SP-B mRNA Research Articles

In utero ventilation induces lung parenchymal and vascular alterations in extremely preterm fetal sheep.

Extremely preterm infants are often exposed to long durations of mechanical ventilation to facilitate gas exchange, resulting in ventilation-induced lung injury (VILI). New lung protective strategies utilizing noninvasive ventilation or low tidal volumes are now common but have not reduced rates of bronchopulmonary dysplasia. We aimed to determine the effect of 24 h of low tidal volume ventilation on the immature lung by ventilating preterm fetal sheep in utero. Preterm fetal sheep at 110 ± 1(SD) days' gestation underwent sterile surgery for instrumentation with a tracheal loop to enable in utero mechanical ventilation (IUV). At 112 ± 1 days' gestation, fetuses received either in utero mechanical ventilation (IUV, n = 10) targeting 3-5 mL/kg for 24 h, or no ventilation (CONT, n = 9). At necropsy, fetal lungs were collected to assess molecular and histological markers of lung inflammation and injury. IUV significantly increased lung mRNA expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) compared with CONT, and increased surfactant protein (SP)-A1, SP-B, and SP-C mRNA expression compared with CONT. IUV produced modest structural changes to the airways, including reduced parenchymal collagen and myofibroblast density. IUV increased pulmonary arteriole thickness compared with CONT but did not alter overall elastin or collagen content within the vasculature. In utero ventilation of an extremely preterm lung, even at low tidal volumes, induces lung inflammation and injury to the airways and vasculature. In utero ventilation may be an important model to isolate the confounding mechanisms of VILI to develop effective therapies for preterm infants requiring prolonged respiratory support.NEW & NOTEWORTHY Preterm infants often require prolonged respiratory support, but the relative contribution of ventilation to the development of lung injury is difficult to isolate. In utero mechanical ventilation allows for mechanistic investigations into ventilation-induced lung injury without confounding factors associated with sustaining extremely preterm lambs ex utero. Twenty-four hours of in utero ventilation, even at low tidal volumes, increased lung inflammation and surfactant protein expression and produced structural changes to the lung parenchyma and vasculature.

Wet lung leading to RDS: the lung ultrasound findings and possible mechanisms - a pilot study from an animal mode

Background Clinically, the lung ultrasound (LUS) showed wet lung could cause respiratory distress syndrome (RDS) in newborns. This work aimed to investigate LUS changes over time and its potential mechanism as alveolar fluid increase in a rabbit model. Methods A total of 35 New Zealand Rabbits were randomly assigned to seven groups. Models of various alveolar fluid levels were induced by infusion of different volumes of normal saline (NS) via the endotracheal tube. LUS was performed before NS infusion, immediately after NS infusion and 4 h after NS infusion. To appraise LUS changes and its potential mechanism as alveolar fluid increase, histopathological examination, the mRNA and protein expression of surfactant protein (SP), and immunohistochemistry (IHC) were performed. The expression levels of SP-B and SP-C proteins were detected using western blotting, and the relative expression levels of SP-B and SP-C mRNA were detected using qRT-PCR. Results The results showed that LUS changed from B-line to lung consolidations accompanied by air-bronchograms in some locations of lungs at 4 h when the injection volume ≥ 6 ml/kg. Histopathological examination showed alveoli collapse, inflammatory cell infiltration and alveolar wall thickened. SP-B and SP-C mRNA and protein expression were statistically significantly reduced when the injection volume ≥6 ml/kg (p < .05). IHC staining displayed the same findings. Conclusions As alveolar fluid increase, LUS changed from wet lung to RDS after 4 h. The possible mechanism was that the SP protein expression was significantly reduced. LUS can be used to guide the administration of exogenous surfactant in this situation.

Organic dust inhibits surfactant protein expression by reducing thyroid transcription factor-1 levels in human lung epithelial cells.

