obvious histological findings of hepatic cirrhosis identical to those in hypoxic rat livers. In contrast, 8 of 10 untreated rats had periportal or septal fibrosis only. The frequency of hepatic cirrhosis in hypoxic rats (P = 0.009) and hyperoxic rats (P = 0.003) was significantly higher than that in untreated rats. In addition, hepatic TGF-β mRNA levels in hyperoxic rats were significantly higher than those in untreated rats. The mean value of the normalized TGF-β mRNA/β-actin expression ratio in the hyperoxic rats was 1.9-fold higher than that in the untreated rats (P = 0.027).. We demonstrated that both hypoxia and hyperoxia accelerated the progression of hepatic fibrosis in rats. Significant up-regulation of hepatic TGF-β in hyperoxic rats suggests that TGF-β is involved in the acceleration of hepatic fibrosis under hyperoxic conditions.

Topics: Animals; Disease Progression; Hyperoxia; Hypoxia; Liver; Liver Cirrhosis; Male; Random Allocation; Rats, Sprague-Dawley; Transforming Growth Factor beta

Human amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury.. Newborn mouse pups were randomized to either normoxia (inspired O2 content (FiO2) = 0.21, n = 60) or hyperoxia (FiO2 = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14.. Hyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-β and platelet-derived growth factor-β). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia.. Intraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.

Topics: Amnion; Animals; Cells, Cultured; Epithelial Cells; Female; Humans; Hyperbaric Oxygenation; Hyperoxia; Infant, Newborn; Interleukin-1alpha; Interleukin-6; Lung Injury; Mice; Mice, Inbred C57BL; Platelet-Derived Growth Factor; Pregnancy; RNA, Messenger; Transforming Growth Factor beta

Curcumin, a potent anti-inflammatory and antioxidant agent, modulates peroxisome proliferator-activated receptor-γ signaling, a key molecule in the etiology of bronchopulmonary dysplasia (BPD). We have previously shown curcumin's acute protection against neonatal hyperoxia-induced lung injury. However, its longer-term protection against BPD is not known. Hypothesizing that concurrent treatment with curcumin protects the developing lung against hyperoxia-induced lung injury long-term, we determined if curcumin protects against hyperoxic neonatal rat lung injury for the first 5 days of life, as determined at postnatal day (PND) 21. One-day-old rat pups were exposed to either 21 or 95% O₂ for 5 days with or without curcumin treatment (5 mg/kg) administered intraperitoneally one time daily, following which the pups grew up to PND21 in room air. At PND21 lung development was determined, including gross and cellular structural and functional effects, and molecular mediators of inflammatory injury. To gain mechanistic insights, embryonic day 19 fetal rat lung fibroblasts were examined for markers of apoptosis and MAP kinase activation following in vitro exposure to hyperoxia for 24 h in the presence or absence of curcumin (5 μM). Curcumin effectively blocked hyperoxia-induced lung injury based on systematic analysis of markers for lung injury (apoptosis, Bcl-2/Bax, collagen III, fibronectin, vimentin, calponin, and elastin-related genes) and lung morphology (radial alveolar count and alveolar septal thickness). Mechanistically, curcumin prevented the hyperoxia-induced increases in cleaved caspase-3 and the phosphorylation of Erk1/2. Molecular effects of curcumin, both structural and cytoprotective, suggest that its actions against hyperoxia-induced lung injury are mediated via Erk1/2 activation and that it is a potential intervention against BPD.

Topics: Animals; Apoptosis; Biomarkers; Cell Proliferation; Curcumin; Elastin; Enzyme Activation; Female; Fibroblasts; Fibronectins; Gene Expression Regulation; Hyperoxia; Lung; MAP Kinase Signaling System; Mesoderm; Protective Agents; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Triglycerides

In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.

Topics: Aged; Aged, 80 and over; Animals; Animals, Newborn; Bronchopulmonary Dysplasia; Cell Adhesion Molecules; Cell Differentiation; DNA; Female; Gene Expression Regulation, Developmental; Gene Knockout Techniques; Humans; Hyperoxia; Hypoventilation; Infant, Newborn; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Myofibroblasts; Phenotype; Pulmonary Alveoli; RNA, Messenger; Transforming Growth Factor beta

Maternally derived inflammatory mediators, such as IL-6 and IL-8, contribute to preterm delivery, low birth weight, and respiratory insufficiency, which are routinely treated with oxygen. Premature infants are at risk for developing adult-onset cardiac, metabolic, and pulmonary diseases. Long-term pulmonary consequences of perinatal inflammation are unclear. We tested the hypothesis that a hostile perinatal environment induces profibrotic pathways resulting in pulmonary fibrosis, including persistently altered lung structure and function. Pregnant C3H/HeN mice injected with LPS or saline on embryonic day 16. Offspring were placed in room air (RA) or 85% O(2) for 14 days and then returned to RA. Pulmonary function tests, microCTs, molecular and histological analyses were performed between embryonic day 18 and 8 wk. Alveolarization was most compromised in LPS/O(2)-exposed offspring. Collagen staining and protein levels were increased, and static compliance was decreased only in LPS/O(2)-exposed mice. Three-dimensional microCT reconstruction and quantification revealed increased tissue densities only in LPS/O(2) mice. Diffuse interstitial fibrosis was associated with decreased micro-RNA-29, increased transforming growth factor-β expression, and phosphorylation of Smad2 during embryonic or early fetal lung development. Systemic maternal LPS administration in combination with neonatal hyperoxic exposure induces activation of profibrotic pathways, impaired alveolarization, and diminished lung function that are associated with prenatal and postnatal suppression of miR-29 expression.

