Surfactant Protein Gene Expression Research Articles

Alveolar-Capillary Barrier Protection In Vitro: Lung Cell Type-Specific Effects and Molecular Mechanisms Induced by 1α, 25-Dihydroxyvitamin D3.

Low serum levels of 1α, 25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS. However, the molecular mechanisms behind this observation are not yet understood. VD3 is known to stimulate lung maturity, alveolar type II cell differentiation, or pulmonary surfactant synthesis and guides epithelial defense during infection. In this study, we investigated the impact of VD3 on the alveolar-capillary barrier in a co-culture model of alveolar epithelial cells and microvascular endothelial cells respectively in the individual cell types. After stimulation with bacterial LPS (lipopolysaccharide), gene expression of inflammatory cytokines, surfactant proteins, transport proteins, antimicrobial peptide, and doublecortin-like kinase 1 (DCLK1) were analyzed by real-time PCR, while corresponding proteins were evaluated by ELISA, immune-fluorescence, or Western blot. The effect of VD3 on the intracellular protein composition in H441 cells was analyzed by quantitative liquid chromatography-mass spectrometry-based proteomics. VD3 effectively protected the alveolar-capillary barrier against LPS treatment, as indicated by TEER measurement and morphological assessment. VD3 did not inhibit the IL-6 secretion by H441 and OEC but restricted the diffusion of IL-6 to the epithelial compartment. Further, VD3 could significantly suppress the surfactant protein A expression induced in the co-culture system by LPS treatment. VD3 induced high levels of the antimicrobial peptide LL-37, which counteracted effects by LPS and strengthened the barrier. Quantitative proteomics identified VD3-dependent protein abundance changes ranging from constitutional extracellular matrix components and surfactant-associated proteins to immune-regulatory molecules. DCLK1, as a newly described target molecule for VD3, was prominently stimulated by VD3 (10 nM) and seems to influence the alveolar-epithelial cell barrier and regeneration.

Regulation of Surfactant Protein Gene Expression by Aspergillus fumigatus in NCl-H441 Cells.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes serious lung diseases in immunocompromised patients. The lung surfactant produced by alveolar type II and Clara cells in the lungs is an important line of defense against A. fumigatus. The surfactant consists of phospholipids and surfactant proteins (SP-A, SP-B, SP-C and SP-D). The binding to SP-A and SP-D proteins leads to the agglutination and neutralization of lung pathogens as well as the modulation of immune responses. SP-B and SP-C proteins are essential for surfactant metabolism and can modulate the local immune response; however, the molecular mechanisms remain unclear. We investigated changes in the SP gene expression in human lung NCI-H441 cells infected with conidia or treated with culture filtrates obtained from A. fumigatus. To further identify fungal cell wall components that may affect the expression of SP genes, we examined the effect of different A. fumigatus mutant strains, including dihydroxynaphthalene (DHN)-melanin-deficient ΔpksP, galactomannan (GM)-deficient Δugm1 and galactosaminogalactan (GAG)-deficient Δgt4bc strains. Our results show that the tested strains alter the mRNA expression of SP, with the most prominent and consistent downregulation of the lung-specific SP-C. Our findings also suggest that secondary metabolites rather than the membrane composition of conidia/hyphae inhibit SP-C mRNA expression in NCI-H441 cells.

The homeodomain transcription factor Ventx2 regulates respiratory progenitor cell number and differentiation timing during Xenopus lung development.

Ventx2 is an Antennapedia superfamily/NK-like subclass homeodomain transcription factor best known for its roles in the regulation of early dorsoventral patterning during Xenopus gastrulation and in the maintenance of neural crest multipotency. In this work we characterize the spatiotemporal expression pattern of ventx2 in progenitor cells of the Xenopus respiratory system epithelium. We find that ventx2 is directly induced by BMP signaling in the ventral foregut prior to nkx2-1, the earliest epithelial marker of the respiratory lineage. Functional studies demonstrate that Ventx2 regulates the number of Nkx2-1/Sox9+ respiratory progenitor cells induced during foregut development, the timing and level of surfactant protein gene expression, and proper tracheal-esophageal separation. Our data suggest that Ventx2 regulates the balance of respiratory progenitor cell expansion and differentiation. While the ventx gene family has been lost from the mouse genome during evolution, humans have retained a ventx2-like gene (VENTX). Finally, we discuss how our findings might suggest a possible function of VENTX in human respiratory progenitor cells.

