Neonatal Rat Lung Research Articles

Pulmonary targeting of sustained release formulation of budesonide in neonatal rats

Systemic corticosteroids are widely used for the treatment/prevention of chronic lung disease (CLD) in premature infants. The use of the inhalation route for delivering corticosteroids has been widely recognized, however so far pre-term babies continue to be treated with oral glucocorticoids, such as dexamethasone. We hypothesize that the pulmonary administration of sustained release formulations of inhaled corticosteroids to pre-term infants will result in a higher benefit/risk ratio as compared to traditional inhalation therapy. To achieve a slow release formulation budesonide particles were coated with a very thin film of polylactic acid using a pulse laser ablation technique. Coated material was characterized with respect to the dissolution behavior and particle size. Ex vivo receptor binding studies were performed to monitor the cumulative lung, liver and brain receptor occupancies after adminstration in neonatal (10–11 days old) rats after intratracheal instillation of either uncoated budesonide or poly (l-lactic acid) (PLA) coated budesonide. The mean dissolution timed for the uncoated and the polymer coated formulations were 1.2 ± 0.5 and 4.7 ± 0.1 h, respectively (p < 0.05). No significant differences in the respirable fraction were found between coated and uncoated formulation (p>0.05). The average receptor occupancies in the lung, liver and brain after administration of uncoated budesonide were 58.4 ± 12.9, 56.4 ± 6.8 and 38.3 ± 6.7%, respectively. However, after administration of PLA coated budesonide, the average AUC estimates in the lung, liver and brain were 75.8 ± 3.7%, 46.6 ± 14.5 and 29 ± 7, respectively. The results from our study suggest sustained receptor occupancy in the lungs of neonatal rats after administration of PLA coated budesonide results in lower systemic exposure (as indicated by low liver receptor occupancy). The data strongly underscore the urgent need to develop sustained release pulmonary-targeted delivery systems of corticosteroids for the treatment of CLD in pre-term infants. The administration of inhaled corticosteroids using targeted drug-delivery systems will potentially result in higher local effects and a reduction in systemic exposure.

Rosiglitazone, a peroxisome proliferator-activated receptor-γ agonist, prevents hyperoxia-induced neonatal rat lung injury in vivo

Molecular disruption of homeostatic alveolar epithelial-mesenchymal interactions results in transdifferentiation of alveolar interstitial lipofibroblasts to myofibroblasts. Although this process was suggested to be a central molecular event in the pathogenesis of bronchopulmonary dysplasia (BPD), so far it has been only demonstrated in vitro; whether it also occurs in vivo is unknown. Our objectives were to determine if exposure to hyperoxia results in pulmonary alveolar lipo-to-myofibroblast transdifferentiation in vivo, and whether treatment with a potent peroxisome proliferator-activated receptor gamma (PPARgamma) (the key lipogenic fibroblast nuclear transcription factor) agonist, rosiglitazone, prevents this process. Newborn Sprague Dawley rat pups were exposed to control (21% O2), hyperoxia alone (95% O2 for 24 hr), or hyperoxia with rosiglitazone (95% O2 for 24 hr + rosiglitazone, 3 mg/kg, administered intraperitoneally) conditions. Subsequently, pups were sacrificed, and lung tissue was analyzed by morphometry, and by reverse transcription-polymerase chain reaction, Western hybridization, and immunohistochemistry for the expression of key lipogenic and myogenic markers. We observed a significant decrease in the expression of lipogenic markers, and a significant increase in the expression of myogenic markers in the hyperoxia-alone group. These hyperoxia-induced morphologic, molecular, and immunohistochemical changes were almost completely prevented by rosiglitazone. This is the first evidence of in vivo lipo-to-myofibroblast transdifferentiation and its almost complete prevention by rosiglitazone, prompting us to conclude that administration of PPARgamma agonists may be a novel, effective strategy to prevent the hyperoxia-induced lung molecular injury that has been implicated in the pathogenesis of BPD.