Topics: Animals; Collagen; Female; Fibrosis; Hyperoxia; Inflammation; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred C3H; MicroRNAs; Models, Animal; Pregnancy; Prenatal Exposure Delayed Effects; Pulmonary Alveoli; Smad2 Protein; Transforming Growth Factor beta

Granulocyte colony stimulating factor (G-CSF) has been known to increase neutrophil production and have anti-inflammatory properties, but the effect of G-CSF on pulmonary system is in controversy. We investigated whether G-CSF treatment could attenuate hyperoxia-induced lung injury, and whether this protective effect is mediated by the down-modulation of inflammatory responses in a neonatal rat model.. Newborn Sprague-Dawley rats (Orient Co., Seoul, Korea) were subjected to 14 days of hyperoxia (90% oxygen) beginning within 10 h after birth. G-CSF (20 μg/kg) was administered intraperitoneally on the fourth, fifth, and sixth postnatal days.. This treatment significantly improved hyperoxia-induced reduction in body weight gain and lung pathology such as increased mean linear intercept, mean alveolar volume, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling positive cells. Hyperoxia- induced activation of nicotinamide adenine dinucleotide phosphate oxidase, which is responsible for superoxide anion production, as evidenced by upregulation and membrane translocation of p67(phox) was significantly attenuated after G-CSF treatment, as were inflammatory responses such as increased myeloperoxidase activity and mRNA expression of transforming growth factor-β. However, the attenuation of other proinflammatory cytokines such as tumor necrosis factor-α and interleukin- 6 was not significant.. In sum, G-CSF treatment significantly attenuated hyperoxia-induced lung injury by down-modulating the inflammatory responses in neonatal rats.

Topics: Animals; Animals, Newborn; Blotting, Western; Female; Granulocyte Colony-Stimulating Factor; Hyperoxia; In Situ Nick-End Labeling; Interleukin-6; Lung; Lung Injury; NADPH Oxidases; Peroxidase; Pregnancy; Random Allocation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Weight Gain

There is no effective intervention to prevent or treat bronchopulmonary dysplasia (BPD). Curcumin has potent antioxidant and anti-inflammatory properties, and it modulates signaling of peroxisome proliferator-activated receptor-γ (PPARγ), an important molecule in the pathobiology of BPD. However, its role in the prevention of BPD is not known. We determined 1) if curcumin enhances neonatal lung maturation, 2) if curcumin protects against hyperoxia-induced neonatal lung injury, and 3) if this protection is mediated by blocking TGF-β. Embryonic day 19 fetal rat lung fibroblasts were exposed to 21% or 95% O(2) for 24 h following 1 h of treatment with curcumin. Curcumin dose dependently accelerated e19 fibroblast differentiation [increased parathyroid hormone-related protein (PTHrP) receptor, PPARγ, and adipocyte differentiation-related protein (ADRP) levels and triolein uptake] and proliferation (increased thymidine incorporation). Pretreatment with curcumin blocked the hyperoxia-induced decrease (PPARγ and ADRP) and increase (α-smooth muscle actin and fibronectin) in markers of lung injury/repair, as well as the activation of TGF-β signaling. In a separate set of experiments, neonatal Sprague-Dawley rat pups were exposed to 21% or 95% O(2) for 7 days with or without intraperitoneal administration of curcumin. Analysis for markers of lung injury/repair [PTHrP receptor, PPARγ, ADRP, fibronectin, TGF-β receptor (activin receptor-like kinase 5), and Smad3] and lung morphology (radial alveolar count) demonstrated that curcumin effectively blocks TGF-β activation and hyperoxia-induced lung injury. Therefore, curcumin accelerates lung maturation by stimulating key alveolar epithelial-mesenchymal interactions and prevents hyperoxia-induced neonatal lung injury, possibly by blocking TGF-β activation, suggesting that it is a potential intervention against BPD.

