Neonatal Rat Lung Research Articles

Platelet activating factor (PAF) receptor expression is increased in lungs of neonatal rats with hypoxia‐induced pulmonary hypertension

Endogenous PAF contributes to high vasomotor tone in the fetal pulmonary circulation and has been implicated in the pathogenesis of chronic‐hypoxia (CH)‐induced pulmonary hypertension (PH). We studied the effect of CH on neonatal rat (pup) body weight, right ventricle/left ventricle + septum ratio (RV/LV+S), and lung PAF receptor (PAF‐r) protein expression by Western blotting. Pups were placed in an air‐tight chamber ventilated with 13% oxygen (hypoxia) or room air (normoxia) from 1d to 22d of age. Three groups were studied (12 pups in each group): Group1, pups in Hypoxia; Group2, pups in hypoxia given 5mg/kg WEB 2170 daily, a PAF‐r antagonist, (Hypoxia +WEB); Group3, pups in Normoxia. We found that: a) Pups in Hypoxia gained 50% less weight than pups in Normoxia; WEB treatment in Hypoxia did not improve weight gain; b) RV/LV+S in Hypoxia group was highest at 0.79; 2‐fold higher than in WEB + Hypoxia and Normoxia groups; c) PAF‐r protein expression in lungs of pups in Hypoxia was >3‐fold higher than in WEB+Hypoxia and Normoxia groups. Our findings show that chronic hypoxia induces PAF‐r expression in lungs and suggest that increased PAF‐r expression may be responsible for the right ventricular hypertrophy and PH. Inhibition of PAF effects with a PAF‐r antagonist, WEB, though it did not improve weight gain of the pups in hypoxia, prevented RV hypertrophy in the pups. HL‐077819.

The PPAR Gamma Agonist Rosiglitazone Protects Against Wnt Signaling In Hyperoxia‐Induced Neonatal Rat Lung Injury

Bronchopulmonary Dysplasia (BPD) is a consequence of abnormally repaired lung damage. Hyperoxia disrupts critical signaling pathways that support normal lung development. We characterized in vivo hyperoxia induced perturbations in lung morphology and Wnt and TGF‐β signaling in postnatal day (PND) 7 rat lung, and tested if the PPARγ agonist rosiglitazone (RGZ) prevents such perturbations. Newborn rats were grouped as normoxic, hyperoxic (95% O2), and hyperoxic/RGZ (3 mg/kg i.p. daily during 95% O2). PND7 lungs were analyzed by immunoblot, morphometry, and immunohistochemistry (IHC). Hyperoxia caused significant up‐regulation of Wnt signaling markers Lef1 and βcatenin, and TGFβ pathway markers pSMAD3 and SMAD7. The myogenic phenotype markers αSMA and calponin were also up‐regulated. There was significantly reduced alveolar septal thickness, radial alveolar count, but larger alveoli. PPARγ expression, however, was significantly down‐regulated. Hyperoxia‐induced changes were almost completely prevented by RGZ treatment. We demonstrate up‐regulation of Lef1, βcatenin, pSMAD3 and SMAD7 in neonatal rat lungs in hyperoxia. Hyperoxia‐induced simplified alveoli, a hallmark of BPD, along with altered molecular changes, strongly implicating these factors in hyperoxia‐induced lung injury. PPARγ agonists may effectively prevent BPD Grant Support‐NIH HL75405 & HL55268 & TRDRP 15IT‐0250 & 14RT‐0013.

Heat Shock Protein 27 (Hsp27) Protects Lung Epithelial Cells from Hyperoxia‐Induced Cell Death

Supplemental oxygen therapy (hyperoxia) is a common life‐saving practice for preterm babies with respiratory distress. Prolonged hyperoxia is one of the major contributing factors in cell death and the development of bronchopulmonary dysplasia (BPD), a chronic lung disease, in preterm babies. Hsp27 is the smallest inducible heat shock chaperone protein that modulates the ability of cells to respond to oxidative stress. Our DNA Superarray analysis revealed that hyperoxia significantly decreased Hsp27 gene expression in newborn rat lungs. Immunohistochemistry of Hsp27 confirmed that Hsp27 protein in epithelial cells of neonatal rat lungs was markedly decreased after hyperoxic exposure. In cultured human lung epithelial cells (A549), prolonged hyperoxia significantly reduced Hsp27 expression and caused cell death. Overexpression of hHsp27 could prevent hyperoxia‐induced cell death in cultured human lung epithelial cells. Our studies suggest that hyperoxia reduces Hsp27 expression in lung epithelial cells and overexpression of hHsp27 confers cytoprotection against hyperoxia‐induced cell death in lung epithelial cells.