Perinatal expression of HSP70 and VEGF in neonatal rat lung vessels exposed to nicotine during gestation

We assessed the influence of maternal nicotine exposure during gestation on perinatal expression of HSP70 and VEGF in rat lung parenchyma and lung vessels. Adult white Sprague-Dawley virgin rats were mated with adult male rats over 2 days, with two females for every male. After confirming pregnancy, 30 gravid rats (dams) were then randomly assigned to two equal groups (one experimental and one control; n=15 in each). Experimental dams were treated with subcutaneus (s.c.) (-)-nicotine tartrate, 3 mg/kg body weight/day, during pregnancy from gestational days 9 through 21. After sacrifice, lungs were removed en bloc and formalin-fixed, and paraffinembedded tissue sections were evaluated by immunohistochemistry using a three-step streptavidin-biotin-peroxidase method with monoclonal antibodies directed against HSP70 or VEGF. HSP70 and VEGF expression was increased in the vascular smooth muscle cells of the experimental group (t1) compared to the control group (t(2))t(1)=7.593, t(2)=4.666, p<0.05). The number of bronchioles that stained positively with HSP70 was higher in the nicotineexposed group than in the control group (t(1)=9.274, t(2)=6.956, p<0.05). In conclusion, gestational nicotine exposure increased the expression of VEGF and HSP70 in rat lung parenchyma, especially in the airway epithelium and vascular smooth muscle cells. In vascular smooth muscle cells, these molecules may contribute to nicotine-related hypoxic pulmonary hypertension.

Effect of passive smoking during pregnancy on the expression of TNF-α and CINC in the lungs of neonatal rats

目的 研究孕鼠被动吸烟对新生大鼠肺组织肿瘤坏死因子α(tumor necrosis factor-α, TNF-α)和细胞因子诱生的中性粒细胞趋化因子(cytokine-induced neutrophil chemoattractant, CINC)表达的影响,探讨产前被动吸烟对新生大鼠肺损伤的机制. 方法分别将SD大鼠于妊娠第1、8和15天置于烟箱内,每天被动吸烟2 h建立孕鼠被动吸烟动物模型,孕鼠每天置于无烟箱内2 h为对照组,于妊娠第20天剖宫取胎鼠测体重,采用逆转录聚合酶链反应(RT-PCR)技术和酶联免疫吸附(ELISA)法检测新生鼠肺组织TNF-α、CINC mRNAs的表达和TNF-α的蛋白水平. 结果不同孕期被动吸烟SD大鼠所产新生鼠体重较对照组明显下降(P<0.05或0.01),肺组织TNF-α mRNA的表达与对照组比较明显增强 (P<0.01),肺组织TNF-α蛋白含量明显增加(P<0.05或0.01);孕鼠妊娠早、中期被动吸烟所产新生鼠肺组织CINC mRNA表达与对照组和晚期组比较明显增强(P<0.01). 结论孕鼠被动吸烟可影响新生大鼠肺组织炎性介质TNF-α和CINC的表达,这可能是引起新生鼠肺损伤的重要原因之一。

Growth hormone (GH) action in the developing lung: Changes in lung proteins after adenoviral GH overexpression

Growth hormone (GH) recently has been shown to be expressed in the neonatal rat lung during alveolarization. The possible functional importance of lung GH in lung function, therefore, has been assessed by determining changes in GH-responsive proteins in the developing rat lung after the overexpression of the GH gene in this tissue. GH overexpression was achieved using an adenovirus that expressed the mouse GH gene. This adenovirus was effective in inducing mouse GH expression in cultured rat lung L2 epithelial cells. It was also shown to be strongly expressed in the alveoli of 14-day-old rat pup lungs 10 days after it was administered by intratracheal injection, during a period of rapid lung development. Expression of the transgene in these pups was accompanied by changes in lung protein concentrations determined by two-dimensional gel electrophoresis and mass spectrometry. The lung concentrations of specific enzymes (nucleotide diphosphate kinase B, Cu/Zn superoxide dismutase, glutathione-S-transferase, and aldehyde reductase-1) were increased by the adenoviral expression of mouse GH, as were the concentrations of beta subunit G-protein calponin 2, beta-5 tubulin, retinoblastoma binding protein 4, and fetuin A. In contrast, the lung concentrations of haptoglobin and major acute phase alpha-1 protein were reduced by adenoviral expression of mouse GH. Although most of these proteins have not previously been identified as GH-responsive proteins, these results demonstrate actions of GH in the rat lung and support the possibility that GH acts as an autocrine/paracrine during early lung development.