Topics: Animals; Animals, Newborn; Blotting, Western; Bronchopulmonary Dysplasia; Cell Differentiation; Curcumin; Female; Fibroblasts; Gene Expression Regulation; Humans; Hyperoxia; Infant, Newborn; Infant, Newborn, Diseases; Lung; Parathyroid Hormone-Related Protein; Peroxisome Proliferator-Activated Receptors; PPAR gamma; Pregnancy; Rats; Rats, Sprague-Dawley; Receptor, Parathyroid Hormone, Type 1; Signal Transduction; Transforming Growth Factor beta

In the ischaemia-reperfused heart, transforming growth factor beta (TGFbeta) proteins trigger the differentiation of cardiac fibroblasts (CFs) contributing to fibrosis. Reoxygenation of the heart, in addition to being a trigger for reperfusion injury, induces tissue remodelling by hyperoxia-sensitive signalling processes involving TGFbeta. Here, we sought to characterize the molecular mechanisms responsible for the O(2)-sensitive transcriptional induction of TGFbeta in murine CF and to test the significance of such findings in the infarcted myocardium in vivo using laser capture microdissection.. All three isoforms of TGFbeta were induced in the CF-rich peri-infarct tissue as well as in CF exposed to hyperoxic challenge. Reporter studies demonstrated that TGFbeta transcription is hyperoxia inducible. Deletion of any one or both of the activating protein-1 (AP-1) binding sites in the TGFbeta reporter construct resulted in loss of O(2) sensitivity, demonstrating that AP-1 confers O(2) sensitivity to TGFbeta transcription. Fos-related AP-1 transcription factor (Fra-2) and Ask-1 (apoptosis signal-regulating kinase-1) were identified as key mediators of AP-1-dependent O(2)-sensitive TGFbeta transcription. Knockdown of Fra-2 significantly blunted O(2)-induced expression of TGFbeta1 as well as TGFbeta3 in CF. Knockdown of Ask-1 blunted hyperoxia-induced Fra-2 gene expression and nuclear localization in CF. Collectively, these observations point towards a central role of Ask-1 and Fra-2 in O(2)-inducible AP-1 activation and induction of TGFbeta.. Taken together with the observation that Fra-2-regulated genes are implicated in fibrosis, identification of Fra-2 as an O(2)-sensitive transcriptional regulator of inducible TGFbeta expression positions Fra-2 as an important player in reoxygenation-induced fibrosis.

Topics: Animals; Binding Sites; Cells, Cultured; Disease Models, Animal; Fibroblasts; Fibrosis; Fos-Related Antigen-2; Hyperoxia; MAP Kinase Kinase Kinase 5; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Oxygen; Promoter Regions, Genetic; RNA Interference; Time Factors; Transcription Factor AP-1; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Up-Regulation

Despite tremendous technological and therapeutic advances, bronchopulmonary dysplasia (BPD) remains a leading cause of respiratory morbidity in very low birth weight infants, and there are no effective preventive and/or therapeutic options. We have previously reported that hyperoxia-induced neonatal rat lung injury might be prevented by rosiglitazone (RGZ). Here, we characterize 1) perturbations in wingless/Int (Wnt) and transforming growth factor (TGF)-beta signaling, and 2) structural aberrations in lung morphology following 7-day continuous in vivo hyperoxia exposure to neonatal rats. We also tested whether treatment of neonatal pups with RGZ, concomitant to hyperoxia, could prevent such aberrations. Our study revealed that hyperoxia caused significant upregulation of Wnt signaling protein markers lymphoid enhancer factor 1 (Lef-1) and beta-catenin and TGF-beta pathway transducers phosphorylated Smad3 and Smad7 proteins in whole rat lung extracts. These changes were also accompanied by upregulation of myogenic marker proteins alpha-smooth muscle actin (alpha-SMA) and calponin but significant downregulation of the lipogenic marker peroxisome proliferator-activated receptor-gamma (PPARgamma) expression. These molecular perturbations were associated with reduction in alveolar septal thickness, radial alveolar count, and larger alveoli in the hyperoxia-exposed lung. These hyperoxia-induced molecular and morphological changes were prevented by systemic administration of RGZ, with lung sections appearing near normal. This is the first evidence that in vivo hyperoxia induces activation of both Wnt and TGF-beta signal transduction pathways in lung and of its near complete prevention by RGZ. Hyperoxia-induced arrest in alveolar development, a hallmark of BPD, along with these molecular changes strongly implicates these proteins in hyperoxia-induced lung injury. Administration of PPARgamma agonists may thus be a potential strategy to attenuate hyperoxia-induced lung injury and subsequent BPD.

Topics: Actins; Acute Lung Injury; Age Factors; Animals; Animals, Newborn; beta Catenin; Calcium-Binding Proteins; Calponins; Cells, Cultured; Fibroblasts; Hyperoxia; Hypoglycemic Agents; Lac Operon; Lymphoid Enhancer-Binding Factor 1; Mice; Mice, Transgenic; Microfilament Proteins; PPAR gamma; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Rosiglitazone; Signal Transduction; Thiazolidinediones; Transforming Growth Factor beta; Wnt Proteins