Exposure to organic dust is a risk factor for the development of respiratory diseases. Surfactant proteins (SP) reduce alveolar surface tension and modulate innate immune responses to control lung inflammation. Therefore, changes in SP levels could contribute to the development of organic-dust-induced respiratory diseases. Because information on the effects of organic dust on SP levels is lacking, we studied the effects of dust from a poultry farm on SP expression. We found that dust extract reduced SP-A and SP-B mRNA and protein levels in H441 human lung epithelial cells by inhibiting their promoter activities, but did not have any effect on SP-D protein levels. Dust extract also reduced SP-A and SP-C levels in primary human alveolar epithelial cells. The inhibitory effects were not due to LPS or protease activities present in dust extract or mediated via oxidative stress, but were dependent on a heat-labile factor(s). Thyroid transcription factor-1, a key transcriptional activator of SP expression, was reduced in dust-extract-treated cells, indicating that its down-regulation mediates inhibition of SP levels. Our study implies that down-regulation of SP levels by organic dust could contribute to the development of lung inflammation and respiratory diseases in humans.

Expression of surfactant protein B is dependent on cell density in H441 lung epithelial cells.

BackgroundExpression of surfactant protein (SP)-B, which assures the structural stability of the pulmonary surfactant film, is influenced by various stimuli, including glucocorticoids; however, the role that cell-cell contact plays in SP-B transcription remains unknown. The aim of the current study was to investigate the impact of cell-cell contact on SP-B mRNA and mature SP-B expression in the lung epithelial cell line H441.MethodsDifferent quantities of H441 cells per growth area were either left untreated or incubated with dexamethasone. The expression of SP-B, SP-B transcription factors, and tight junction proteins were determined by qPCR and immunoblotting. The influence of cell density on SP-B mRNA stability was investigated using the transcription inhibitor actinomycin D.ResultsSP-B mRNA and mature SP-B expression levels were significantly elevated in untreated and dexamethasone-treated H441 cells with increasing cell density. High cell density as a sole stimulus was found to barely have an impact on SP-B transcription factor and tight junction mRNA levels, while its stimulatory ability on SP-B mRNA expression could be mimicked using SP-B-negative cells. SP-B mRNA stability was significantly increased in high-density cells, but not by dexamethasone alone.ConclusionSP-B expression in H441 cells is dependent on cell-cell contact, which increases mRNA stability and thereby potentiates the glucocorticoid-mediated induction of transcription. Loss of cell integrity might contribute to reduced SP-B secretion in damaged lung cells via downregulation of SP-B transcription. Cell density-mediated effects should thus receive greater attention in future cell culture-based research.

Association of Surfactant Protein with Expression of Hoxa5 and Hoxb5 in Rabbit Fetal Lung

Hox genes regulate organ formation and identity of the embryo, and expressed in specific temporo-spatial patterns in the developing embryo. We compared the expression levels of the Hoxa5, Hoxb5, surfactant protein (SP)-A, and SP-B genes in immature and mature rabbit fetal lung tissues, and to uncover roles for Hoxa5, Hoxb5, SP-A, and SP-B. Cesarean sections were performed after rabbits were divided into two groups of 30-31 days of gestation (term group, n = 24) and 26-27 days of gestation (preterm group, n = 24). mRNA levels of Hoxa5, Hoxb5, SP-A, and SP-B were compared by quantitative reverse transcriptase polymerase chain reaction, and protein expression of Hoxa5 and Hoxb5 was compared by western blot analysis. Fetal lung tissue histology was observed by hematoxylin and eosin (H&E) staining. The relative expression ratios of SP-A and SP-B mRNA in the term to preterm groups were 2.45:1 and 2.94:1, respectively. Hoxb5 mRNA and protein levels decreased in the term group, with a relative expression ratio of 0.48:1 and 0.50:1, however, Hoxa5 mRNA and protein levels increased in the term group with a relative expression ration of 2.99:1 and 2.33:1, respectively, for the term to preterm groups. Moreover, a significant positive correlation was found between the expression of Hoxa5 and SP-A, SP-B in the term group. Hoxa5 gene may be essential for the expression of SP-A and SP-B in term rabbits. The Hoxb5 gene may be an important factor for lung maturation in preterm rabbits.

