Surfactant Protein mRNA Expression Research Articles

Surfactant protein a attenuates generalized and localized neuroinflammation in neonatal mice.

Surfactant protein A (SP-A) has important roles in innate immunity and modulation of pulmonary and extrapulmonary inflammation. Given SP-A has been detected in rat and human brain, we sought to determine if SP-A has a role in modulating inflammation in the neonatal mouse brain. Neonatal wildtype (WT) and SP-A-deficient (SP-A-/-) mice were subjected to three models of brain inflammation: systemic sepsis, intraventricular hemorrhage (IVH) and hypoxic-ischemic encephalopathy (HIE). Following each intervention, RNA was isolated from brain tissue and expression of cytokine and SP-A mRNA was determined by real-time quantitative RT-PCR analysis. In the sepsis model, expression of most cytokine mRNAs was significantly increased in brains of WT and SP-A-/- mice with significantly greater expression of all cytokine mRNA levels in SP-A-/- mice compared to WT. In the IVH model, expression of all cytokine mRNAs was significantly increased in WT and SP-A-/- mice and levels of most cytokine mRNAs were significantly increased in SP-A-/- mice compared to WT. In the HIE model, only TNF-α mRNA levels were significantly increased in WT brain tissue while all pro-inflammtory cytokine mRNAs were significantly increased in SP-A-/- mice, and all pro-inflammatory cytokine mRNA levels were significantly higher in SP-A-/- mice compared to WT. SP-A mRNA was not detectable in brain tissue of adult WT mice nor in WT neonates subjected to these models. These results suggest that SP-A-/- neonatal mice subjected to models of neuroinflammation are more susceptible to both generalized and localized neuroinflammation compared to WT mice, thus supporting the hypothesis that SP-A attenuates inflammation in neonatal mouse brain.

Development and Functional Characterization of Fetal Lung Organoids.

Preterm infants frequently suffer from pulmonary complications due to a physiological and structural lung immaturity resulting in significant morbidity and mortality. Novel in vitro and in vivo models are required to study the underlying mechanisms of late lung maturation and to facilitate the development of new therapeutic strategies. Organoids recapitulate essential aspects of structural organization and possibly organ function, and can be used to model developmental and disease processes. We aimed at generating fetal lung organoids (LOs) and to functionally characterize this in vitro model in comparison to primary lung epithelial cells and lung explants ex vivo. LOs were generated with alveolar and endothelial cells from fetal rat lung tissue, using a Matrigel-gradient and air-liquid-interface culture conditions. Immunocytochemical analysis showed that the LOs consisted of polarized epithelial cell adhesion molecule (EpCAM)-positive cells with the apical membrane compartment facing the organoid lumen. Expression of the alveolar type 2 cell marker, RT2-70, and the Club cell marker, CC-10, were observed. Na+ transporter and surfactant protein mRNA expression were detected in the LOs. First time patch clamp analyses demonstrated the presence of several ion channels with specific electrophysiological properties, comparable to vital lung slices. Furthermore, the responsiveness of LOs to glucocorticoids was demonstrated. Finally, maturation of LOs induced by mesenchymal stem cells confirmed the convenience of the model to test and establish novel therapeutic strategies. The results showed that fetal LOs replicate key biological lung functions essential for lung maturation and therefore constitute a suitable in vitro model system to study lung development and related diseases.

Comparison of survival of preterm newborn rabbits at 25-28 days of gestation with perinatal therapies at birth transition.