Recent evidence suggests mesenchymal stem cells (MSCs) can downmodulate bleomycin-induced lung injury, and umbilical cord blood (UCB) is a promising source for human MSCs. This study examined whether intratracheal or intraperitoneal transplantation of human UCB-derived MSCs can attenuate hyperoxia-induced lung injury in immunocompetent newborn rats. Wild-type Sprague-Dawley rats were randomly exposed to 95% oxygen or air from birth. In the transplantation groups, a single dose of PKH26-labeled human UCB-derived MSCs was administered either intratracheally (2 x 10(6) cells) or intraperitoneally (5 x 10(5) cells) at postnatal day (P) 5. At P14, the harvested lungs were examined for morphometric analyses of alveolarization and TUNEL staining, as well as the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and transforming growth factor (TGF)-beta mRNA, alpha-smooth muscle actin (SMA) protein, and collagen levels. Differentiation of MSCs to the respiratory epithelium was also evaluated both in vitro before transplantation and in vivo after transplantation. Despite one fourth dosage of MSCs, significantly more PKH26-labeled donor cells were recovered with intratracheal administration than with intraperitoneal administration both during normoxia and hyperoxia. The hyperoxia-induced increase in the number of TUNEL-positive cells, myeloperoixdase activity, and the level of IL-6 mRNA were significantly attenuated with both intratracheal and intraperitoneal MSCs transplantation. However, the hyperoxia-induced impaired alveolarization and increased the level of TNF-alpha and TGF-beta mRNA, alpha-SMA protein, and collagen were significantly attenuated only with intratracheal MSCs transplantation. MSCs differentiated into respiratory epithelium in vitro and a few PKH26-positive donor cells were colocalized with pro surfactant protein C in the damaged lungs. In conclusion, intratracheal transplantation of human UCB-derived MSCs is more effective than intraperitoneal transplantation in attenuating the hyperoxia-induced lung injury in neonatal rats.

Topics: Animals; Animals, Newborn; Cell Differentiation; Cells, Cultured; Cord Blood Stem Cell Transplantation; Female; Humans; Hyperoxia; Interleukin-6; Lung Injury; Mesenchymal Stem Cell Transplantation; Peroxidase; Pregnancy; Rats; Rats, Sprague-Dawley; Respiratory Mucosa; Transforming Growth Factor beta; Transplantation, Heterologous; Tumor Necrosis Factor-alpha

Hyperoxic rats treated with inosine during oxygen exposure have increased levels of active transforming growth factor (TGF)-beta in the bronchoalveolar lavage (BAL), yet alveolar epithelial type 2 cells (AEC2) isolated from these animals demonstrate less hyperoxia-induced DNA damage and increased expression of active Smad2. To determine whether TGF-beta1 signaling per se protected AEC2 against hyperoxic damage, freshly isolated AEC2 from hyperoxic rats were incubated with TGF-beta1 for 24 h and assayed for DNA damage by fluorescein-activated cell sorter analysis of TdT-mediated dUTP nick end labeling. TGF-beta1 was protective over a concentration range similar to that in BAL of inosine-treated hyperoxic animals (50-5,000 pg/ml). TGF-beta1 also augmented hyperoxia-induced DNA repair activity and cell migration, stimulated autocrine secretion of fibronectin, accelerated closure of a monolayer scratch wound, and restored hyperoxia-depleted VEGF secretion by AEC2 to normoxic levels. The TGF-beta receptor type I activin-like kinase-4, -5, and -7 inhibitor peptide SB-505124 abolished the protective effect of TGF-beta on hyperoxic DNA damage and increased TdT-mediated dUTP nick end labeling in normoxic cells. These data suggest that endogenous TGF-beta-mediated Smad signaling is required for AEC2 homeostasis in vitro, while exogenous TGF-beta1 treatment of hyperoxia-damaged AEC2 results in a cell that is equipped to survive, repair, migrate, secrete matrix, and induce new blood vessel formation more efficiently than AEC2 primed by hyperoxia alone.

Topics: Animals; Benzodioxoles; Cell Culture Techniques; Cell Cycle; Cell Survival; Hyperoxia; Imidazoles; In Situ Nick-End Labeling; Male; Pulmonary Alveoli; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Transforming Growth Factor beta; Respiratory Mucosa; Transforming Growth Factor beta