Inhibition of Prostaglandin F2α Receptors Exaggerates HCl-Induced Lung Inflammation in Mice.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe respiratory disorders that are caused by aspiration, sepsis, trauma, and pneumonia. A clinical feature of ALI/ARDS is the acute onset of severe hypoxemia, and the mortality rate, which is estimated at 38–50%, remains high. Although prostaglandins (PGs) are detected in the bronchoalveolar lavage fluid of patients with ALI/ARDS, the role of PGF2α in ALI remains unclear. We aimed to clarify the role of PGF2α/PGF2α receptor (FP) signaling in acid-induced ALI using an FP receptor antagonist, AL8810. Intratracheal injection of hydrochloric acid (HCl) increased neutrophil migration into the lungs, leading to respiratory dysfunction. Pre-administration of AL8810 further increased these features. Moreover, pre-treatment with AL8810 enhanced the HCl-induced expression of pro-inflammatory cytokines and neutrophil migratory factors in the lungs. Administration of HCl decreased the gene expression of lung surfactant proteins, which was further reduced by co-administration of AL8810. Administration of AL8810 also increased lung edema and reduced mRNA expression of epithelial sodium channel in the lungs, indicating that AL8810 reduced fluid clearance. Furthermore, AL8810 also increased lipopolysaccharide-induced expression of adhesion molecules such as intracellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells. These results indicate that inhibition of FP receptors by AL8810 exacerbated HCl-induced ALI.

The Effect of Modified Porcine Surfactant Alone or in Combination with Polymyxin B on Lung Homeostasis in LPS-Challenged and Mechanically Ventilated Adult Rats.

The study aimed to prove the hypothesis that exogenous surfactant and an antibiotic polymyxin B (PxB) can more effectively reduce lipopolysaccharide (LPS)-induced acute lung injury (ALI) than surfactant treatment alone, and to evaluate the effect of this treatment on the gene expression of surfactant proteins (SPs). Anesthetized rats were intratracheally instilled with different doses of LPS to induce ALI. Animals with LPS 500 μg/kg have been treated with exogenous surfactant (poractant alfa, Curosurf®, 50 mg PL/kg b.w.) or surfactant with PxB 1% w.w. (PSUR + PxB) and mechanically ventilated for 5 hrs. LPS at 500 μg/kg increased lung edema, oxidative stress, and the levels of proinflammatory mediators in lung tissue and bronchoalveolar lavage fluid (BALF). PSUR reduced lung edema and oxidative stress in the lungs and IL-6 in BALF. This effect was further potentiated by PxB added to PSUR. Exogenous surfactant enhanced the gene expression of SP-A, SP-B, and SP-C, however, gene expression for all SPs was reduced after treatment with PSUR + PxB. In mechanically ventilated rats with LPS-induced ALI, the positive effect of exogenous surfactant on inflammation and oxidative stress was potentiated with PxB. Due to the tendency for reduced SPs gene expression after surfactant/PxB treatment topical use of PxB should be considered with caution.

Complementary Effect of Maternal Sildenafil and Fetal Tracheal Occlusion Improves Lung Development in the Rabbit Model of Congenital Diaphragmatic Hernia.