Effects of Bufei Yishen Granules Combined with Acupoint Sticking Therapy on Pulmonary Surfactant Proteins in Chronic Obstructive Pulmonary Disease Rats.

Our previous studies have demonstrated the beneficial effects of Bufei Yishen granules combined with acupoint sticking therapy (the integrated therapy) in chronic obstructive pulmonary disease (COPD), but the underlying mechanism remains unclear. Dysfunction of pulmonary surfactant proteins (SPs, including SP-A, SP-B, SP-C, and SP-D) may be included in pathophysiology of COPD. This study aimed to explore the mechanism of the integrated therapy on SPs. COPD rat models were established. The treatment groups received Bufei Yishen granules or acupoint sticking or their combination. Using aminophylline as a positive control drug. The levels of SPs in serum, BALF, and lung were measured. The results showed that the integrated therapy markedly reduced the levels of SPs in serum and increased these indicators in the lung. The integrated therapy was better than aminophylline in reducing the levels of SPs and was better than Bufei Yishen granules in reducing SP-A, SP-C, and SP-D in serum. The integrated therapy was better than aminophylline and Bufei Yishen granules in increasing SP-A, SP-B, and SP-D mRNA in the lung. SP-A and SP-D in BALF were positively correlated with PEF and EF50. The levels of SPs are associated with airway limitation. The beneficial effects of the integrated therapy may be involved in regulating pulmonary surfactant proteins.

Expression of lung surfactant proteins SP-B and SP-C and their modulating factors in fetal lung of FGR rats.

This study investigated the expression of lung surfactant proteins SP-B and SP-C, and their modulating factors TTF-1 and PLAGL2 in the fetal lung of rats with fetal growth restriction (FGR). The rat FGR model was established by prenatal hypoxia in the first stage of pregnancy, 180 rats for experiment served as hypoxia group, and 197 healthy rats served as normal control group. The FGR incidence in hypoxia was compared with that in normal control group. The histological changes in the fetal lung were observed under the light microscope and electronic microscope in two groups. The SP-B, SP-C, TTF-1 and PLAGL2 proteins were determined in the fetal lung of two groups immunohistochemically. The expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung of two groups were detected by using Western blotting and RT-PCR respectively. The FGR rat model was successfully established by using hypoxia. Pathologically the fetal lung developed slowly, and the expression levels of SP-B, SP-C, TTF-1 and PLAGL2 protein and mRNA in the fetal lung were significantly reduced in hypoxia group as compared with those in normal control group. It was suggested that maternal hypoxia in the first stage of pregnancy could induce FGR, and reduce the expression of SP-B and SP-C, resulting in the disorder of fetal lung development and maturation.

DPP4 inhibitors increase differentially the expression of surfactant proteins in Fischer 344 rats.

Intact surface active agent (surfactant) composed of surfactant-associated proteins (SPs) and lipids is necessary for respiration and prevents alveoli from collapsing. CD26, a transmembrane glycoprotein exerting dipeptidyl peptidase activity (DPP4), highly expressed in lung parenchyma, is involved in inflammatory processes. A pharmacological inhibition of DPP4 influenced not only the inflammation but also elevated the SPs. Thus, DPP4 inhibitors may be a novel drug for treatment of diseases with surfactant deficiency. Therefore, we tested firstly the hypothesis that DPP4 inhibitors increase the expression of SPs in healthy rats. SP mRNA and protein expression were determined different times after nebulization of aerosolized DPP4 inhibitors [L-isoleucine-thiazolidide (L-Ile-Thia), L-valine-pyrrolidide (L-Val-Pyrr)], budesonide, saline or stereoisomers. Compared with negative controls (1) L-Ile-Thia as well as budesonide led to a significant higher and L-Val-Pyrr had the tendency to a significant higher expression of SP-A mRNA 6 h after nebulization, (2) the expression of SP-D mRNA increased significantly 6 h after nebulization with L-Ile-Thia and 3 and 6 h after nebulization with Val-pyrr, (3) SP-B mRNA levels showed significantly higher values 3 and 6 h after nebulization with L-Val-Pyrr, (4) protein levels of SP-A, SP-B and SP-C were elevated significantly 6 h after nebulization with L-Val-Pyrr as well as with budesonide, and (5) phospholipids were also increased in response to DPP4 inhibition; the minimal surface tension was comparable. DPP4 inhibition influence differently the expression of surfactant proteins in healthy rats and may be suitable to elevate surfactant synthesis in different diseases accompanied with surfactant deficiencies.