Eligibility of ventilated preterm rabbit model to investigate extreme pulmonary immaturity at birth transition is unknown. By extending this model to early saccular stage of fetal lung development, we evaluated efficacy in survival, lung maturation, and underlying mechanisms of contemporary perinatal therapies. Pregnant New Zealand White rabbit does were given dexamethasone (DEX), or sham injection as control (NDEX), 48 and 24 h before delivery at gestational age (GA) of 25-28 days. At birth, newborn rabbits were anesthetized and randomly allocated to four groups receiving either surfactant or nonsurfactant for both DEX and NDEX, and mechanically ventilated within low tidal volumes. Ranges of time to maintain survival rate ≥ 50% in GA 25-28 days were 59-136, 138-259, 173-288, and 437 to ≥600 min, respectively, each across the four groups. The benefits of DEX and/or surfactant for survival were more obvious in GA 25-26 days, as judged by improved lung mechanics, lower lung injury scores, higher lung surfactant phospholipid pools, and surfactant protein mRNA expression, with DEX-surfactant combination being the most optimal for the outcome. In contrast, those of GA 27-28 days had variable but meaningful responses to the treatment. Cox regression analysis revealed GA, DEX, and surfactant being independently protective factors whereas pneumothorax was a risk factor. The extremely preterm rabbits at GA 25-26 days markedly responded to the perinatal therapies for longer survival, lung maturation and injury alleviation, and were relevant for study of preterm birth transition-associated morbidities and underlying mechanisms.NEW & NOTEWORTHY An extremely preterm rabbit model with gestational age of 25-26 (term 31) days was established by mechanical ventilation with individually adjusted tidal volume at lower ranges. The administration of antenatal glucocorticoids and/or postnatal surfactant achieved significantly longer duration to maintain 50% survival and facilitated lung maturation and protection at early saccular stage. The usefulness of this model should be validated in future investigation of perinatal and neonatal morbidity and mortality at extremely preterm birth transition.

Paracrine stimulation of perinatal lung functional and structural maturation by mesenchymal stem cells

BackgroundMesenchymal stem cells (MSCs) were shown to harbor therapeutic potential in models of respiratory diseases, such as bronchopulmonary dysplasia (BPD), the most common sequel of preterm birth. In these studies, cells or animals were challenged with hyperoxia or other injury-inducing agents. However, little is known about the effect of MSCs on immature fetal lungs and whether MSCs are able to improve lung maturity, which may alleviate lung developmental arrest in BPD.MethodsWe aimed to determine if the conditioned medium (CM) of MSCs stimulates functional and structural lung maturation. As a measure of functional maturation, Na+ transport in primary fetal distal lung epithelial cells (FDLE) was studied in Ussing chambers. Na+ transporter and surfactant protein mRNA expression was determined by qRT-PCR. Structural maturation was assessed by microscopy in fetal rat lung explants.ResultsMSC-CM strongly increased the activity of the epithelial Na+ channel (ENaC) and the Na,K-ATPase as well as their mRNA expression. Branching and growth of fetal lung explants and surfactant protein mRNA expression were enhanced by MSC-CM. Epithelial integrity and metabolic activity of FDLE cells were not influenced by MSC-CM. Since MSC’s actions are mainly attributed to paracrine signaling, prominent lung growth factors were blocked. None of the tested growth factors (VEGF, BMP, PDGF, EGF, TGF-β, FGF, HGF) contributed to the MSC-induced increase of Na+ transport. In contrast, inhibition of PI3-K/AKT and Rac1 signaling reduced MSC-CM efficacy, suggesting an involvement of these pathways in the MSC-CM-induced Na+ transport.ConclusionThe results demonstrate that MSC-CM strongly stimulated functional and structural maturation of the fetal lungs. These effects were at least partially mediated by the PI3-K/AKT and Rac1 signaling pathway. Thus, MSCs not only repair a deleterious tissue environment, but also target lung cellular immaturity itself.

Glucocorticoids Equally Stimulate Epithelial Na+ Transport in Male and Female Fetal Alveolar Cells.

Preterm infants frequently suffer from respiratory distress syndrome (RDS), possibly due to lower expression of epithelial Na+ channels (ENaC). RDS incidence is sex-specific, affecting males almost twice as often. Despite the use of antenatal glucocorticoids (GCs), the sex difference persists. It is still controversial whether both sexes benefit equally from GCs. We previously showed that Na+ transport is higher in female compared with male fetal distal lung epithelial (FDLE) cells. Since GCs increase Na+ transport, we hypothesized that their stimulating effect might be sex-specific. We analyzed FDLE cells with Ussing chambers and RT-qPCR in the presence or absence of fetal serum. In serum-free medium, GCs increased the ENaC activity and mRNA expression, independent of sex. In contrast, GCs did not increase the Na+ transport in serum-supplemented media and abolished the otherwise observed sex difference. Inhibition of the GC receptor in the presence of serum did not equalize Na+ transport between male and female cells. The GC-induced surfactant protein mRNA expression was concentration and sex-specific. In conclusion, female and male FDLE cells exhibit no sex difference in response to GCs with regard to Na+ transport, and GR activity does not contribute to the higher Na+ transport in females.