The role of IL‐6 and IL‐11 in hyperoxic injury in developing lung

We examined the cytoprotective effect of interleukin-6 (IL-6) and interleukin-11 (IL-11) during oxidant injury in neonatal lung and the regulators of cell death in vitro and in vivo after oxidant exposure. Type II cells from day 21 fetal neonatal rat lungs were treated with varying concentrations of either IL-6 or IL-11 for 24 hr prior to exposure to H(2)O(2). Three-day-old transgenic lung-specific IL-11 and IL-6 overexpressing and wild type (WT) mouse pups were exposed to hyperoxia or room air for 3 days. Type II cells exposed to either IL-6 or IL-11 prior to oxidant injury exhibited improved survival compared to controls, 67% +/- 2.6 survivals in IL-6 pretreated cells compared to 48% +/- 1.6 in control; 63% +/- 3 survivals in IL-11 pretreated cells compared to 49% +/- 2.6 in control. The number of TUNEL positive cells in hyperoxia-exposed lungs was increased compared to room air animals (27 +/- 0.9 vs. 4 +/- 0.4; mean +/- SEM; P < 0.05). In contrast, the number of TUNEL positive cells was reduced in hyperoxia-exposed lungs from IL-11 (+) mice (15.2 +/- 2.2; mean +/- SEM; P < 0.05). There was an enhanced accumulation of Bcl-2 and reduction of Bax protein in hyperoxia-exposed IL-11 (+) compared to room air-exposed mice. This was not seen in hyperoxia-exposed IL-6 (+) pups. An increase in caspase-3 was seen in hyperoxia-exposed lungs of WT pups compared to IL-11 (+) pups. IL-11 and IL-6 provide protective effects against oxidant-mediated injury in fetal type II cells and IL-11 provides protection in vivo by down-regulation of caspase-mediated cell death.

Effects of Antenatal Dexamethasone on Antioxidant Enzymes and Nitric Oxide Synthase in the Rat Lung

We investigated the effects of prenatal dexamethasone (DEX) administration on antioxidant enzymes (AOEs) and nitric oxide synthase (NOS) in fetal and neonatal rat lungs. DEX (1 mg/kg, s.c., for 2 days) or vehicle alone was administered to pregnant rats, and the lungs of fetuses on days 19 and 21 of gestation and of 1- and 3-day-old neonates were examined. We measured protein levels of the AOEs manganese superoxide dismutase and copper-zinc super-oxide dismutase (Mn SOD and Cu-Zn SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and inducible and endothelial nitric oxide synthase (i-NOS and e-NOS). Mn SOD, GSH-Px, and e-NOS expression gradually increased with increasing gestational and postnatal age in the lungs of the control groups. Cu-Zn SOD, CAT, and i-NOS expression did not change with increasing gestational and postnatal age in the lungs of the control groups. DEX administration had significant effects on i-NOS and e-NOS protein and mRNA expression. The increased Mn SOD, GSH-Px, and e-NOS expressions during the perinatal period suggests that antenatal developmental changes in AOEs in the lungs of premature fetuses could be reduced by reactive oxygen species–mediated injury at birth. Furthermore, antenatal glucocorticoid treatment may accelerate the development of lungs via the two types of NOS.

THE CALCITRIOL ANALOGUE EB1089 IMPAIRS ALVEOLARIZATION AND INDUCES LOCALIZED REGIONS OF INCREASED FIBROBLAST DENSITY IN NEONATAL RAT LUNG