In vitro differentiation of rat amniotic fluid-derived mesenchymal stem cells into type II alveolar epithelial cells

To explore the in vitro differentiation of rat amniotic fluid-derived mesenchymal stem cells (AF-MSCs) into type II alveolar epithelial cells (AECII). Flow cytometry was used to analyze the phenotypes of AF-MSCs from 10 pregnant Sprague-Dawley rats. And the Oct-4 mRNA expression level was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Rat embryonic stem cell was used as a positive control. According to different culturing methods, AF-MSCs were randomly divided into 5 groups of A (control group), B, C, D and E. After in vitro differentiation, SPA, SPB, SPC, SPD and TTF1 mRNA expressions were detected by qRT-PCR, SPA and SPC protein expressions measured by immunofluorescence and lamellar bodies observed by transmission electron microscopy. AF-MSCs could grow spirally in L-DMEM medium containing 20% fetal bovine serum and 4 µg/L basic fibroblast growth factor. The expressions of such surface antigens of AF-MSCs (third passage) as CD29 (99.1 ± 7.9)%, CD44 (99.2 ± 7.4)%, CD73 (75.6 ± 5.2)%, CD90 (98.9 ± 8.1)%, CD105 (92.9 ± 7.3)% and CD166 (89.3 ± 6.7)% were positive while CD34 and CD45 were negative. And the expression of Oct-4 mRNA (relative quantity: 0.690 ± 0.059) was significantly lower than rat embryonic stem cells (relative quantity: 1.000 ± 0.002) positive control group (P < 0.01). After in vitro differentiation, the expressions of SPA, SPB, SPC, SPD and TTF1 mRNA and SPA and SPC protein were negative in group A and positive in group B. The expressions of SPA, SPB, SPC, SPD and TTF1 mRNA (relative quantity: 0.426 ± 0.043, 0.368 ± 0.028, 0.492 ± 0.058, 0.327 ± 0.024 and 0.183 ± 0.018) and SPA and SPC protein in group B were significantly higher than other groups (all P < 0.01). Lamellar bodies could be found in the differentiated cells of group B. Rat AF-MSCs from amniotic fluid may differentiated into AECII like cells in vitro.

Effects of antenatal dexamethasone and ambroxol on expression of surfactant protein-B mRNA and ;thyroid transcription factor-1 in pulmonary tissue of premature rats