Adverse impact of ambient PM2.5 on expression and trafficking of surfactant protein A through reactive oxygen species damage to lamellar bodies

Particulate matter with a diameter of less than 2.5 μm (PM2.5) easily deposits on lung alveoli and degrades human health. Surfactant protein A (SP-A) is the most abundant pulmonary surfactant protein stored in lamellar bodies (LBs) of alveolar epithelial type II cells. The impacts of PM2.5 on SP-A are multifaceted and intractable, and the underlying mechanism remains unclear. In this study, the expression and distribution of SP-A in Balb/c mice and A549 cells under PM2.5 exposure were investigated. The results showed that the low and medium concentration of PM2.5 gradually enhanced SP-A protein and mRNA expression, whereas the high concentration of PM2.5 conspicuously decreased SP-A protein but not its mRNA compared with the control. The trafficking of SP-A to LBs was gradually disturbed, and concomitantly, the lesions of LBs responsible for the transport and storage of SP-A protein were exacerbated with increased PM2.5 concentration. Reactive oxygen species production abundantly increased upon PM2.5 exposure, and it was antagonized by the oxidant inhibitor N-acetylcysteine. Subsequently, the injured LBs and the decrease in SP-A expression under exposure to the high concentration of PM2.5 were well rescued. The present study provides a new perspective to investigate the adverse effects of PM2.5 or diesel exhaust particles on other proteins transported to and stored in LBs.

Normalisation of surfactant protein -A and -B expression in the lungs of low birth weight lambs by 21 days old.

Intrauterine growth restriction (IUGR) induced by placental restriction (PR) in the sheep negatively impacts lung and pulmonary surfactant development during fetal life. Using a sheep model of low birth weight (LBW), we found that there was an increase in mRNA expression of surfactant protein (SP)-A, -B and -C in the lung of LBW lambs but no difference in the protein expression of SP-A or -B. LBW also resulted in increased lysosome-associated membrane glycoprotein (LAMP)-3 mRNA expression, which may indicate an increase in either the density of type II Alveolar epithelial cells (AEC) or maturity of type II AECs. Although there was an increase in glucocorticoid receptor (GR) and 11β-hydroxysteroid dehydrogenase (11βHSD)-1 mRNA expression in the lung of LBW lambs, we found no change in the protein expression of these factors, suggesting that the increase in SP mRNA expression is not mediated by increased GC signalling in the lung. The increase in SP mRNA expression may, in part, be mediated by persistent alterations in hypoxia signalling as there was an increase in lung HIF-2α mRNA expression in the LBW lamb. The changes in the hypoxia signalling pathway that persist within the lung after birth may be involved in maintaining SP production in the LBW lamb.

Vitamin D Depletion in Pregnancy Decreases Survival Time, Oxygen Saturation, Lung Weight and Body Weight in Preterm Rat Offspring.