To evaluate whether human mesenchymal stem cells (hMSCs) administration alter the clinical course of hyperoxia-induced lung injury.. hMSCs were obtained from bone marrow aspirates from healthy donors after informed consent was signed, hMSCs were separated, cultured, amplified, identified and labeled with BrdU. For BrdU labeling, a sterile stock solution was added to the culture medium 48 h before the end of culture, at a final concentration of 10 micromol/L. Thirty-two 3-day old SD rats from four litters were randomly divided into four groups, as hyperoxia exposed + hMSC group (A), air-exposed + hMSC group (B), hyperoxia exposed group (C), and air-exposed group (D). The rats from the group A and the group C were placed in a sealed Plexiglas chamber with a minimal in- and outflow, providing six to seven exchanges per hour of the chamber volume and maintaining O2 levels above 95%, while the rats in the group B and the group D were only exposed to room air. Seven days later, all of them were taken out of the chamber, rats in the group A and B were injected intraperitoneally with hMSCs (1 x 10(5) in 50 microl of PBS) immediately, while the rats in the group C and D were only treated with 50 microl of PBS 3 days later. All the animals were sacrificed by an injection of sodium pentobarbital (120 mg/kg), perfused with cold 0.9% NaCl, and the left lungs were removed, the upper lobes of which were ground as tissue homogenates and used for ELISA, while the inferior lobes were stored at -70 degrees C until use for RT-PCR. The right lungs were fixed in situ for 2 h by the intratracheal instillation with 10% neutral formalin and then postfixed for 24 h. Sagittal sections (4-microm) of paraffin-embedded middle lobe and upper lobe of the right lung were used for immunohistochemistry and histology, respectively.. (1) There was a significant difference in the value of RAC (raditive alveoli coant) among the 4 groups (11.145 +/- 1.331, 13.941 +/- 0.985, 9.595 +/- 0.672, 14.819 +/- 1.080, F = 43.234, P = 0.000). RAC in group A and C were significantly reduced compared with subjects in group D (P < 0.05, P < 0.05); and there was also a significant difference between group A and group C (P < 0.05), but not between group B and D subjects (P > 0.05). (2) There were significant differences in the levels of both TNFalpha and TGFbeta(1) in the homogenate of lungs among the 4 groups (142.933 +/- 24.017, 79.033 +/- 11.573, 224.088 +/- 41.915, 76.500 +/- 10.373, F = 59.970, P = 0.000; 1726.484 +/- 91.086, 1530.359 +/- 173.441, 2047.717 +/- 152.057, 1515.777 +/- 131.049, F = 24.977, P = 0.000). The levels of TNFalpha and TGFbeta1 were significantly elevated in both group A and group C when compared with subjects in group D (P < 0.05 for both). Concentrations of TNFalpha and TGFbeta1 were both significantly decreased in group A versus group C (P < 0.05 for both). There was no significant difference between group B and D subjects in the fields of TNFalpha and TGFbeta(1) (P > 0.05 for both). (3) BrdU-labelled cells were observed at alveolar wall and bronchioles in both group A and group B, and there was a significant difference in BrdU-labeled cells between two groups (0.230 +/- 0.026, 0.190 +/- 0.015; t = 3.769, P = 0.002), but none was found in group C and group D. Electrophoresis of the PCR products showed a 224 bp band, specific for Alu mRNA, in 7 of 8 rats of group A and 5 of 8 rats of group B, respectively, but no such band was found in group C and group D.. hMSCs administered by intraperitoneal injection could be implanted in the lungs of newborn rats, and they could effectively protect the rats against damage to the lungs caused by hyperoxia.

Topics: Animals; Animals, Newborn; Bone Marrow Cells; Bromodeoxyuridine; Cell Communication; Cell Differentiation; Cells, Cultured; Hematopoietic Stem Cells; Humans; Hyperoxia; Infant, Newborn; Lung; Lung Injury; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Oxygen; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Topics: Animals; Animals, Newborn; Apoptosis; Bone Morphogenetic Proteins; Bronchopulmonary Dysplasia; Cell Proliferation; Disease Models, Animal; Epithelial Cells; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Humans; Hyperoxia; Infant, Newborn; Lung Diseases; Mice; Myocytes, Smooth Muscle; NIH 3T3 Cells; Protein Transport; Pulmonary Alveoli; Pulmonary Artery; Respiration; RNA, Messenger; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

Pulmonary injury is associated with the disruption of alveologenesis in the developing lung and causes bronchopulmonary dysplasia (BPD) in prematurely born infants. Transforming growth factor (TGF)-beta is an important regulator of cellular differentiation and early lung development, and its levels are increased in newborn lung injury. Although overexpression of TGF-beta in the lungs of newborn animals causes pathological features that are consistent with BPD, the role of endogenous TGF-beta in the inhibition of the terminal stage of lung development is incompletely understood. In this investigation, the hypothesis that O(2)-induced injury of the maturing lung is associated with TGF-beta-mediated disruption of alveologenesis and microvascular development was tested using a murine model of BPD. Here we report that treatment of developing mouse lungs with TGF-beta-neutralizing antibodies attenuates the increase in pulmonary cell phospho-Smad2 nuclear localization, which is indicative of augmented TGF-beta signaling, associated with pulmonary injury induced by chronic inhalation of 85% oxygen. Importantly, the neutralization of the abnormal TGF-beta activity improves quantitative morphometric indicators of alveologenesis, extracellular matrix assembly, and microvascular development in the injured developing lung. Furthermore, exposure to anti-TGF-beta antibodies is associated with improved somatic growth in hyperoxic mouse pups and not with an increase in pulmonary inflammation. These studies indicate that excessive pulmonary TGF-beta signaling in the injured newborn lung has an important role in the disruption of the terminal stage of lung development. In addition, they suggest that anti-TGF-beta antibodies may be an effective therapy for preventing some important developmental diseases of the newborn lung.