To explore the effects and mechanism of dexamethasone and ambroxol on expression of surfactant protein （SP）-B mRNA and thyroid transcription factor （TTF）-1 in premature rat lung. Methods Sixteen pregnant Sprague-Dawley rats were randomly divided into four equal groups： two doses of dexamethasone （0.2 mg/kg injected intramuscularly on Day 17 and 18 of pregnancy respectively）;single dose of dexamethasone （0.2 mg/kg injected intramuscularly on Day 18 of pregnancy）;ambroxol group （100 mg/kg injected intraperitoneally on Day 16, 17 and 18 of pregnancy respectively）; and control group （normal saline injected intraperitoneally on Day 16, 17 and 18 of pregnancy respectively）. There were four pregnant rats in each group. All of the fetal rats were taken out on Day 19 of pregnancy as the preterm birth model, and 20 fetal rats from each group were randomly selected. The ratio of body weight to fetal lung weight of newborn rats was calculated. Changes in lung morphology were observed under light microscopy and the ratio of alveoli surface area to alveolar septae surface area was calculated. Expression of TTF-1 protein was determined by immunohistochemistry. Expression of SP-B mRNA was detected by reverse transcriptase-polymerase chain reaction. One-way analysis of variance, Student-Newman-Keuls method and Pearson correlation analysis were applied as statistical methods. Results （1） The ratio of body weight to fetal lung weight was （6.5±0.6）, （7.9±0.8）, （9.5±0.8） and （9.5±0.9） mg/g in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=67.69,P〈0.01）. The ratio of two doses and one dose of dexamethasone group was lower than that of control group （q=17.143 and 9.143, all P〈0.01） and ambroxol group （q=17.143 and 9.143, all P〈0.01）. The ratio of two doses dexamethasone group was lower than that of one dose dexamethasone group （q=8.000, P〈0.01）. （2） The ratio of alveoli surface area to alveolar septae surface area was 2.19±0.15, 1.70±0.18, 1.67±0.13 and 1.08±0.12 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=190.85, P〈0.01）. The ratio of two doses of dexamethasone group, one dose of dexamethasone group and ambroxol group were higher than that of the control group （q=33.639, 18.788 and 17.879, all P〈0.01）. The ratio of two doses dexamethasone group was higher than that of one dose dexamethasone group （q=14.848, P〈0.01）. （3） Expression of TTF-1 protein was 0.311±0.018, 0.224±0.019, 0.196±0.013 and 0.191±0.018 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=211.69,P〈0.01）. TTF-1 protein expression of two doses and one dose of dexamethasone group were higher than that of control group （q=30.000 and 8.250, all P〈0.01） and ambroxol group （q=28.750 and 7.000, all P〈0.01）. TTF-1 protein expression of two doses dexamethasone group was higher than that of one dose dexamethasone group （q=21.750, P〈0.01）. （4） Expression of SP-B mRNA was 1.25±0.13, 1.15±0.12, 1.10±0.10 and 1.01±0.12 in two doses of dexamethasone group, one dose of dexamethasone group, ambroxol group and control group respectively （F=14.48, P〈0.01）. SP-B mRNA expression of two doses of dexamethasone group, one dose of dexamethasone group and ambroxol group were higher than that of control group （q=9.231, 5.385 and 3.462, all P〈0.01）. SP-B mRNA expression of two doses of dexamethasone group was higher than that of ambroxol group （q=5.769, P〈0.01） and one dose of dexamethasone group （q=3.846, P〈0.01）. （5）TTF-1 expression in two doses of dexamethasone group, one dose of dexamethasone group and control groups was positively correlated with SP-B mRNA expression （r=0.512, 0.597 and 0.449, respectively, all P〈0.05）. Conclusions Ambroxol can accelerate the maturation of fetal lung with minimal adverse effects on fetal lung weight. Ambroxol might be an alternative to dexamethasone to prevent neonatal respiratory distress syndrome. Key words: Dexamethasone; Ambroxol; Pulmonary surfactant-associated protein B; Nuclear proteins; Transcription factors; Infant,premature

Effects of histologic chorioamnionitis induced by lipopolysaccharide intra-amniotic injection on fetal lung maturation and development in preterm fetal rats