Animal studies suggest a role of vitamin D in fetal lung development although not studied in preterm animals. We tested the hypothesis that vitamin D depletion aggravates respiratory insufficiency in preterm rat offspring. Furthermore, the effects of vitamin D depletion on growth and lung surfactant were investigated. Female Sprague-Dawley rats were randomly assigned low vitamin D (VDL) or control diet before mating and followed with serum 25-hydroxyvitamin D (s-25(OH)D) determinations. After cesarean section at gestational day 19 (E19) or day 22 (E22), placental weight, birth weight, crown-rump-length (CRL), oxygenation (SaO2) at 30 min and survival time were recorded. The pup lungs were analyzed for phospholipid levels, surfactant protein A-D mRNA and the expression of the vitamin D receptor (VDR). S-25(OH)D was significantly lower in the VDL group at cesarean section (12 vs. 30nmol/L, p<0.0001). Compared to the controls, E19 VDL pups had lower birth weight (2.13 vs. 2.29g, p<0.001), lung weight (0.09 vs. 0.10g, p = 0.002), SaO2 (54% vs. 69%, p = 0.002) as well as reduced survival time (0.50 vs. 1.25h, p<0.0001). At E22, the VDL-induced pulmonary differences were leveled out, but VDL pups had lower CRL (4.0 vs. 4.5cm, p<0.0001). The phospholipid levels and the surfactant protein mRNA expression did not differ between the dietary groups. In conclusion, Vitamin D depletion led to lower oxygenation and reduced survival time in the preterm offspring, associated with reduced lung weight and birth weight. Further studies of vitamin D depletion in respiratory insufficiency in preterm neonates are warranted.

Ex-Vivo Uterine Environment (EVE) Therapy Induced Limited Fetal Inflammation in a Premature Lamb Model.

Introduction Ex-vivo uterine environment (EVE) therapy uses an artificial placenta to provide gas exchange and nutrient delivery to a fetus submerged in an amniotic fluid bath. Development of EVE may allow us to treat very premature neonates without mechanical ventilation. Meanwhile, elevations in fetal inflammation are associated with adverse neonatal outcomes. In the present study, we analysed fetal survival, inflammation and pulmonary maturation in preterm lambs maintained on EVE therapy using a parallelised umbilical circuit system with a low priming volume.MethodsEwes underwent surgical delivery at 115 days of gestation (term is 150 days), and fetuses were transferred to EVE therapy (EVE group; n = 5). Physiological parameters were continuously monitored; fetal blood samples were intermittently obtained to assess wellbeing and targeted to reference range values for 2 days. Age-matched animals (Control group; n = 6) were surgically delivered at 117 days of gestation. Fetal blood and tissue samples were analysed and compared between the two groups.ResultsFetal survival time in the EVE group was 27.0 ± 15.5 (group mean ± SD) hours. Only one fetus completed the pre-determined study period with optimal physiological parameters, while the other 4 animals demonstrated physiological deterioration or death prior to the pre-determined study end point. Significant elevations (p<0.05) in: i) inflammatory proteins in fetal plasma; ii) selected cytokine/chemokine mRNA expression levels in fetal tissues; and iii) histological inflammatory score in fetal lung, were observed in the EVE group compared to the Control group. There was no significant difference (p>0.05) in surfactant protein mRNA expression level between the two groups.ConclusionIn this study, we achieved limited fetal survival using EVE therapy. Despite this, EVE therapy only induced a modest fetal inflammatory response and did not promote lung maturation. These data provide additional insight into markers of treatment efficacy for the assessment of future studies.

Intrafetal glucose infusion alters glucocorticoid signaling and reduces surfactant protein mRNA expression in the lung of the late-gestation sheep fetus.

Increased circulating fetal glucose and insulin concentrations are potential inhibitors of fetal lung maturation and may contribute to the pathogenesis of respiratory distress syndrome (RDS) in infants of diabetic mothers. In this study, we examined the effect of intrafetal glucose infusion on mRNA expression of glucose transporters, insulin-like growth factor signaling, glucocorticoid regulatory genes, and surfactant proteins in the lung of the late-gestation sheep fetus. The numerical density of the cells responsible for producing surfactant was determined using immunohistochemistry. Glucose infusion for 10 days did not affect mRNA expression of glucose transporters or IGFs but did decrease IGF-1R expression. There was reduced mRNA expression of the glucocorticoid-converting enzyme HSD11B-1 and the glucocorticoid receptor, potentially reducing glucocorticoid responsiveness in the fetal lung. Furthermore, surfactant protein (SFTP) mRNA expression was reduced in the lung following glucose infusion, while the number of SFTP-B-positive cells remained unchanged. These findings suggest the presence of a glucocorticoid-mediated mechanism regulating delayed maturation of the surfactant system in the sheep fetus following glucose infusion and provide evidence for the link between abnormal glycemic control during pregnancy and the increased risk of RDS in infants of uncontrolled diabetic mothers.