322 Prenatal Glucocorticoids: Effects on Lung of Newborn Rats Exposed to Hypoxia and Recovered in Hyperoxia or Air.

Aims: To examine the morphometric changes produced on lung of neonatal rats treated with prenatal administration of betamethasone or dexamethasone, and test the hypothesis if glucocorticoids would produce any difference when pups are exposed to hypoxia and its recovery with oxygen or air ambient. Material and Methods: Dexamethasone, Bethametasone or equivalent saline solution was administered i.v. To gestant Wistar rats on 20 and 21 day of gestation. All rats delivered their pups within 48 hours after the last injection. The groups of rat pups were randomly assigned to either. At 4–8 hours of life newborn rats were exposed in sequential way during 2 hours in hypoxia, and recovered in > 95% oxygen or 21% oxygen. Lung and body weight were measured. For the morphometric study the lungs were fixed by intratracheal instilation of formol at a pressure of 20 cm.H2O. Quantitative morphometric assessment was done on coded slides with 400x magnification and a eye piece with a sample square grid pattern (model CPLW 1018, Zeiss Optical, Hannover Md). Morphmetric changes are shown in table 1. Statystical Analyses: ANOVA test was performed followed by Bonferroni test. P< 0,05 was considered. Result: Comparative wet lung weight/dry lung weight and morphometric study is show in table I DEX (dexamethasone) BETA (bethametasone) W.(weight), Sa (alveolar surface), SV (surface density of alveolar walls), Nv (numerical density of alveoli), rv (alveolar radius) * Significant differences with saline group. Conclusion: Prenatal administration of dexametasone and bethametasone causes a reduction in alveolar number in newborn rats. Lungs of treated pups showed a longer alveoli radius, lower values of surface density of alveolar walls and lower numerical density of alveoli than the control group. Probably, prenatal glucocorticoids could produce a partial suppression of the formation of secondary septa and decreased final alveoli number.

Modulation of IGF-Binding Protein-2 and -3 in Hyperoxic Injury in Developing Rat Lung

Retinoids play an important role in lung development and repair. We showed that retinoic acid (RA) inhibits O(2)-induced fibroblast proliferation in rat lung explants. IGF-1, which enhances the proliferation of human fetal lung fibroblasts and stimulates collagen production during lung injury, has an important role in the lung injury/repair process. Interactions of IGF-1 with its receptor are modulated by IGF-binding proteins IGFBPs. We hypothesized that RA alters IGFBP-2 and -3 in hyperoxia-exposed neonatal lung and alters collagen production. Neonatal rat lungs were cultured in room air or 95% O(2) and 5% CO(2) for 3 d with or without RA. IGFBP-2 and -3 were measured both in culture medium and in lung tissue. Type I collagen and procollagen propeptide were analyzed in the lung tissue. Hyperoxia induced an increase in type I collagen that was significantly inhibited in the presence of RA. IGFBP-2 and IGFBP-3 in the lungs were decreased in hyperoxia but significantly increased in hyperoxia plus RA. In the culture medium, IGFBP-2 and -3 were not increased with hyperoxia but significantly increased in the presence of RA plus hyperoxia. There was no increase in IGFBP-3 RNA transcript after RA treatment in either room air or O(2) exposure. In conclusion, RA modulates the secreted IGFBP-2 and -3 during O(2) exposure and inhibits the increase in collagen that occurs during lung injury. We speculate that RA protects against O(2)-induced neonatal lung injury through modulation of the IGFBPs.

Dynamic Changes in Glottal Resistance during Exposure to Severe Hypoxia in Neonatal Rats In Situ

The neural control of respiratory airflow via the vocal fold is characterized by inspiratory abduction and postinspiratory (early expiratory) adduction causing decreases and increases in glottal resistance, respectively. The postinspiratory increase in glottal resistance plays a major role in braking the speed of expiratory airflow, to act against the high recoil pressure of the neonatal rat lung. In the present study, we investigated changes in upper airway patency during severe hypoxia in neonatal rats. We measured dynamic changes in subglottal pressure during normoxic and hypoxic conditions in an arterially perfused brainstem preparation in which we could control gas tensions accurately. Initially, hypoxia (5% O(2), 5% CO(2), and 90% nitrogen) produced an excitatory response in phrenic nerve activity accompanied by augmentation of both inspiratory-related glottal dilation and postinspiratory glottal constriction. Later, during the early stages of hypoxia-induced respiratory depression and initiation of gasping, we observed a massive reduction of the respiratory modulation of glottal resistance. In most preparations, this was transient and replaced by a paradoxic inspiratory-related glottal constriction. We propose that during severe hypoxia in the in situ preparation, paradoxic inspiratory glottal constriction can be observed during gasping, and this may impair ventilation despite the persistence of rhythmic contractions of the respiratory muscles. The latter is of clinical interest, because this may relate to the finding of cot death victims who died as a result of upper airway obstruction but without apparent apnea or rebreathing.