To evaluate the effect of combining antenatal sildenafil with fetal tracheal occlusion (TO) in fetal rabbits with surgically induced congenital diaphragmatic hernia (CDH). Although antenatal sildenafil administration rescues vascular abnormalities in lungs of fetal rabbits with CDH, it only partially improves airway morphometry. We hypothesized that we could additionally stimulate lung growth by combining this medical treatment with fetal TO. CDH was created on gestational day (GD)23 (n=54). Does were randomized to receive either sildenafil 10 mg/kg/d or placebo by subcutaneous injection from GD24 to GD30. On GD28, fetuses were randomly assigned to TO or sham neck dissection. At term (GD30) fetuses were delivered, ventilated, and finally harvested for histological and molecular analyses. Unoperated littermates served as controls. The lung-to-body-weight ratio was significantly reduced in sham-CDH fetuses either (1.2 ± 0.3% vs 2.3 ± 0.3% in controls, P=0.0003). Sildenafil had no effect on this parameter, while CDH fetuses undergoing TO had a lung-to-body-weight ratio comparable to that of controls (2.5 ± 0.8%, P<0.0001). Sildenafil alone induced an improvement in the mean terminal bronchiolar density (2.5 ± 0.8 br/mm2 vs 3.5 ± 0.9 br/mm2, P=0.043) and lung mechanics (static elastance 61 ± 36 cmH2O /mL vs 113 ± 40 cmH2O/mL, P=0.008), but both effects were more pronounced in fetuses undergoing additional TO (2.1 ± 0.8 br/mm2, P=0.001 and 31 ± 9 cmH2O/mL, P<0.0001 respectively). Both CDH-sham and CDH-TO fetuses treated with placebo had an increased medial wall thickness of peripheral pulmonary vessels (41.9 ± 2.9% and 41.8 ± 3.2%, vs 24.0 ± 2.9% in controls, P<0.0001). CDH fetuses treated with sildenafil, either with or without TO, had a medial thickness in the normal range (29.4% ± 2.6%). Finally, TO reduced gene expression of vascular endothelial growth factor and surfactant protein A and B, but this effect was counteracted by sildenafil. In the rabbit model for CDH, the combination of maternal sildenafil and TO has a complementary effect on vascular and parenchymal lung development.

Short-Term versus Long-Term Culture of A549 Cells for Evaluating the Effects of Lipopolysaccharide on Oxidative Stress, Surfactant Proteins and Cathelicidin LL-37.

Alveolar epithelial type II (ATII) cells and their proper function are essential for maintaining lung integrity and homeostasis. However, they can be damaged by lipopolysaccharide (LPS) during Gram-negative bacterial infection. Thus, this study evaluated and compared the effects of LPS on short and long-term cultures of A549 cells by determining the cell viability, levels of oxidative stress and antimicrobial peptide cathelicidin LL-37 and changes in the expression of surfactant proteins (SPs). Moreover, we compared A549 cell response to LPS in the presence of different serum concentrations. Additionally, the effect of N-acetylcysteine (NAC) on LPS-induced oxidative stress as a possible treatment was determined. Our results indicate that A549 cells are relatively resistant to LPS and able to maintain integrity even at high LPS concentrations. Their response to endotoxin is partially dependent on serum concentration. NAC failed to lower LPS-induced oxidative stress in A549 cells. Finally, LPS modulates SP gene expression in A549 cells in a time dependent manner and differences between short and long-term cultures were present. Our results support the idea that long-term cultivation of A549 cells could promote a more ATII-like phenotype and thus could be a more suitable model for ATII cells, especially for in vitro studies dealing with surfactant production.