Keratinocyte growth factor‐2 intratracheal instillation significantly attenuates ventilator‐induced lung injury in rats

Preservation or restoration of normal alveolar epithelial barrier function is crucial for pulmonary oedema resolution. Keratinocyte growth factor-2 (KGF-2), a potent epithelial cell mitogen, may have a role in preventing ventilator-induced lung injury (VILI), which occurs frequently in mechanically ventilated patients. The aim of the study was to test the role of KGF-2 in VILI in rats. Forty healthy adult male Sprague-Dawley rats were randomly allocated into four groups, where rats in Groups HVZP (high-volume zero positive end-expiratory pressure) and HVZP+KGF-2 were given intratracheally equal PBS and 5 mg/kg KGF-2 72 hrs before 4 hrs HVZP ventilation (20 ml/kg), respectively, while PBS and KGF-2 were administered in the same manner in Groups Control and KGF-2, which underwent tracheotomy only with spontaneous breathing. Inflammatory cytokines (tumour necrosis factor-α, macrophage inflammatory protein 2), neutrophil and total protein levels in bronchoalveolar lavage fluid and surfactant protein mRNA expression in lung tissue were detected; the number of alveolar type II cells, lung water content and lung morphology were also evaluated. The results indicate that pre-treatment with KGF-2 showed dramatic improvement in lung oedema and inflammation compared with HVZP alone, together with increased surfactant protein mRNA and alveolar type II cells. Our results suggest that KGF-2 might be considered a promising prevention for human VILI or other acute lung injury diseases.

Effects of dexamethasone and oxidative stress on surfactant protein A, B, C and D expressions of H441 cells

Objective To explore the effects of dexamethasone on the expressions of surfactant protein(SP) A,B,C and D in H441 cells injured by oxidative stress.Methods H441 cells were challenged with hydrogen peroxide as oxidative stress damage cell model.Then the cells were divided into control group,groups on different dexamethasone levels (1,10 and 100 umol/L),H2O2 (100 μmol/L) injury group and dexamethasone (100 μmol/L)+ H2 O2 (100 μmol/1) group.All cells were cultured for 24 hours,then the expressions of SP mRNA were analyzed by quantitative real timepolymerase chain reaction,and proteins were detected by Western blot.One-way variant analysis and Dunneu's T3 test were applied as statistical methods.Results (1) The mRNA level of SP-A in H2 O2 injury group was higher than that in the control group (4.0016±0.3618 vs 0.9914±0.4170),while mRNA of SP C and SP-D were lower than those in the control group (SP C:0.2840±0.0586 vs 0.9719±0.1022;SP-D:0.6199±0.0074 vs0.9518±0.0614) (all P＜0.05).Compared with H2O2injury group,the mRNA level of SP-A in dexamethasone + H2O2 group was down-regulated (2.5191±0.3463),but SP-D mRNA was up-regulated (0.9076±0.0167)(all P＜0.05).(2) The SP-A protein level in H2O2 injury group was higher than that in the control group (2.1795±0.0645 vs 0.9728±0.009),while SP-B、SP-C and SP-D protein levels were lower than those in the control group (SP-B:0.2593±0.0086 vs 0.7253±0.0134;SP-C:0.5592±0.0111 vs 0.9472±0.0052;SP-D:1.1550±0.0237 vs 1.2991±0.0298).Compared with H2O2 injury group,the protein level of SP-A in dexamethasone+ H2 O2 group was down-regulated(1.4974 ± 0.0185),while SP-B,SP-C and SP-D levels were up-regulated (0.7389±0.0182,0.9244±0.0282 and 1.3339±0.0136).The differences were statistically significant respectively (all P＜0.05).Conclusions Dexamethasone may protect H441 cells from being damaged by H2O2 through regulating SP mRNA and protein expression. Key words: Oxidative stress; Cell line, tumor; Pulmonary surfactant-associated proteins; Dexamethasone