Expression of Epidermal Growth Factor-Like Domain 7 in Neonatal Rat Lungs During Normoxia and Hyperoxia.

Purpose of Study: Preterm babies treated with ventilator support and supplemental oxygen frequently develop chronic lung disease (CLD) that has significant mortality and morbidity. Oxygen toxicity plays an important role in CLD etiology. Several lines of evidence have suggested that impairment of pulmonary angiogenesis is implicated in alveolization and the development of CLD. Epidermal growth factor-like domain 7 (EGFL-7) is a recently identified protein secreted from vascular endothelial cells and it regulates vascular tubulogenesis (Nature 2004;428:754). Aim of this study was to measure EGFL-7 expression in the neonatal lung during normoxia and hyperoxia. Methods: Rat pups at 4 days of age were randomly assigned to normoxic and hyperoxic groups. The rats in the normoxic and hyperoxic groups were treated with room air and 95% O2 for 3, 6, and 10 days, respectively. The lung tissues were collected for total RNA isolation. EGFL-7 mRNA expression was measured by quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR). Separately, human umbilical vein endothelial cells (HUVEC) were cultured in 37°C, 5% CO2 incubator, and were exposed to normoxia (room air) or hyperoxia (95% O2). Results EGFL-7 mRNA in normoxic neonatal rat lung was consistently expressed from 7 days to 2 months of age (n = 3) at each time. EGFL-7 mRNA expression in the hyperoxic group was significantly decreased after oxygen exposure for 3, 6 and 10 days; it decreased 2.1 fold at day 3 (n = 3); 4.1 fold at day 6 (n = 3); and 3.1 fold at day 10 (n = 3) compared to time-matched normoxic group results, respectively. EGFL-7 mRNA expression in the hyperoxic group returned to nearly normal levels 2 weeks (n = 3) after discontinuing oxygen exposure, and it remained at normal levels during the 2 month recovery period (n = 2–3). In cultured HUVEC, EGFL-7 mRNA expression also decreased 2.6 fold after 95% O2 exposure for 48 hours. Conclusions: Oxygen exposure is associated with the decrease of EGFL-7 mRNA expression in the neonatal rat lung and the expression level returns to normal after oxygen treatment. These findings imply that reduced levels of EGFL-7 at a critical lung development stage may be a contributing factor in the impairment of pulmonary angiogenesis and alveolization after hyperoxic lung injury.

Significance of transforming growth factor β1 expression in the lung of neonatal premature rats exposed to hyperoxia

目的 探讨转化生长因子β1(TGF-β1)在新生未成熟大鼠高氧肺损伤不同时点肺组织中的表达及意义. 方法将99只新生未成熟大鼠随机分为高氧组和空气组,于日龄3、7和14 d随机分批处死并取肺组织HE染色进行病理观察,免疫组化检测TGF-β1在各组中的表达,秩和检验组间THF-β1阳性强度表达差异. 结果高氧各组鼠肺组织TGF-β1的表达较同期空气组广泛,且在肺泡和支气管上皮细胞、肺内间质中的表达强度差别始终强于对照组(u值分别为:3 d时:49.0、14.0、63.0;7 d时:34.0、2.0、45.0;14 d时:32.0、13.5、33.0,P值均<0.05),日龄14 d时,高氧组鼠肺泡巨噬细胞中可见TGF-β1的表达,而空气组鼠肺泡巨噬细胞中未见TGF-β1的表达,两组比较u值为13.0,P<0.05. 结论肺组织中的TGF-β1表达增强以及肺泡巨噬细胞中表达TGF-β1,可能与高氧肺损伤有内在联系。

Ultrastructural changes in the lungs of neonatal rats intratracheally inoculated with meconium.