Strain-dependent effects on lung structure, matrix remodeling, and Stat3/Smad2 signaling in C57BL/6N and C57BL/6J mice after neonatal hyperoxia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of preterm infants, characterized by lung growth arrest and matrix remodeling. Various animal models provide mechanistic insights in the pathogenesis of BPD. Since there is increasing evidence that genetic susceptibility modifies the response to lung injury, we investigated strain-dependent effects in hyperoxia (HYX)-induced lung injury of newborn mice. To this end, we exposed newborn C57BL/6N and C57BL/6J mice to 85% O2 (HYX) or normoxia (NOX; 21% O2) for 28 days, followed by lung excision for histological and molecular measurements. BL/6J-NOX mice exhibited a lower body and lung weight than BL/6N-NOX mice; hyperoxia reduced body weight in both strains and increased lung weight only in BL/6J-HYX mice. Quantitative histomorphometric analyses revealed reduced alveolar formation in lungs of both strains after HYX, but the effect was greater in BL/6J-HYX mice than BL/6N-HYX mice. Septal thickness was lower in BL/6J-NOX mice than BL/6N-NOX mice but increased in both strains after HYX. Elastic fiber density was significantly greater in BL/6J-HYX mice than BL/6N-HYX mice. Lungs of BL/6J-HYX mice were protected from changes in gene expression of fibrillin-1, fibrillin-2, fibulin-4, fibulin-5, and surfactant proteins seen in BL/6N-HYX mice. Finally, Stat3 was activated by HYX in both strains; in contrast, activation of Smad2 was markedly greater in lungs of BL/6N mice than BL/6J mice after HYX. In summary, we demonstrate strain-dependent differences in lung structure and matrix, alveolar epithelial cell markers, and Smad2 (transforming growth factor β) signaling in neonatal HYX-induced lung injury. Strain-dependent effects and genetic susceptibility need be taken into consideration for reproducibility and reliability of results in animal models.

MicroRNA-19b Mediates Lung Epithelial-Mesenchymal Transition via Phosphatidylinositol-3,4,5-Trisphosphate 3-Phosphatase in Response to Mechanical Stretch.

Lung epithelial-mesenchymal transition (EMT) plays an important role in ventilation-associated lung fibrosis, which may contribute to the poor outcome of patients with acute respiratory distress syndrome. Because microRNAs control and modulate normal physiological and pathophysiological processes, we investigated the role of microRNAs in the development of acute respiratory distress syndrome-associated EMT in response to mechanical stress. In the current study, primary human alveolar epithelial type II (AEII) cells were subjected to cyclic stretch that resulted in EMT profiles with decreased gene expression of cytokeratin-8, E-cadherin, and surfactant protein B, and increased expression of vimentin, α-smooth muscle actin, and N-cadherin. Microarray analysis revealed that the expression of microRNA-19b (miR-19b) was up-regulated in the AEII cells, and real-time polymerase chain reaction showed that the expression of miR-19b increased in both the AEII cells and the primary human small-airway epithelial cells. Overexpression of miR-19b in small-airway epithelial cells promoted the mechanical stretch-induced EMT phenotypes, whereas inhibition of miR-19b attenuated it. The inhibitory effect of miR-19b was attributed to enhanced signaling of phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), leading to inactivation of the AKT pathway. Restoration of PTEN expression or inhibition of AKT phosphorylation suppressed the mechanical stretch-induced EMT phenotypes. We further demonstrated that the mechanical stretch-induced miR19 expression was regulated by the focal adhesion kinase-Rho pathway. In conclusion, we found that miR-19b plays a key role in the development of the EMT phenotype through down-regulation of PTEN in human lung epithelial cells in response to mechanical stretch. The miR-19b-PTEN signaling pathway may serve as a novel therapeutic target in the context of ventilator-associated lung fibrosis.

Comparison of the effects of betamethasone and dexamethasone on surfactant protein A mRNA expression in human lung cells

Objective: While prenatal administration of synthetic corticosteroids stimulates both fetal lung development and expression of pulmonary surfactant, the specific effects may depend on the corticosteroid formulation used. We compared the dose-dependent effects of various concentrations of two synthetic corticosteroids, betamethasone and dexamethasone, on steady state levels of surfactant protein A (SP-A) mRNA in human lung cells. Methods: Cultured human NCI-H441 bronchoalveolar epithelial cells were exposed to varying concentrations of betamethasone or dexamethasone (10−7 to 10−12 M) for 48 h alone or in combination with dibutyryl cAMP (1 mM), which augments surfactant protein gene expression. RNA was harvested and SP-A mRNA levels were quantified by real-time quantitative reverse transcriptase polymerase chain reaction analysis. Results were compared using the Kruskal–Wallis test. Results: A dose-dependent modification in SP-A mRNA levels was demonstrated with both dexamethasone and betamethasone. Cells treated with cAMP expressed higher levels of SP-A mRNA than untreated cells. A biphasic curve in the SP-A mRNA response to corticosteroids was elicited only in the presence of cAMP: at lower concentrations (10−10 through 10−12 M), SP-A mRNA levels were upregulated, whereas at higher concentrations (10−7 and 10−8 M), SP-A mRNA levels were reduced. Dexamethasone was more effective than betamethasone in inducing these changes. Conclusions: Our results support a biphasic effect on SP-A mRNA levels after exposure to corticosteroids in combination with cAMP. At higher corticosteroid concentrations, betamethasone is less inhibitory than dexamethasone on SP-A mRNA.