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, or calcitriol), is a potent mitogen for fibroblasts cultured from rat lungs at postnatal day 4 (P4), during the peak of septation (P3 to P7). In light of the key role of fibroblasts in alveolar septation, the authors conducted studies to measure the extent to which 1,25-(OH)2D3 affects lung maturation in vivo, as well as its ability to influence the stimulatory activity of all-trans retinoic acid (RA). To identify a calcitriol analogue with maximal mitogenic activity and low systemic toxicity, two compounds with reduced calcemic activity (EB1089 and CB1093) and a superagonist (MC1288) were evaluated in neonatal rat lung fibroblast cultures. All 3 analogues were more potent mitogens than 1,25-(OH)2D3 itself (MC1288 ∼ CB1093 > EB1089 > 1,25-(OH)2D3). In addition, each was more effective than 1,25-(OH)2D3(EB1089 > CB1093 > MC1288 > 1,25-(OH)2D3) in the activation of a vitamin D response element from the platelet-derived growth factor (PDGF)-A gene, whose expression is essential for normal alveolarization. Daily administration of EB1089 to rats 4 to 12 days of age caused an increase in mean alveolar chord length (P <.0001), and also elicited prominent regions of fibroblast hypercellularity, as defined in terms of a vimentin-positive, factor VIII–negative phenotype. EB1089 and RA each induced the expression of 2 important lung structural proteins, collagen and elastin. Regions of fibroblast hypercellularity induced by EB1089 were strongly positive for expression of the alveolarization-relevant growth factors, PDGF-AA and vascular endothelial growth factor (VEGF). These studies demonstrate that 1,25-(OH)2D3 disrupts the overall alveolarization process in the neonatal lung, although it stimulates expression of some proteins associated with lung morphogenesis.

Influence of maternal nicotine exposure during gestation and lactation on lactate dehydrogenase isoenzyme profile and transcript levels in developing neonatal rat lung

In this study we investigated the effect of (a) aging on rat lung lactate dehydrogenase (LDH) mRNA expression and isoenzyme profile and, (b) the effect of maternal nicotine exposure during gestation and lactation on LDH isoenzyme profile and mRNA expression in the lungs of the offspring. Pregnant rats were injected subcutaneously (1 mg nicotine/kg body weight/day) from day 1 after mating. Lung tissue to determine LDH isoenzyme profile and mRNA expression was obtained from the offspring. LDH mRNA expression and isoenzyme profile was determined on postnatal days 1, 7, 14, 21 and 49. Generally the %Densities of all the isoenzymes decreased between postnatal days 1 and 49. Between postnatal days 7 and 14, a period associated with rapid alveolar formation, all isoenzymes except LDH-1, decreased ( P < 0.01). During this period all the isoforms in nicotine-exposed lung increased ( P < 0.05). In both control and nicotine-exposed lung LDH-1 is the dominant isoenzyme. The LDH-M and LDH-H isoenzyme levels are higher ( P < 0.001) in the lungs of the nicotine exposed rats. Maternal nicotine exposure during gestation and lactation changed the metabolic status of the nicotine-exposed lungs to become more glycolytic compared to that of the control lung. These changes in LDH isoenzyme profiles and mRNA expression are irreversible.

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.

Developmental expression of the receptor for advanced glycation end-products (RAGE) and its response to hyperoxia in the neonatal rat lung

BackgroundThe receptor for advanced glycation end products (mRAGE) is associated with pathology in most tissues, while its soluble form (sRAGE) acts as a decoy receptor. The adult lung is unique in that it expresses high amounts of RAGE under normal conditions while other tissues express low amounts normally and up-regulate RAGE during pathologic processes. We sought to determine the regulation of the soluble and membrane isoforms of RAGE in the developing lung, and its expression under hyperoxic conditions in the neonatal lung.ResultsFetal (E19), term, 4 day, 8 day and adult rat lung protein and mRNA were analyzed, as well as lungs from neonatal (0–24 hrs) 2 day and 8 day hyperoxic (95% O2) exposed animals. mRAGE transcripts in the adult rat lung were 23% greater than in neonatal (0–24 hrs) lungs. On the protein level, rat adult mRAGE expression was 2.2-fold higher relative to neonatal mRAGE expression, and adult sRAGE protein expression was 2-fold higher compared to neonatal sRAGE. Fetal, term, 4 day and 8 day old rats had a steady increase in both membrane and sRAGE protein expression evaluated by Western Blot and immunohistochemistry. Newborn rats exposed to chronic hyperoxia showed significantly decreased total RAGE expression compared to room air controls.ConclusionTaken together, these data show that rat pulmonary RAGE expression increases with age beginning from birth, and interestingly, this increase is counteracted under hyperoxic conditions. These results support the emerging concept that RAGE plays a novel and homeostatic role in lung physiology.