Meconium aspiration syndrome has been for many years an important cause of neonatal respiratory distress in newborn babies and sporadically reported in animals. This investigation was designed to study the ultrastructural and morphometric changes in the lungs of neonatal rats following the intratracheal inoculation of meconium. Seven-day-old Fischer-344 rats (n = 24) were randomly allocated in two groups. One group was intratracheally inoculated with saline solution and the second group received homologous meconium. Neonates were euthanatized at 1, 3 and 7 postinoculation days (PID) and lungs were examined by light and electron microscopy. Saline solution did not induce any ultrastructural changes in the lung. In contrast, meconium induced deciliation, recruitment of neutrophils and pulmonary alveolar macrophages to the bronchoalveolar space, intravascular sequestration of neutrophils and aggregation of platelets at PID 1 and 3. Other ultrastructural changes at PID 1 and 3 included interstitial edema and escape of red cells and fibrin into the alveolar space and interstitium. Interstitial edema and sequestration of neutrophils were responsible for the significant increase in thickness of alveolar septa. At PID 7 there was hyperplasia and enlargement of type II pneumocytes as well as interstitial proliferation of mesenchymal cells with intra-alveolar fibrosis. It was concluded that intratracheal inoculation of meconium in neonatal rats induces acute ultrastructural changes followed by a reparative response.

Regulation of elastin gene transcription by interleukin-1 beta-induced C/EBP beta isoforms.

We previously showed that interleukin (IL)-1beta decreases elastin gene transcription through activation of the NF-kappaB subunit p65 in neonatal rat lung fibroblasts. The present study was undertaken to further explore the molecular mechanisms responsible for the inhibitory effect of IL-1beta on elastin gene transcription. We found that cycloheximide blocked IL-1beta-induced downregulation of elastin mRNA but did not inhibit IL-1beta-induced translocation of p65 into the nucleus. IL-1beta treatment increased CCAAT/enhancer-binding protein (C/EBP)beta mRNA and protein levels including liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP), which was cycloheximide sensitive. C/EBPbeta isoforms bound a GCAAT-containing sequence in the proximal elastin promoter as determined by electrophoretic gel shift studies and confirmed by using specific anti-C/EBPbeta antibodies and by competition studies with oligonucleotides. Transient transfection of LIP expression vectors strongly decreased the transcriptional activity of the cotransfected elastin promoter and decreased levels of endogenous elastin mRNA. We demonstrated that IL-1beta-induced downregulation of elastin mRNA is dependent on NF-kappaB activation and C/EBPbeta expression. These results indicate that IL-1beta treatment activates NF-kappaB, which subsequently induces LIP expression and inhibition of elastin gene transcription.

IS MATERNAL COPPER SUPPLEMENTATION DURING ALVEOLARIZATION PROTECTING THE DEVELOPING RAT LUNG AGAINST THE ADVERSE EFFECTS OF MATERNAL NICOTINE EXPOSURE? A Morphometric Study

In a previous study, it was shown that maternal nicotine exposure during gestation and lactation interfered with alveolarization and resulted in gradual deterioration of the lung parenchyma, resulting in microscopic emphysema. The aim of this study was thus to investigate the long-term effects of maternal nicotine exposure (1 mg/kg body weight/day, subcutaneous [sc] from the onset of the phase of rapid alveolarization, which occur from postnatal day 4 in rats, on (1) the development of the gas-exchange area of the lungs of the offspring and, (2) whether maternal copper supplementation (1 mg/kg body weight/day, SC) during the same period of time will prevent the effect of maternal nicotine exposure on the development of the neonatal rat lung. Nicotine administration lasted until weaning on postnatal day 21. The day of birth was designated day 0. The offspring were exposed to nicotine via the mother's milk only. The experimental animals received no nicotine or copper after postnatal day 21. The lung tissue of the neonates was collected on postnatal days 14, 21, and 42 and prepared for morphometry. The results obtained show that maternal nicotine exposure had no influence on body weight, chest circumference, crown-rump length, and lung volume, but resulted in bigger alveolar volumes and suppressed alveolarization in the lungs of the offspring. Copper supplementation during this period of lung development reduced the adverse effect of maternal nicotine exposure on neonatal lung development. Even though copper reduced the adverse effects of maternal nicotine exposure during this phase of lung development, it did not prevent the induction of microscopic emphysema.

Microscopic changes induced by the intratracheal inoculation of amniotic fluid and meconium in the lung of neonatal rats.