Effect of tobacco extract on surfactant synthesis and its reversal by retinoic acid—role of cell–cell interactions in vitro

Tobacco induces oxidative stress in the alveolar epithelium and causes its damage. Retinoic acid (RA) has a cardinal role in alveolar cell growth, differentiation, and maturation. The aim of the study was to investigate the role of cell-cell interactions and whether RA could reverse the effect of tobacco extract on epithelial function as expressed by surfactant synthesis. For this, an in vitro model, which provides multiple cell type interactions, as seen in vivo, was used. We had used the major lung cell types, alveolar epithelial and mesenchymal cells represented by the cell lines A549 (human lung adenocarcinoma cell line), and human fetal lung fibroblast-1 (HFL-1) for developing the monoculture and co-culture systems and studied the effect of tobacco extract and retinoic acid. The effect of tobacco and retinoic acid both singly and in combination on proliferation and surfactant synthesis was analyzed. Retinoic acid induced proliferation and upregulated surfactant synthesis in monocultures and co-cultures. Tobacco extract at 100μg/ml concentration decreased A549 proliferation and upregulated surfactant protein mRNA expression. In co-cultures treated with tobacco extract (100μg/ml), retinoic acid (1μM), regulated cell proliferation, and surfactant protein mRNA expression vis-à-vis the monoculture system. This clearly points to the fact that cell-cell interactions modulate the effect of additives or stimulants and help in assessing the in vivo combinatorial responses in vitro and that the retinoic acid effect is regenerative.

Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

BackgroundLipoteichoic acid (LTA), a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS), could induce surfactant protein-A (SP-A) production in human alveolar epithelial (A549) cells.ObjectivesIn this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms.MethodsA549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF)-κB, extracellular signal-regulated kinase 1/2 (ERK1/2), and mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1 were determined.ResultsExposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA.ConclusionsTherefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

Alcohol exposure during late gestation: multiple developmental outcomes in sheep.

Alcohol consumption during pregnancy remains common in many countries. Exposure to even low amounts of alcohol (i.e. ethanol) in pregnancy can lead to the heterogeneous fetal alcohol spectrum disorders (FASD), while heavy alcohol consumption can result in the fetal alcohol syndrome (FAS). FAS is characterized by cerebral dysfunction, growth restriction and craniofacial malformations. However, the effects of lower doses of alcohol during pregnancy, such as those that lead to FASD, are less well understood. In this article, we discuss the findings of recent studies performed in our laboratories on the effects of fetal alcohol exposure using sheep, in which we investigated the effects of late gestational alcohol exposure on the developing brain, arteries, kidneys, heart and lungs. Our studies indicate that alcohol exposure in late gestation can (1) affect cerebral white matter development and increase the risk of hemorrhage in the fetal brain, (2) cause left ventricular hypertrophy with evidence of altered cardiomyocyte maturation, (3) lead to a decrease in nephron number in the kidney, (4) cause altered arterial wall stiffness and endothelial and smooth muscle function and (5) result in altered surfactant protein mRNA expression, surfactant phospholipid composition and pro-inflammatory cytokine mRNA expression in the lung. These findings suggest that fetal alcohol exposure in late gestation can affect multiple organs, potentially increasing the risk of disease and organ dysfunction in later life.

Effects of recombinant human keratinocyte growth factor on surfactant, plasma, and liver phospholipid homeostasis in hyperoxic neonatal rats