In vivo effects of TGF-β1 in lung surfactant regulation, lung meachanics amd structure

Transforming growth factor beta 1 (TGF-β1) is a signalling protein with a wide range of biological activities. TGF-β1 is thought to have a pivotal role in fibrogenesis, where TGF-β1 induces myofibroblast migration and increases extracellular matrix synthesis, including collagen. Moreover, it has been described that TGF-β1 is a negative modulator of the regulation of surfactant associated proteins A (SP-A), B (SP-B) and C (SP-C) in vitro. Pulmonary surfactant is a lipid-protein complex that lowers surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. SP-B and SP-C deficiency has been found in patients suffering from lung diseases and related to potential mechanical stress of the lung epithelium. We have characterized lung surfactant protein composition 1 and 2 weeks after adenoviral mediated gene transfer of active TGF-β1 into lungs. Gene expression of surfactant proteins is down-regulated pointing at deficient transcriptional regulation that might include a deficient activity of TTF-1, during TGF-β1 overexpression. Deficiency on SP-B and SP-C at early stages correlates with high surface tension under dynamic cycling of isolated surfactant in Captive Bubble Surfactometry (CBS). In addition, high surface tension correlates with decreased quasistatic lung compliance and increased collapsibility of distal airspaces. Stereological data demonstrate a correlation between septal wall thickness and quasistatic compliance 2 weeks after gen-transfer. At the ultrastructural level thickening of septal walls, could be attributed to an increase in interstitial cells, formation of dense alveolar oedema and increase in profiles of epithelial type II (AEII) cells. The latter could be also attributed to Epithelial to Mesenchymal Transition (EMT), indicated by down-regulation of epithelial markers associated to up-regulation of mesenchymal molecular markers. We can conclude that in vivo TGF-β1 is a strong negative regulator of surfactant metabolism, originating a mechanical stress that may contribute to EMT at following stages.

Heterogeneous Mixed-Lineage Differentiation of Mouse Embryonic Stem Cells Induced by Conditioned Media from A549 Cells

Conditioned media (CM) of transformed cells, such as the human lung-derived A549 cells, is a useful tool for directing differentiation of embryonic stem cells (ESCs). Previous work indicates that A549-CM induced pulmonary differentiation of mouse ESCs (mESCs). In this study, we compared the effects of A549-CM treatment on the differentiation of mESCs organized in monolayer or embryoid bodies. We analyzed the cultures treated with A549-CM using specific lineage markers by quantitative polymerase chain reaction (qPCR) and lineage-focused PCR arrays and demonstrated heterogeneous CM-induced differentiation. We then constructed bioinformatics-based gene networks to establish correlations between the upregulated lineage-specific genes and proteins in the A549-CM identified by proteomic analysis. Network analysis supported the phenotypic and genotypic heterogeneic differentiation of mESCs into multiple cell lineages via enriched stemness, cardiovascular, neuronal, and lung development gene ontologies (GOs). The significant enrichment toward lung ontologies was specific for treatment with A549-CM, but not CM of liver (HepG2) and pancreas (Capan-1) cells. Based on network analysis, we identified laminin alpha5, prosaposin, lamin A/C, dickkopf homolog 1, clusterin, and calreticulin as the most relevant proteins related to the enrichment of lung GOs. We validated the effects of laminin isoforms on mESC differentiation in vitro and found enriched differential induction of surfactant protein gene expression. Our data suggest that A549-CM can be used for identifying secreted proteins for the heterogeneous mixed-lineage differentiation of mESCs toward a variety of lung-relevant cells. Such a heterogeneous cell population will be required for the in vitro generation of complex lung tissue and mixed cell populations for regenerative pulmonary therapy.