Retinoic Acid Combined with Vitamin A Synergizes to Increase Retinyl Ester Storage in the Lungs of Newborn and Dexamethasone-Treated Neonatal Rats

Background: Retinyl esters (REs), the major storage form of vitamin A (retinol), provide substrates for the production of bioactive retinoids, including retinoic acid (RA), which are known to promote lung development and maturation. We previously showed that the nutrient-metabolite combination VARA (molar ratio 10 vitamin A to 1 RA), synergistically increased REs in the lungs of 1-week-old rats, compared to vitamin A or RA alone. Objectives: To test the hypotheses, first, that VARA is more effective in increasing lung RE than is vitamin A in newborn rats prior to alveolarization, and, second, that the effect of VARA is maintained during concurrent treatment with the glucocorticoid, dexamethasone (Dex). Methods: Newborn rats were treated with VARA, vitamin A alone, or oil (C) on postnatal days (P) 1–3, and RE in the lungs was quantified on P4, and again on P8 to assess retention. Additionally, neonatal rats were treated on P5–7 with VARA with and without Dex, and the lung and liver REs were quantified on P8. Results and Conclusions: Lung RE was nearly 8-fold higher in VARA compared to vitamin A-treated rats on P4 (p < 0.01) and 2.5-fold higher on P8. In neonates co-treated with Dex and VARA on P5–7, the elevation in lung RE on P8 by VARA was not antagonized by Dex, although Dex reduced growth. Lung morphology and development were not significantly altered. The VARA combination may significantly increase lung RE content even during concurrent Dex therapy. Because lung retinoids are important for lung maturation and repair, increasing lung RE may possibly have clinical benefit.

ERp57, an Endoplasmic Reticulum (ER) Protein, Reduces Hyperoxia‐Induced ER Stress in Lung Epithelial Cells

Purpose of Study: ERp57 is an ER chaperone protein with protein disulfide isomerase activity. Our previous studies have demonstrated that hyperoxia down-regulates ERp57 protein expression in the neonatal rat lung. In this study, we tested whether over-expression of ERp57 in lung epithelial cells would reduce hyperoxia-induced ER stress. Methods: Human ERp57 cDNA was subcloned into pcDNA3 plasmid and then transfected into A549 lung epithelial cells. ERp57 over-expressing cells (ERp57-A549) were selected by G418 and used for the experiments. Hyperoxia treatment for cultured cells was carried out in a chamber flushed with 95% O2 and 5% CO2. Results: We found that prolonged hyperoxic treatment for 24, 48 and 72 hours significantly increased phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), one of the known ER stress markers, in A549 cells. However, ERp57 over-expression decreased hyperoxia-induced eIF2α phosphorylation in ERp57-A549 cells. In addition, over-expression of human ERp57 decreased reactive oxygen species (ROS) production caused by hyperoxia, suggesting that ERp57 may be involved in regulation of cellular redox. Conclusions: Our results reveal that hyperoxia induces eIF2α phosphorylation and ER stress. Over-expression of ERp57 in human lung epithelial cells reduces ROS generation and ER stress under hyperoxic condition.

Retinoic Acid Inducibility of the Human PDGF-A Gene Is Mediated by 5′-Distal DNA Motifs That Overlap With Basal Enhancer and Vitamin D Response Elements

Retinoic acid (RA) upregulates expression of PDGF ligands and receptors in neonatal rat lung fibroblasts, a process likely to promote maturation of the lung alveolus and possibly microstructures of other organs. A mutational analysis of the gene encoding the PDGF-A ligand has identified a complex retinoic acid response element (RARE) located far upstream of the transcription start site, in a 5'-distal enhanceosome region previously shown to harbor basal and vitamin D-inducible enhancer activity. Maximal RA responsiveness (fourfold) was conferred by nucleotide sequence located between -7064 and -6787, with a variety of deletion and point mutations revealing the importance of at least three nuclear receptor half-sites within the enhancer region (-6851 to -6824), as well as nucleotides located further upstream. Recombinant human retinoic acid receptor/retinoid-X receptor heterodimers bound with high affinity and sequence specificity to multiple regions within the RARE, as demonstrated by electrophoretic mobility shift and DNase I footprinting assays. The addition of RARE activity to previously described functions of the 5'-distal enhanceosome underscores the importance of this region as a key integration point for regulatory control of PDGF-A expression.