Respiratory distress and bronchopulmonary dysplasia (BPD) are major problems in preterm infants that are often addressed by glucocorticoid treatment and increased oxygen supply, causing catabolic and injurious side effects. Recombinant human keratinocyte growth factor (rhKGF) is noncatabolic and antiapoptotic and increases surfactant pools in immature lungs. Despite its usefulness in injured neonatal lungs, the mechanisms of improved surfactant homeostasis in vivo and systemic effects on lipid homeostasis are unknown. We therefore exposed newborn rats to 85% vs. 21% oxygen and treated them systemically with rhKGF for 48 h before death at 7 days. We determined type II pneumocyte (PN-II) proliferation, surfactant protein (SP) mRNA expression, and the pulmonary metabolism of individual phosphatidylcholine (PC) species using [D(9)-methyl]choline and tandem mass spectrometry. In addition, we assessed liver and plasma lipid metabolism, addressing PC synthesis de novo, the liver-specific phosphatidylethanolamine methyl transferase (PEMT) pathway, and triglyceride concentrations. rhKGF was found to maintain PN-II proliferation and increased SP-B/C expression and surfactant PC in both normoxic and hyperoxic lungs. We found increased total PC together with decreased [D(9)-methyl]choline enrichment, suggesting decreased turnover rather than increased secretion and synthesis as the underlying mechanism. In the liver, rhKGF increased PC synthesis, both de novo and via PEMT, underlining the organotypic differences of rhKGF actions on lipid metabolism. rhKGF increased the hepatic secretion of newly synthesized polyunsaturated PC, indicating improved systemic supply with choline and essential fatty acids. We suggest that rhKGF has potential as a therapeutic agent in neonates by improving pulmonary and systemic PC homeostasis.

Prostaglandins mediate the fetal pulmonary response to intrauterine inflammation

Intra-amniotic (IA) lipopolysaccharide (LPS) induces intrauterine and fetal lung inflammation and increases lung surfactant and compliance in preterm sheep; however, the mechanisms are unknown. Prostaglandins (PGs) are inflammatory mediators, and PGE(2) has established roles in fetal lung surfactant production. The aim of our first study was to determine PGE(2) concentrations in response to IA LPS and pulmonary gene expression for PG synthetic [prostaglandin H synthase-2 (PGHS-2) and PGE synthase (PGES)] and PG-metabolizing [prostaglandin dehydrogenase (PGDH)] enzymes and PGE(2) receptors. Our second study aimed to block LPS-induced increases in PGE(2) with a PGHS-2 inhibitor (nimesulide) and determine lung inflammation and surfactant protein mRNA expression. Pregnant ewes received an IA saline or LPS injection at 118 days of gestation. In study 1, fetal plasma and amniotic fluid were sampled before and at 2, 4, 6, 12, and 24 h after injection and then daily, and fetuses were delivered 2 or 7 days later. Amniotic fluid PGE(2) concentrations increased (P < 0.05) 12 h and 3-6 days after LPS. Fetal lung PGHS-2 mRNA and PGES mRNA increased 2 (P = 0.0084) and 7 (P = 0.014) days after LPS, respectively. In study 2, maternal intravenous nimesulide or vehicle infusion began immediately before LPS or saline injection and continued until delivery 2 days later. Nimesulide inhibited LPS-induced increases in PGE(2) and decreased fetal lung IL-1β and IL-8 mRNA (P ≤ 0.002) without altering lung inflammatory cell infiltration. Nimesulide decreased surfactant protein (SP)-A (P = 0.05), -B (P = 0.05), and -D (P = 0.0015) but increased SP-C mRNA (P = 0.023). Thus PGHS-2 mediates, at least in part, fetal pulmonary responses to inflammation.

Expression of pulmonary surfactant protein A mRNA in lungs of fetal rats with maternal intrahepatic cholestasis

Objective To investigate the pulmonary morphologic changes and pulmonary surfactant protein A (SP-A) mRNA expression in fetal rats with maternal intrahepatic cholestasis of pregnancy (ICP).Methods Animal models of intrahepatic cholestasis of pregnancy were induced by 17-α-ethinylestradiol and progesterone.Histopathologic examination of the fetal lungs was performed under light and electronic microscopes.The expression of SP-A mRNA in the fetal lungs was measured by reverse transcription-polymerase chain reaction technique.Results (1) Compared with the control group,histopathologic examination showed that fetal pulmonary tissues had dilated and congested interstitial lung capillaries,thickened alveolar septum,mild focal inflammatory exudation and focal hemorrhage in alveolus.Furthermore,reduced microvilli,mitochondrion vacuolization,cytoplasm disintegration and increased lamellar body evacuation were observed in alveolar epithelial cell II in ICP group under light and electronic microscopes.(2) Expression of SP-A mRNA in fetal lungs of ICP group (0.71 + 0.10) was significantly lower than that in control group (1.00±0.27),t=3.093,P＜0.05.Conclusions Hyperbileacidemia in ICP maternal rat might lead to pathological changes in fetal pulmonary tissues.Low expression of SP-A mRNA might contribute to lesions of fetal lung with ICP. Key words: Pregnancy complications; Cholestasis,intrahepatic; Pulmonary surfactantassociated protein A; RNA,messenger; Rats