Does lung development differ in male and female fetuses?

ABSTRACTPreterm male infants have a higher incidence of morbidity and mortality due to respiratory insufficiency than females of the same gestational age. This male disadvantage could be due to differences in lung architecture; however, few studies have compared lung architecture in male and female fetuses during late gestation. Our principal objectives were to compare the morphology of the fetal lung and the maturity of the surfactant system in preterm male and female fetuses. Lungs from male (n = 9) and female (n = 11) fetal sheep were collected at 0.9 of term (131 days of the 145-day gestation) for morphological and molecular analyses. In separate groups, tracheal liquid was obtained from male (n = 9) and female (n = 9) fetuses at 0.9 of term for determination of surfactant phospholipid composition. We found no sex-related differences in body weight, lung weight, right lung volume, lung tissue and airspace fractions, mean linear intercept, septal crest density, septal thickness, the proportion of proliferating and apoptotic cells, and the percentages of collagen or elastin. The gene expression of surfactant protein -A, -B, -C, and -D and tropoelastin was similar between sexes. There were no differences in the proportion of the major phospholipid classes in the tracheal liquid between sexes; however there was a significantly higher percentage of the phospholipid species phosphatidylinositol 38:5 in males. The greater morbidity and mortality in preterm male lambs do not appear to be related to differences in lung structure or surfactant phospholipid synthesis before birth, but may relate to physiological adaptation to air-breathing at birth.

Deletion of STK40 Protein in Mice Causes Respiratory Failure and Death at Birth*

STK40 is a putative serine/threonine kinase and was shown to induce extraembryonic endoderm differentiation from mouse embryonic stem cells. However, little is known about its physiological function in vivo. Here, we generate Stk40 knock-out mice and demonstrate that loss of the Stk40 gene causes neonatal lethality at birth. Further examination reveals that the respiratory distress and atelectasis occur in the homozygous mutants. The maturation of lung and alveolar epithelium is delayed in the mutant, as indicated by narrowed air spaces, thickened interstitial septa, and increased glycogen content in the lungs of Stk40(-/-) mice. The reduction in levels of T1-α, SP-B, and SP-C indicates delayed maturation of both type I and type II respiratory epithelial cells in Stk40(-/-) lungs. Moreover, Stk40 is found to be most highly expressed in lungs of both fetal and adult mice among all organs tested. Mechanistically, a genome-wide RNA microarray analysis reveals significantly altered expression of multiple genes known to participate in lung development. The expression of some genes involved in lipid metabolism, immune response, and glycogen metabolism is also disrupted in the lung of Stk40(-/-) mice. Protein affinity purification identifies RCN2, an activator of ERK/MAPK signaling, as an STK40-associated protein. Consistently, Stk40 deficiency attenuates the ERK/MAPK activation, and inhibition of ERK/MAPK activities reduces surfactant protein gene expression in lung epithelial cells. Collectively, this study uncovers an important role of STK40 for lung maturation and neonatal survival. STK40 may associate with RCN2 to activate ERK/MAPK signaling and control the expression of multiple key regulators of lung development.

Decreased Expression of Surfactant Protein Genes Is Associated with an Increased Expression of Forkhead Box M1 Gene in the Fetal Lung Tissues of Premature Rabbits