The components of VARA, a nutrient-metabolite combination of vitamin A and retinoic acid, act efficiently together and separately to increase retinyl esters in the lungs of neonatal rats.

Retinoic acid (RA), produced from vitamin A (VA, retinol), is required for normal lung development and postnatal lung maturation. The concentration of retinyl ester (RE), the major storage form of retinol, decreases in the lungs in the perinatal period. Previously, we tested VARA, a nutrient-metabolite combination of VA and RA (10:1 molar ratio), on lung RE formation in postnatal rats and showed that the components of VARA acted synergistically to increase lung RE, as compared with the effects of equal amounts of VA and RA given alone. In this study, we first determined the equivalency of orally administered VARA in comparison to a standard oral supplement of VA, with respect to lung and liver RE storage. In a dose-dilution study, VARA was 4 times as effective as the standard dose of VA (VARA-25% did not differ from VA-100%). The synergistic effect of VARA was selective for the lungs, compared with the liver, in which VA and VARA had equal effects. Secondly, we tested whether the 2 components of VARA must be coadministered to exert their synergistic effect on lung RE content. RA and VA and were administered separately and together as VARA. Although RA alone had no effect on lung RE in this 24-h experiment, RA synergized with VA administered either 12 h before RA or 12 h after RA, as well as when coadministered as VARA. We infer that VA and RA are both limiting for lung RE formation in neonates. Given the importance of bioactive retinoids in cell differentiation and lung development, assuring an adequate lung RE content postnatally could be of benefit for lung maturation.

Role of adipose differentiation-related protein in lung surfactant production: a reassessment

Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) dependent cytoskeletal tension during lung organogenesis

There is growing evidence for the role of CFTR (cystic fibrosis transmembrane conductance regulator) in lung development and differentiation. The mechanism by which the chloride channel could affect lung organogenesis, however, is unknown. In utero CFTR gene transfer in the fetal lungs of mice, rats, and non-human primates was shown previously to alter lung structure and function. A study of the genes altered in the fetal rat lung following CFTR overexpression was initiated in an effort to determine the molecular mechanism for CFTR-dependent differentiation. In utero gene transfer with recombinant adenoviruses carrying either a reporter gene or CFTR resulted in the increased expression of a number of genes upon microarray analysis. The majority of the genes overexpressed in the CFTR-treated lungs were primarily associated with muscle structure and function. Histological and biochemical characterization of these proteins including myosin heavy chain, heat shock protein 27, and isoforms of myosin light chain showed that CFTR overexpression had a profound effect on smooth muscle contraction-related proteins. The CFTR-dependent regulation of smooth muscle contraction related proteins was shown to be related to chloride and extracellular ATP and was dependent upon the PI3 Kinase and Phospholipase C pathways. The changes in smooth muscle proteins were consistent with CFTR-dependent contractions of the embryonic airway smooth muscle. An assay was developed using fluorescent polystyrene beads to show that CFTR did indeed increase amniotic fluid flow into the fetal lung. Increased amniotic fluid pressure was shown previously to be associated with stretch-induced differentiation of the lung. Evaluation of neonatal respiratory function showed that CFTR-dependent muscle contractions and increased amniotic fluid pressure resulted in accelerated maturation of the neonatal rat lung. In addition, these CFTR-dependent changes were shown to be sufficient to reverse the lung phenotype of the CFTR knockout mouse. Mechanical forces influence lung development through pulmonary distension. CFTR overexpression in the fetal lung altered differentiation and development in the lung. These results are consistent with CFTR influencing lung development by regulating the muscle contractions associated with cytoskeletal tension and stretch induced differentiation. Deficiency of CFTR altering lung development would contribute significantly to the Cystic Fibrosis disease phenotype.

Apoptosis and the activity of ceramide, Bax and Bcl-2 in the lungs of neonatal rats exposed to limited and prolonged hyperoxia