Objective With various doses of lipopolysaccharide (LPS) intra-amniotic injection in pregnant rats to establish histologic chorioamnionitis (HCA) model,and to determine the effects of HCA on fetal lung maturation and development in preterm fetal rats.Methods Thirty pregnant Sprague-Dawley rats were randomly assigned to four groups.On 19 days of gestation,rats in three model groups (n=8) were given intra-amniotic injection of LPS 2,8,10 μg,respectively,and those in the only one control group (n=6)was given normal saline (NS) 40 μl.Fetal rats were taken out 48 hours after LPS injection.Placenta,fetal membrane,fetal lung and umbilical cord were collected for pathological examination.mRNAs of surfactant protein (SP)-A,B and C and keratinocyte growth factor (KGF) in lung were determined by quantitative real-time polymerase chain preaction.KGF protein expression in lung was measured by immunohistochemistry.Morphologic observation of lung was performed on postnatal day 3.R× C table Chi-square test,KruskalWallis test and analysis of variance were used as statistical methods.Results (1) Fetal rat mortality in control,LPS2,LPS8 and LPS10 group was 0.0％ (0/55),22.5％ (18/80),51.4％ (36/70) and 87.5％(35/40),respectively,which increased with increasing dose of LPS (x2=46.183,P＜0.005).(2) HCA,fetal lung inflammation and umbilical vasculitis were induced in LPS groups,and the severity was dosedependent (fetal lung inflammation:Hc=39.84,HCA:Hc=41.13,umbilical vasculitis:Hc=41.52,all P＜0.01).(3) The expression of SP-A,SP-B,SP-C and KGF mRNAs in the four groups (control,LPS2,8,10) had statistical differences (SP-A mRNA:0.99±0.28,2.48±0.34,1.09±0.31 and 0.09±0.09,F=78.051; SP-B mRNA:0.99±0.34,2.23±0.53,1.49±0.51 and 0.14±0.06,F=28.327; SP-C mRNA:1.20±0.39,2.00±0.20,1.04±0.37 and 0.12±0.16,F=39.546; KGF mRNA:0.97±0.19,2.18±0.61,0.93±0.09 and 0.21±0.11,F=37.544; all P＜0.01).All mRNA expressions in LPS2 group were higher than those in the control group and those in LPS10 group was lower (all P＜0.05).(4) The expression of KGF protein in LPS2 group was significantly higher than that in other groups (0.60±0.20 vs 0.28±0.12,0.37±0.22,0.24±0.12,F=17.280,all P＜0.01).(5) Alveolarization was significantly inhibited in LPS8 group on postnatal day 3,and less maturity of pulmonary tissue was observed.Conclusions Various doses of LPS intra-amniotic injection in rats could induce HCA,fetal lung inflammation and umbilical vasculitis with different degrees.Histological inflammation would be worse with increasing LPS dosage.Moderate inflammation could promote lung maturation without inducing bronchopulmonary dysplasia,and KGF may play a role in this process. Key words: Chorioamnionitis; Infant, premature; Lung; Fetal organ maturity; Fibroblast growth factor7; Disease models, animal; Lipopolysaccharides

Effect of SP-A/B in lipoic acid on acute paraquat poisoning.

This study was undertaken to observe the concentration of SP-A/B and the pulmonary surfactant in the lung tissue of rats with acute lung injury/acute respiratory distress syndrome caused by paraquat poisoning after the treatment of metabolic antioxidant-lipoic acid and whether its influence was related to TNF-α. Sixty-six male Sprage-Dawley rats were randomly divided into three groups: normal control group (NS group), 6 rats; paraquat poisoning group (PQ group), 30 rats; and paraquat+lipoic acid treatment group (LA group), 30 rats. The rats in the PQ and LA groups were subdivided into 3-, 6-, 12-, 24-, 48-hour subgroups, with 6 rats in each group. After the rats were sacrificed, lung tissue from the same part was taken from the rats. After HE staining, histological changes were observed in the tissue under a light microscope. Lung tissue was also taken to test the levels of superoxide dismutase (SOD) and malondialdehyde (MDA). Whole blood (0.8 mL) without anticoagulant was drawn from the tail vein of rats for the determination of the TNF-α level. The total RNA of the lung tissue was collected, and the Rt-PCR method was used to measure the levels of SP-A and SP-B mRNA. HE staining showed that histopathological changes were milder in the LA group than in the PQ group. There were significant differences in MDA and SOD levels between different intervals both in intergroups and intragroups except the 3-hour subgroup (P<0.01). Likewise, the significant differences in the levels of TNF-α were also present between the three groups and between different intervals (P<0.01). The significant differences in SP-A mRNA and SP-B mRNA amplification ratio were seen between the three groups at the same intervals (P<0.01), but the differences between different intervals in the PQ group were statistically significant (P<0.05). The differences between different intervals in the LA group were statistically significant (P<0.01). Lipoic acid in acute paraquat poisoning could diminish lung tissue damage by regulating directly tumor necrosis factor and indirectly the content of pulmonary surfactant so as to reduce pulmonary edema, improve lung compliance, and finally protect lung tissues.