Meconium aspiration syndrome is a major contributor to neonatal respiratory distress in infants and it has been sporadically recognized in neonatal animals. This investigation was designed to study the short and long term effects of meconium and amniotic fluid in the lungs of neonatal rats. Seven-day-old rats (n = 123) divided in three groups were intratracheally inoculated with saline solution, amniotic fluid or meconium. Rats were euthanatized on 1, 3, 7, 14, 28, 56 and 112 postinoculation days (PID) and the lungs were examined by light microscopy. Saline solution did not induce any change while amniotic fluid elicited only a mild foreign body response which disappeared by PID 14. In contrast, meconium induced an exudative alveolitis characterized by recruitment of neutrophilsn in the bronchoalveolar spaces. Meconium also induced atelectasis, hyperinflation and thickening of alveolar septa all of which had disappeared by PID 14. Starting at PID 7, neutrophils were progressively replaced by macrophages, giant cells, and some fibroblasts. There were sporadic foci of mineralization starting at PID 14 and lasting up to PID 112. Some mineralized foci became lined with cuboidal epithelial cells at PID 28. Meconium was slowly degraded but still evident by PID 112. It was concluded that inoculation of meconium in neonatal rats induces acute microscopic changes typical of meconium aspiration syndrome. The long term lesions induced by meconium consisted of persistent multifocal histiocytic alveolitis and bronchiolitis reaction with occasional foci of calcification.

Regulation of TGF-beta ligand and receptor expression in neonatal rat lungs exposed to chronic hypoxia.

Long-term effects of hypoxia are largely due to its modulatory effects on proliferation and differentiation of epithelial and endothelial cells, processes also regulated by the transforming growth factor (TGF)-beta system. We investigated the effects of hypoxia on the TGF-beta system in rat lungs from different developmental stages. Sprague-Dawley rats were exposed to 9.5% oxygen during either the first 2 wk of life or adulthood. Analysis revealed an arrest of alveolarization in hypoxic postnatal day 14 rats. Bioactive TGF-beta levels in bronchoalveolar lavage fluid were increased in these animals, and Western blot analysis revealed upregulation of TGF-beta receptor (TbetaR) I and II. None of these changes was observed in hypoxic adults. Hypoxia did, however, lead to decreased expression of TbetaRIII in both postnatal day 14 and adult rats. Immunohistochemical analysis localized TbetaRI-III predominantly to bronchiolar and alveolar epithelium; these patterns did not change with hypoxia. Thus we observed changes in TGF-beta activity and TbetaR isotype expression in rat lung that parallel the arrest in alveolarization seen with chronic hypoxia in early development. These alterations may partly explain the morphological changes observed in hypoxia.

Regulation of 1-cys peroxiredoxin expression in lung epithelial cells.

1-cys peroxiredoxin (1-cys Prx), the only member of a Prx subfamily that contains a single conserved cysteine residue, is abundant in lung. This bifunctional protein has both glutathione peroxidase and phospholipase A2 activities compatible with a role both in protection against lung oxidant injury and also in lung phospholipid metabolism. Here we studied the developmental expression of 1-cys Prx in rat lungs and hormonal effects on protein expression in human and rat lung cells. There was little change in 1-cys Prx expression during the prenatal period, but a marked increase in expression immediately after birth. Enzymatic (peroxidase and phospholipase) activities increased gradually after birth and reached adult level at 7-14 postnatal days. Expression of the protein was induced in the presence of dexamethasone (Dex) in cultured human and rat lung epithelial cells and also was upregulated in neonatal rat lung in vivo. cAMP treatment had no effect on expression, although there was a modest synergistic effect when combined with Dex in human fetal lung epithelial cells. The increased expression of 1-cys Prx at birth may be important for surfactant phospholipid turnover related to the phopholipase A2 activity of the protein and for antioxidant defense based on its peroxidase function.

NF-kappaB induced by IL-1beta inhibits elastin transcription and myofibroblast phenotype.

Interleukin (IL)-1beta released after lung injury regulates the production of extracellular matrix components. We found that IL-1beta treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibroblasts. Deletion analysis of the rat elastin promoter detected a cis-acting element located at -118 to -102 bp that strongly bound Sp1 and Sp3 but not nuclear factor (NF)-kappaB. This element mediated IL-1beta-induced inhibition of the elastin promoter. IL-1beta treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-kappaB. Overexpression of p65 decreased elastin promoter activity and markedly reduced elastin mRNA. Immunoprecipitation studies indicated an interaction between the p65 subunit and Sp1 protein. Microarray analysis of mRNA isolated after overexpression of p65 or treatment with IL-1beta revealed downregulation of alpha-smooth muscle actin and calponin mRNAs. Expression of these genes is associated with the myofibroblast phenotype. These results indicate that IL-1beta activates the nuclear localization of NF-kappaB that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype.