BackgroundThe aim of the study is to examine the effect of limited and prolonged hyperoxia on neonatal rat lung. This is done by examining the morphologic changes of apoptosis, the expression of ceramide, an important mediator of apoptosis, the expression of inflammatory mediators represented by IL-1β and the expression of 2 proto-oncogenes that appear to modulate apoptosis (Bax and Bcl-2).MethodsNewborn rats were placed in chambers containing room air or oxygen above 90% for 7 days. The rats were sacrificed at 3, 7 or 14 days and their lungs removed. Sections were fixed, subjected to TUNEL, Hoechst, and E-Cadherin Staining. Sections were also incubated with anti-Bcl-2 and anti-Bax antisera. Bcl-2 and Bax were quantitated by immunohistochemistry. Lipids were extracted, and ceramide measured through a modified diacylglycerol kinase assay. RT-PCR was utilized to assess IL-1β expression.ResultsTUNEL staining showed significant apoptosis in the hyperoxia-exposed lungs at 3 days only. Co-staining of the apoptotic cells with Hoechst, and E-Cadherin indicated that apoptotic cells were mainly epithelial cells. The expression of Bax and ceramide was significantly higher in the hyperoxia-exposed lungs at 3 and 14 days of age, but not at 7 days. Bcl-2 was significantly elevated in the hyperoxia-exposed lungs at 3 and 14 days. IL-1β expression was significantly increased at 14 days.ConclusionExposure of neonatal rat lung to hyperoxia results in early apoptosis documented by TUNEL assay. The early rise in Bax and ceramide appears to overcome the anti-apoptotic activity of Bcl-2. Further exposure did not result in late apoptotic changes. This suggests that apoptotic response to hyperoxia is time sensitive. Prolonged hyperoxia results in acute lung injury and the shifting balance of ceramide, Bax and Bcl-2 may be related to the evolution of the inflammatory process.

Dynamic study on acute injury and oxidative stress in lung of neonatal rats induced by hyperoxia

目的 研究新生鼠暴露于高浓度氧不同时间后肺组织的病理改变,以及肺组织氧化应激反应状况.方法新生鼠暴露于＞95%O2和正常空气(21%O2)中,取暴露12、24、48和72 h 4个时相点的肺组织,观察其病理变化,检测肺组织匀浆中丙二醛(malondiadehyde,MDA)含量、总抗氧化能力(total anti-oxygen capability,TAOC).结果新生鼠暴露于＞95%O212 h即可引起肺组织损伤,肺组织MDA含量显著升高(P＜0.01),TAOC显著降低(P＜0.01);随着氧暴露时间延长,MDA含量逐渐增高,TAOC逐渐降低,肺损伤加重,肺组织病理学检查可见肺组织微血管扩张、充血,肺泡内蛋白渗出,炎症细胞浸润,肺组织结构紊乱,肺大泡数量增多,Ⅱ型肺泡上皮细胞脱落等.结论新生鼠暴露于＞95%O2 12 h即可导致肺损伤和肺组织氧化应激反应的发生,高氧肺损伤与肺组织氧化应激反应有关。

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.

Fibroblast Growth Factor-2 and Receptor-1α(IIIc) Regulate Postnatal Rat Lung Cell Apoptosis

Fibroblast growth factor receptor-1alpha(IIIc) [FGF-R1alpha(IIIc)] regulates recovery of neonatal rat lung growth, after 95% oxygen-mediated growth arrest. Its role in normal postnatal alveologenesis is unknown. To determine if FGF-R1alpha(IIIc) regulates normal postnatal alveologenesis. Truncated soluble FGF-R1alpha(IIIc) or neutralizing antibodies to FGF-1 or FGF-2 were injected intraperitoneally into 3-d-old rats. The pups were killed at Day 7 for studies of alveolar development. Injected, truncated soluble FGF-R1alpha(IIIc) inhibited phosphorylation of the endogenous FGF-R1, and downstream pathway, and paradoxically increased lung DNA content and tissue fraction while inhibiting lung cell DNA synthesis. The increase in tissue thickness was due to reduced apoptosis, as indicated by reductions in cleaved effector caspases 3 and 7. Inhibition of the intrinsic apoptosis pathway was suggested by decreases in the proapoptotic protein Bax and mitochondrial cytochrome c release, and an increase in the antiapoptotic protein Bcl-x(L). Injected antibodies to FGF-1 and FGF-2 had no effect on DNA synthesis, but both increased Bcl-x(L) content and decreased cytochrome c release and cleaved caspase-7 protein expression. However, only injection of the antibody to FGF-2 replicated the increased tissue fraction and inhibited apoptosis observed with the injection of truncated soluble FGF-R1alpha(IIIc). Inhibition of ligand binding, most likely of FGF-2, to the FGF-R1alpha(IIIc) inhibits normal postnatal lung cell apoptosis.