PurposeRecently, Forkhead box M1 (FoxM1) was reported to be correlated with lung maturation and expression of surfactant proteins (SPs) in mice models. However, no study has been conducted in rabbit lungs despite their high homology with human lungs. Thus, we attempted to investigate serial changes in the expressions of FoxM1 and SP-A/B throughout lung maturation in rabbit fetuses.Materials and MethodsPregnant New Zealand White rabbits were grouped according to gestational age from 5 days before to 2 days after the day of expected full term delivery (F5, F4, F3, F2, F1, F0, P1, and P2). A total of 64 fetuses were enrolled after Cesarean sections. The expressions of mRNA and proteins of FoxM1 and SP-A/B in fetal lung tissue were tested by quantitative reverse-transcriptase real-time PCR and Western blot. Furthermore, their correlations were analyzed.ResultsThe mRNA expression of SP-A/B showed an increasing tendency positively correlated with gestational age, while the expression of FoxM1 mRNA and protein decreased from F5 to F0. A significant negative correlation was found between the expression levels of FoxM1 and SP-A/B (SP-A: R=-0.517, p=0.001; SP-B: R=-0.615, p<0.001).ConclusionPreterm rabbits demonstrated high expression of FoxM1 mRNA and protein in the lungs compared to full term rabbits. Also, the expression of SP-A/B was inversely related with serial changes in FoxM1 expression. This is the first report to suggest an association between FoxM1 and expression of SP-A/B and lung maturation in preterm rabbits.

Long-Term Pulmonary Effects of Intrauterine Exposure to Endotoxin Following Preterm Birth in Sheep

Our aim was to determine whether fetal exposure to intraamniotic lipopolysaccharide (LPS) persistently alters the lungs following moderate preterm birth. Fetal sheep were exposed to LPS (1 mg/d) or saline from 0.75 to preterm birth at 0.90 of gestation. Eleven weeks after preterm birth, lung structure was unaltered. Interleukin (IL)-1β messenger RNA (mRNA) levels were elevated in lungs of LPS-exposed lambs (P < .05) but IL-1β protein levels were unaltered. Lung mRNA levels of IL-6, IL-8 and tumor necrosis factor α, and percentage of inflammatory cells were not different between groups. Surfactant protein (SP)-A and SP-C mRNA levels and SP-B tissue protein expression were higher in LPS-exposed lambs than controls (all P < .05); however, expression of SP-A and SP-C proteins was reduced. Prenatal LPS exposure causes a persistent increase in gene expression of proinflammatory mediators and surfactant proteins and a decrease in lung tissue SP-A and -C protein expression after preterm birth, which may affect lung immunity.

234 Effectiveness of Fetal Perflurocarbon Therapy for Lung Hypoplasia in Diaphragmatic Hernia

AimsTo assess the effects of fetal, intratracheal perfluoroctylbromid (PFOB) instillation on lung-mechanics and gene expression of Surfactant and developmental proteins in a newborn rabbit model of lung hypoplasia.MethodsOn day 23/31,...

The effects of intrauterine growth restriction and antenatal glucocorticoids on ovine fetal lung development

Intrauterine growth restriction (IUGR) is associated with high rates of neonatal morbidity. IUGR babies are often born preterm and are, therefore, exposed to antenatal glucocorticoids. Antenatal glucocorticoids significantly improve overall survival rates of preterm infants, but there is a paucity of information about their effects on IUGR Infants. We induced IUGR in sheep by single umbilical artery ligation (SUAL), or sham in control fetuses. To half the ewes, we administered betamethasone (BM) on d 5 (BM1) and 6 (BM2) following surgery, and collected fetal lung tissue on d 7. SUAL alone was associated with higher circulating fetal cortisol levels (2.8 ± 0.4 vs. 1.0 ± 0.4, P = 0.001) as compared with controls but not with changes in lung morphology or surfactant protein (SP) gene expression. BM was associated with a significant reduction in lung tissue density (P = 0.048). There were no significant differences between groups in lung DNA concentration or septal crest density. SP-A, SP-B, and SP-C gene expressions were significantly increased in control and SUAL fetuses that were administered BM. These results show that in SUAL fetuses, maternal BM is associated with acceleration of fetal lung structure, as occurs in normally grown fetuses, and that BM induces SP production, an effect not observed in SUAL-induced IUGR fetuses alone.