Topics: Animals; Animals, Newborn; Antibodies; Cell Nucleus; Elastin; Female; Hyperoxia; Lung Diseases; Mice; Mice, Inbred C57BL; Neovascularization, Physiologic; Neutralization Tests; Phosphoproteins; Protein Isoforms; Protein Transport; Pulmonary Alveoli; Smad2 Protein; Transforming Growth Factor beta

Lung fibrosis is the ultimate outcome of hyperoxia-induced chronic lung diseases (CLD) and connective tissue growth factor (CTGF) is correlated with fibrosis. This study investigated the role of CTGF in hyperoxia-induced CLD.. Fifty premature rats were randomly exposed to hyperoxia (Model group) and to room air (Control group) (n = 25 each). CLD was induced by hyperoxia exposure. The expression of CTGF was detected by immunohistochemical method at 1, 3, 7, 14 and 21 days after exposure. The severity of pulmonary fibrosis was evaluated.. In the Control group there was a slight expression of CTGF in the bronchial epithelial cells and vascular endothelial cells. The intensity and range of CTGF expression in the Model group were similar to the Control group on days 1, 3 and 7 of exposure. On the 14th day, CTGF was expressed in some alveolar epithelial cells, fibroblasts and interstitial cells, and the intensity of CTGF expression increased significantly compared with the Control group, with the IODT of CTGF of 10.53 +/- 4.24 vs 5.58 +/- 1.18 (P < 0.01). On day 21, the expression intensity and range of CTGF in the Model group (IODT: 16.61 +/- 5.39) increased compared with that of Control group (P < 0.01). The expression of CTGF was correlated with the degree of fibrosis in the Model group on days 14 and 21 (r = 0.903, r = 0.926 respectively, P < 0.01).. The CTGF expression increased with the time of hyperoxia exposure and the development of fibrosis. CTGF is closely related to the development of hyperoxia-induced pulmonary fibrosis.

Topics: Animals; Chronic Disease; Connective Tissue Growth Factor; Female; Hyperoxia; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lung; Pulmonary Fibrosis; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

Inosine, a naturally occurring purine with anti-inflammatory properties, was assessed as a possible modulator of hyperoxic damage to the pulmonary alveolar epithelium. Rats were treated with inosine, 200 mg/kg ip, twice daily during 48-h exposure to >90% oxygen. The alveolar epithelial type 2 cells (AEC2) were then isolated and cultured. AEC2 isolated from inosine-treated hyperoxic rats had less DNA damage and had increased antioxidant status compared with AEC2 from hyperoxic rats. Inosine treatment during hyperoxia also reduced the proportion of AEC2 in S and G2/M phases of the cell cycle and increased levels of the DNA repair enzyme 8-oxoguanine DNA glycosylase. Bronchoalveolar lavage (BAL) recovered from hyperoxic, inosine-treated rats contained threefold higher levels of active transforming growth factor-beta than BAL from rats exposed to hyperoxia alone, and Smad2 was activated in AEC2 isolated from these animals. ERK1/2 was activated both in freshly isolated and 24-h-cultured AEC2 by in vivo inosine treatment, whereas blockade of the MAPK pathway in vitro reduced the protective effect of in the vivo inosine treatment. Together, the data suggest that inosine treatment during hyperoxic exposure results in protective signaling mediated through pathways downstream of MEK. Thus inosine may deserve further evaluation for its potential to reduce hyperoxic damage to the pulmonary alveolar epithelium.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Cycle; Cells, Cultured; Cytoprotection; DNA Damage; DNA Glycosylases; DNA-Binding Proteins; Enzyme Activation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Glutathione; Hyperoxia; Inosine; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Smad2 Protein; Trans-Activators; Transforming Growth Factor beta; Uric Acid

IL-13 is a major stimulator of inflammation and tissue remodeling at sites of Th2 inflammation. In Th2-dominant inflammatory disorders such as asthma, IL-11 is simultaneously induced. However, the relationship(s) between IL-11 and IL-13 in these responses has not been defined, and the role(s) of IL-11 in the genesis of the tissue effects of IL-13 has not been evaluated. We hypothesized that IL-11, signaling via the IL-11Ralpha-gp130 receptor complex, plays a key role in IL-13-induced tissue responses. To test this hypothesis we compared the expression of IL-11, IL-11Ralpha, and gp130 in lungs from wild-type mice and transgenic mice in which IL-13 was overexpressed in a lung-specific fashion. We simultaneously characterized the effects of a null mutation of IL-11Ralpha on the tissue effects of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of IL-11 and IL-11Ralpha. They also demonstrate that IL-13 is a potent stimulator of inflammation, fibrosis, hyaluronic acid accumulation, myofibroblast accumulation, alveolar remodeling, mucus metaplasia, and respiratory failure and death in mice with wild-type IL-11Ralpha loci and that these alterations are ameliorated in the absence of IL-11Ralpha. Lastly, they provide insight into the mechanisms of these processes by demonstrating that IL-13 stimulates CC chemokines, matrix metalloproteinases, mucin genes, and gob-5 and stimulates and activates TGF-beta1 via IL-11Ralpha-dependent pathways. When viewed in combination, these studies demonstrate that IL-11Ralpha plays a key role in the pathogenesis of IL-13-induced inflammation and remodeling.