Regulation of Cell Proliferation by Insulin-like Growth Factor 1 in Hyperoxia-Exposed Neonatal Rat Lung

Hyperoxic exposure of the developing lung leads to characteristic peribronchial and mesenchymal fibroproliferative changes. We hypothesize that O2-induced changes in the neonatal lung are mediated by Insulin-like growth factor 1 (IGF-1) and IGF-1 receptor (IGF-1R). Lung explant cultures were prepared from 3-day-old neonatal rat pups and exposed to room air or 95% O2 for 72 h. Western blots and immunohistochemistry were used to determine if hyperoxia stimulated IGF-1 and IGF-1R, and to identify the cell types involved. Retinoic acid was used to learn if this would inhibit oxygen-induced cell proliferation. Hyperoxia induced a significant increase in thymidine incorporation (control, 54 ± 9; hyperoxia, 254 ± 24 dpm/nM DNA; mean ± SEM; N = 3; P < 0.05). This was inhibited by 5 × 10−5 M RA (149 ± 18 dpm/nM DNA; P < 0.05) and by anti-IGF-1 antibody (115 ± 25 dpm/nM DNA; P < 0.05; N = 3). BrdU labeling in the mesenchymal cells was significantly increased in mesenchymal cells after exposure to oxygen (91% higher than the room air control) but not in epithelial cells. This increase was inhibited in the presence of retinoic acid. Western blots showed IGF-1 protein was increased after 72 h of O2 exposure compared to room air exposure (57 ± 7 compared to 32 ± 5 densitometric units; P < 0.05; N = 3). The increase was inhibited when the cultures were exposed to 95% O2 in the presence of anti-IGF-1 antibody (28 ± 4; P < 0.05; N = 3). IGF-1 protein decreased in the presence of retinoic acid after oxygen exposure but not in room air. Immunostaining of O2-exposed lung showed IGF-1 was most abundant in airway and alveolar epithelial cells. We conclude that hyperoxia increases cell proliferation by stimulating IGF-1 in the neonatal rat lung. Interaction of IGF-1 and IGF-1R is an important cell-cell communication mechanism in the developmental and repair processes of hyperoxic neonatal lung injury.

Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung.

Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1-10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.

Expression of Peroxiredoxin I and II in Neonatal and Adult Rat Lung Exposed to Hyperoxia

Background : In mammals, the activity of antioxidant enzymes is increased in adult lung to adapt to hyperoxia. The increase of these activities is augmented in neonates and is known as an important mechanism of tolerance to high oxygen levels. Peroxiredoxin(Prx) is an abundant and ubiquitous intracellular antioxidant enzyme. Prx I and II are major cytosolic subtypes. The aim of this study was to examine th Prx I and II mRNA and protein expression levels in adult rat lungs and to compare then with those of neonatal rat lungs exposed to hyperoxia. Materials and Methods : Adult Sprague-Dawley rats and neonates that were delivered from timed pregnant Sprague-Dawley rat were randomly exposed to normoxia or hyperoxia. After exposure to high oxygen level for a set time, the bronchoalveolar lavage fluid and lung tissue were obtained. The Prx I and II protein expression levels were measured by western blot analysis using polyclonal rabbit anti-Prx I or anti-Prx II antibodies and the relative expression of the Prx I and Prx II per Actin protein were obtained as an internal standard. The Prx I and II mRNA expression levels were measured by northernblot analysis using Prx I and Prx II-specific cDNA prepared from pCRPrx I and pCRPrx II, and the relative Prx I and Prx II expression levels per Actin mRNA were obtained as an internal standard. Results : Hyperoxia induced some peak increase in the Prx I mRNA levels after 24 hour in adult rats. Interestingly, hyperoxia induced a marked increase of Prx I mRNA 24 hour in neonatal rats. However, hyperoxia did not induce an alteration in the expression of Prx II mRNA in both the adult and neonatal rat lungs. Hyperoxia did not induce an alteration in the expression of the Prx I and Prx II protein in both the adult and neonatal rat lungs. Hyperoxia did not induce an alteration in the amount of Prx I and Prx II protein all the times in the bronchoalveolar fluid of adult rats. Conclusion : Prx I and II is differently regulated by hyperoxia in adult and neonatal rat lung at the transcriptional level. The prominent upregulation of Prx I mRNA in neonates compared to those in adults by hyperoxia may be another mechanism of resistance to high oxygen levels in neonate.