MAPK and JAK-STAT Signaling Pathways are Involved in the Oxidative Stress – Induced Decrease in Expression of Surfactant Protein Genes

Oxidative stress is generated by reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂), hydroxyl radical (•OH ) and superoxide anion (O^2––), which are produced as by-products of cellular metabolism. An imbalance in cellular redox status is a potent pathogenic factor that contributes to various chronic inflammatory diseases. In this study, we demonstrate that H₂O₂decreases surfactant protein A, B and ABCA3 mRNA level, and increases SP-D mRNA level in human pulmonary lung epithelial cells. The decreased mRNA level of SP-A and SP-B were significant with a maximum inhibition of 79 and 87%, respectively by 150 µM H₂O₂after 24 hrs of incubation. In addition, ABCA3 mRNA level was decreased with a maximum inhibition of 55% by 150 µM H₂O₂after 12 hrs of incubation. In contrast, 150 µM H₂O₂caused the SP-D mRNA level to increase to 200% of control after 8 hrs of incubation. The H₂O₂-induced gene repression or activation of SP-A, SP-B, SP-D and ABCA3 was blocked by pretreatment with the antioxidants N-acetyl-L-cysteine (NAC) and catalase. Furthermore, the inhibition of SP-A and SP-B was associated with reduced thyroid transcription factor -1 (TTF-1) DNA binding activity, and this reduced TTF-1 binding activity may be due to decreased TTF-1 protein expression level. The analyses of signal transduction pathways that may play a role in the regulation of gene expression by H₂O₂using several specific inhibitors showed that U0126, an inhibitor of ERK1/2 upstream kinase MEK1/2, blocked both H₂O₂-induced inhibition of SP-A and SP-B gene expression, whereas SB203580, an inhibitor of p38 MAPK, partially blocked H₂O₂-mediated inhibition of SP-A gene expression but not SP-B expression. In contrast, AG-490, a specific inhibitor of JAK-STAT pathway, blocked H₂O₂-mediated inhibition of SP-B gene expression but not SP-A expression. Immunoblot analyses using specific phosphor-antibodies demonstrated that ERK1/2, p38 MAPK and STAT3 are phosphorylated by oxidative stress suggesting that H₂O₂-induced inhibition of SP-A and SP-B gene expression is associated with MAPK and JAKSTAT signaling pathway. These data, therefore, suggest that H₂O₂affects SP-A and SP-B gene regulation by reducing TTF-1 DNA binding activity via MAPKs or STAT signaling pathways.

Is antenatal Administration of Retinoic Acid Effective on Pulmonary Development in Preterm Mice with Hypoxia-Induced Intrauterine Growth Restriction?

Background: Preterm newborns with intrauterine growth retardation (IUGR) are at increased risk for the development of pulmonary disorders. Vitamin A and its active form, all-trans retinoic acid (ATRA), are essential for growth and development of cells and tissues. Retinoids play an important role in the lung and respiratory epithelium. The fetal lung maturation has been demonstrated to be enhanced by vitamin A supplementation in animal models.Aim: We tested the hypothesis whether antenatal administration of ATRA effects the fetal lung development in preterm mice with IUGR.Methods: We exposed date-mated pregnant C57BL/6 mice (Charles River, Sulzfeld, Germany) by chance to hypoxia (FiO2 0.10) or to normoxia starting from gestational day 14 (E14). On day E15 the animals were randomly allocated to receive intraperitoneally a single dose of ATRA (10mg/kg) or its solvent (0.1ml/kg oil). Newborns were delivered preterm at day E17.5. Fetal lungs tissues were used for histomorphometric analyses. Expression of surfactant proteins (SP)-B and C was determined by quantitative real-time PCR as biochemical indicators for lung maturation.Results: Histomorphometric analyses revealed no differences between groups. The mRNA expression of surfactant proteins was found to be without significant difference between newborns with IUGR and normoxia controls. However, treatment with ATRA increased significantly the pulmonary expression of SP-B and SP-C by the factor of 1.8 in the normoxia group [p< 0.01].Conclusion: Maternal hypoxia led to IUGR in preterm mice without histomorphometric or transcriptional alterations of lung development. Antenatal treatment with ATRA effects SP gene expression in non-restricted newborns.