Topics: Animals; Chemokines, CC; Fibroblasts; Hyaluronic Acid; Hyperoxia; Inflammation; Interleukin-11; Interleukin-11 Receptor alpha Subunit; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Lung; Matrix Metalloproteinases; Metaplasia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Mucins; Protein Subunits; Pulmonary Alveoli; Pulmonary Fibrosis; Receptors, Interleukin; Receptors, Interleukin-11; Receptors, Interleukin-13; Respiratory Insufficiency; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factor beta1

In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to approximately 1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the PO2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac fibroblasts (CFs) isolated from adult murine ventricle were cultured in 10% or 21% O2 (hyperoxia relative to the PO2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or 21% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myofibroblasts. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p21, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming growth factor-beta1, (2) lowered telomerase activity, and (3) activation of transforming growth factor-beta1 and p38 mitogen-activated protein kinase. CFs deficient in p21 were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

Topics: Animals; Cell Differentiation; Cell Division; Cell Hypoxia; Cell Separation; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Fibroblasts; Heart Ventricles; Hyperoxia; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitogen-Activated Protein Kinases; Oxidation-Reduction; Oxidative Stress; Oxygen; p38 Mitogen-Activated Protein Kinases; Reactive Oxygen Species; Signal Transduction; Telomerase; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

Supplemental oxygen is often required in the treatment of critically ill patients. The impact of hyperoxia on pulmonary host defense is not well-established. We hypothesized that hyperoxia directly impairs pulmonary host defense, beyond effects on alveolar wall barrier function. C57BL/6 mice were kept in an atmosphere of >95% O(2) for 4 days followed by return to room air. This exposure does not lead to mortality in mice subsequently returned to room air. Mice kept in room air served as controls. Mice were intratracheally inoculated with Klebsiella pneumoniae and followed for survival. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage after 4 days of in vivo hyperoxia for ex vivo experiments. Mortality from pneumonia increased significantly in mice exposed to hyperoxia compared with infected mice in room air. Burden of organisms in the lung and dissemination of infection were increased in the hyperoxia group whereas accumulation of inflammatory cells in the lung was impaired. Hyperoxia alone had no impact on AM numbers, viability, or ability to phagocytize latex microbeads. However, following in vivo hyperoxia, AM phagocytosis and killing of Gram-negative bacteria and production of TNF-alpha and IL-6 in response to LPS were significantly reduced. AM surface expression of Toll-like receptor-4 was significantly decreased following in vivo hyperoxia. Thus sublethal hyperoxia increases Gram-negative bacterial pneumonia mortality and has a significant adverse effect on AM host defense function. Impaired AM function due to high concentrations of supplemental oxygen may contribute to the high rate of ventilator-associated pneumonia seen in critically ill patients.

Topics: Animals; Cell Count; Cell Survival; Chemokines; Hyperoxia; Immunity, Innate; Inflammation Mediators; Interleukin-10; Interleukin-6; Klebsiella Infections; Klebsiella pneumoniae; Lung; Macrophages, Alveolar; Membrane Glycoproteins; Mice; Phagocytosis; Pneumonia, Bacterial; Receptors, Cell Surface; RNA, Messenger; Toll-Like Receptors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The final stage of lung development, alveolarization, continues after birth in humans and rodents. Clinical interventions, such as oxygen therapy, in the first week of life can adversely impact alveolar formation. We compared alveolarization in the rat neonate under normal vs. hyperoxic conditions, examining gelatinase, transforming growth factor (TGF)-beta, and the protease urokinase-type plasminogen activator (uPA) activities in whole lung and cultured type II alveolar epithelial cells (AEC2). The dynamic induction of gelatinase, TGF-beta, and uPA activities seen in neonatal lungs during the first days of life was significantly impacted by hyperoxia. In whole lung, gelatinase and TGF-beta activities were increased, while uPA activity was decreased. At the level of the epithelium, AEC2 isolated from hyperoxic rat pups early in life secreted less active TGF-beta, less active gelatinases, and less active uPA than control neonatal AEC2. AEC2 from hyperoxic pups also expressed increased levels of proliferating cell nuclear antigen early in life compared with control neonatal AEC2, suggesting that oxygen-induced proliferation and/or repair were occurring. The developmental profile of neonatal lung was perturbed within a day of initiating oxygen treatment, suggesting that putative palliative treatments should be coadministered with oxygen therapy.