Presenilin-1 processing of ErbB4 in fetal type II cells is necessary for control of fetal lung maturation

Maturation of pulmonary fetal type II cells to initiate adequate surfactant production is crucial for postnatal respiratory function. Little is known about specific mechanisms of signal transduction controlling type II cell maturation. The ErbB4 receptor and its ligand neuregulin (NRG) are critical for lung development. ErbB4 is cleaved at the cell membrane by the γ-secretase enzyme complex whose active component is either presenilin-1 (PSEN-1) or presenilin-2. ErbB4 cleavage releases the 80 kDa intracellular domain (4ICD), which associates with chaperone proteins such as YAP (Yes-associated protein) and translocates to the nucleus to regulate gene expression. We hypothesized that PSEN-1 and YAP have a development-specific expression in fetal type II cells and are important for ErbB4 signaling in surfactant production. In primary fetal mouse E16, E17, and E18 type II cells, PSEN-1 and YAP expression increased at E17 and E18 over E16. Subcellular fractionation showed a strong cytosolic and a weaker membrane location of both PSEN-1 and YAP. This was enhanced by NRG stimulation. Co-immunoprecipitations showed ErbB4 associated separately with PSEN-1 and with YAP. Their association, phosphorylation, and co-localization were induced by NRG. Confocal immunofluorescence and nuclear fractionation confirmed these associations in a time-dependent manner after NRG stimulation. Primary ErbB4-deleted E17 type II cells were transfected with a mutant ErbB4 lacking the γ-secretase binding site. When compared to transfection with wild-type ErbB4, the stimulatory effect of NRG on surfactant protein mRNA expression was lost. We conclude that PSEN-1 and YAP have crucial roles in ErbB4 signal transduction during type II cell maturation.

STUDY OF PULMONARY SURFACTANT AND SURFACTANT PROTEIN IN RATS WITH LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY

INTRODUCTION: The abnormal metabolism of pulmonary surfactant (PS) may have some relationship to acute lung injury (ALI). OBJECTIVE: The objective of this study was to examine the alteration trend of PS and surfactant-associated protein (SP) in rats with lipopolysaccharide (LPS)-induced ALI. METHODS: Fifty-six adult Wistar rats were randomly divided into the normal saline (NS) group and the ALI group. The levels of mRNA of surfactant protein A (SP-A) and SP-B were measured by reverse-transcription polymerase chain reaction during intravenous LPS administration at 1, 3, 5, and 7 hours. The content and component of PS in the bronchoalveolar lavage fluid (BALF) were measured by high-performance liquid chromatography. In addition, lung dry/wet weight ratio, the protein content of BALF, alveolar oxygen partial pressure, and histologic changes were detected. RESULTS: Compared with the NS group, the ALI group developed severe lung damage; edema, hemorrhage, and inflammation were found. Total phospholipids in BALF at 1, 3, 5, and 7 hours were lower than those in the NS group; phosphatidylcholine at 3, 5, and 7 hours was lower than that in the NS group, whereas lysophosphatidylcholine at 1, 3, 5, and 7 hours was higher than that in the NS group. The expression of SP-A and SP-B mRNA at 3, 5, and 7 hours was less than that in the NS group. CONCLUSIONS: The changed metabolism of PS may be responsible for the pathogenesis of ALI. It is mainly demonstrated by the decrease in total phospholipids and phosphatidylcholine and the decreased expression of SP-A and SP-B mRNA. Decrease in content and change in components of PS may play an important role in severe hypoxemia in ALI.