Topics: Animals; Animals, Newborn; Bronchoalveolar Lavage; Cell Division; Cells, Cultured; Hyperoxia; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Proliferating Cell Nuclear Antigen; Pulmonary Alveoli; Rats; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Urokinase-Type Plasminogen Activator

The epithelium of the lung alveolus is a major target for oxidant injury, and its proper repair after injury is dependent on the proliferative response of the alveolar epithelial type 2 cells. Recently, we have provided evidence that retinoic acid (RA) stimulates proliferation of type 2 cells. In the present study, we examined the effects of RA on the proliferative response of alveolar type 2 cells exposed to elevated oxygen (O(2)). We showed that pretreatment by RA was able to prevent the growth arrest and cell loss of O(2)-exposed cells. To gain insights into the mechanisms involved, we studied the effects of RA on the cyclin-dependent kinase (CDK) system. The activity of cyclin E-CDK2 complex was found to be decreased in O(2)-exposed cells. Interestingly, this decrease was no longer observed when cells were pretreated with RA. Analysis of p21(CIP1), an inhibitor of CDK, revealed an increased expression in O(2)-exposed cells that was no longer observed in cells treated with RA. These effects were associated with a reduced association of p21(CIP1) with cyclin E-CDK2 complexes in the presence of RA. In addition, studies of Smad activity strongly suggest that the mechanisms through which RA preserves late G(1) cyclin-CDK complex activity may involve interference with the transforming growth factor-beta signaling pathway.

Topics: Animals; CDC2-CDC28 Kinases; Cell Cycle; Cell Division; Cells, Cultured; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; G1 Phase; Hyperoxia; Oxygen; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Rats; Respiratory Mucosa; Signal Transduction; Transforming Growth Factor beta; Tretinoin

Proteoglycans are extracellular matrix components that appear to play important roles in lung development and in the response to injury. Decorin, a small extracellular matrix-associated proteoglycan, is known to be involved in collagen fibrillogenesis and is a likely participant in the pathogenesis of lung injury. We hypothesized that chronic exposure of the developing lung to hyperoxia would result in temporal and spatial changes in decorin expression. To determine the expression of decorin in normal and oxygen-injured lung, newborn rats were exposed to hyperoxia for 6 wk. Decorin mRNA abundance was determined using Northern hybridization analyses, and decorin expression was localized by in situ hybridization and immunohistochemistry. Decorin mRNA expression in type II pneumocytes was studied using reverse transcription-polymerase chain reaction. Oxygen exposure is associated with a 77% reduction in decorin mRNA in whole lung and a decrease in decorin immunoreactivity in connective tissues surrounding large airways and blood vessels, but an increase in decorin mRNA and protein expression at the tips of alveolar septa. Studies using isolated cells indicate that macrophages and polymorphonuclear neutrophils contain decorin core protein but not decorin mRNA. Type II pneumocytes do not contain either decorin mRNA or core protein. These findings demonstrate that hyperoxic lung injury is associated with localized changes in decorin expression, changes that are not reflected in whole lung RNA studies. It is likely that regional changes in lung decorin expression are influenced by factors produced and acting locally, and that such changes may contribute to the morphologic alterations characteristic of oxygen-induced lung injury.

Topics: Animals; Cloning, Molecular; Decorin; Extracellular Matrix Proteins; Hyperoxia; Lung; Proteoglycans; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta

Topics: Cell Division; Cytokines; DNA; DNA Damage; Endothelium, Vascular; Humans; Hyperoxia; Interleukin-1; Interleukin-8; Oxygen; Reactive Oxygen Species; RNA, Messenger; Thymidine; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbilical Veins

Hyperoxia causes a reproducible pattern of lung injury and recovery, characterized by proliferation of type II alveolar epithelial cells (AEC2) during the recovery phase. We measured TGF-beta peptide production by AEC2 and macrophages from lungs of adult male rats exposed to 100% oxygen for 48 h and then allowed to recover for up to 72 h in room air. TGF-beta peptide activity levels were measured using the PAI-1 promoter-luciferase mink lung epithelial cell assay and characterized with peptide specific inhibitory antibodies. Control AEC2 produced 997 +/- 54 pg active TGF-beta x 10(6) cells-1.24h-1 (mean +/- SD), of which > 70% was TGF-beta 3, while cultured macrophages produced 58 +/- 17 pg active TGF-beta x 10(6) macrophages-1.24 h-1, > 80% of which was TGF-beta 1. During hyperoxia and recovery, active TGF-beta 3 production by AEC2 decreased by 75%, with a nadir at 24 h recovery (P < 0.005). In contrast, TGF-beta peptide activity increased from undetectable levels in lung lavage from control rats to a peak of 1,470 +/- 743 pg/rat after 48 h oxygen exposure and 24 h recovery, while lavaged macrophage TGF-beta production in culture also increased threefold to a peak of 150 +/- 5 pg. 10(6) cells-1. 24 h-1 after 48 h oxygen exposure (P < 0.005). The nadir of active TGF-beta 3 production by AEC2 coincided with the peak of the AEC2 proliferative phase of repair as determined by BrdU incorporation and FACS analysis of freshly isolated AEC2. We conclude that active TGF-beta 3 production by AEC2 is dynamically downregulated during the proliferative phase of recovery from acute hyperoxic injury in rat. We speculate that decreased autocrine negative regulation of AEC2 proliferation by TGF-beta 3 may facilitate AEC2 proliferation during recovery from acute hyperoxic injury.

Topics: Acute Disease; Animals; Bronchoalveolar Lavage Fluid; Cell Division; Cells, Cultured; DNA; Epithelium; Hyperoxia; Isomerism; Macrophages, Alveolar; Male; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Thymidine; Time Factors; Transforming Growth Factor beta