Prenatal Hypoxia Downregulates the Expression of Pulmonary Vascular Endothelial Growth Factor and Its Receptors in Fetal Mice

Background: Previous reports showed that prenatal hypoxia delays the process of lung maturation. Vascular endothelial growth factor (VEGF) and its receptors were important for lung development. However, the role of VEGF and VEGF receptors in altered fetal lung development and maturation induced by prenatal hypoxia remains unknown. Objectives: To elucidate the role of VEGF and VEGF receptors in altered fetal lung development and maturation induced by prenatal hypoxia. Methods: Lung sections of control and maternal hypoxic fetal mice were used for the determination of lung development and total RNA isolated from lung homogenates were used for determination of the expression patterns of VEGF, Flt-1, Flk-1, hypoxia-inducible factor (HIF)-1α, HIF-2α, surfactant protein (SP)-A, SP-B, SP-C, and SP-D by quantitative real-time RT-PCR. Results: Prenatal hypoxia resulted in fetal mice body weight gain impairment, delayed fetal pulmonary aeration and maturation. Pulmonary SP-A, SP-B, SP-C, and SP-D mRNA were all decreased in the prenatal hypoxia group. In addition, we demonstrated that prenatal hypoxia inhibited the developmental increase of pulmonary HIF-1α and HIF-2α expression and resulted in decreasing VEGF and its receptors (Flt-1 and Flk-1) at the mRNA expression level and VEGF protein level in fetal lungs. These inhibitory effects persisted and progressed even when the dams were returned to air. Conclusions: We suggest that prenatal hypoxia insults, at least in late gestation, influence pulmonary VEGF and VEGF receptor expression through the down-regulation of HIF pathways and impair fetal lung growth and maturation.

Pulmonary GLP-1 Receptor Increases at Birth and Exogenous GLP-1 Receptor Agonists Augmented Surfactant-Protein Levels in Litters From Normal and Nitrofen-Treated Pregnant Rats

The glucagon-like peptide-1 receptor (GLP-1R) is found in a variety of tissues outside of the pancreas. For example, GLP-1R is expressed in the lung, where it has been implicated in the regulation of the lipid fraction of surfactants, suggesting it fulfills an important role in lung function. Here, we show that GLP-1R expression is strongly up-regulated immediately after birth in neonatal rats, particular in male offspring. Moreover, administering long half-life GLP-1R agonists to the mother from gestational day 14 to birth (exendin-4 or liraglutide) increased surfactant protein (SP)-A and SP-B mRNA expression and the amount of SPs in the amniotic fluid at the end of pregnancy. These effects were similar or more potent to those induced by the glucocorticoid dexamethasone, which also increased GLP-1R expression in fetuses just before delivery. Lir increased fetal SP-A and GLP-1R expression in control rats and in a nitrofen-induced model of lung hypoplasia. Moreover, lung size increased in controls after Lir administration, which also prevented the decrease in lung weight and the poor neonatal survival of the offspring from nitrofen-treated dams, effects that were not produced by dexamethasone. Taken together, our results demonstrate the importance of the GLP-1 system in regulating SP production and lung development.

Infant formula alters surfactant protein A (SP‐A) and SP‐B expression in pulmonary epithelial cells

Surfactant proteins A (SP-A) and SP-B are critical in the ability of pulmonary surfactant to reduce alveolar surface tension and provide innate immunity. Aspiration of infant milk formula can lead to lung dysfunction, but direct effects of aspirated formula on surfactant protein expression in pulmonary cells have not been described. The hypothesis that infant formula alters surfactant protein homeostasis was tested in vitro by assessing surfactant protein gene expression in cultured pulmonary epithelial cell lines expressing SP-A and SP-B that were transiently exposed (6 hr) to infant formula. Steady-state levels of SP-A protein and mRNA and SP-B mRNA in human bronchiolar (NCI-H441) and mouse alveolar (MLE15) epithelial cells were reduced in a dose-dependent manner 18 hr after exposure to infant formula. SP-A mRNA levels remained reduced 42 hr after exposure, but SP-B mRNA levels increased 10-fold. Neither soy formula nor non-fat dry milk affected steady-state SP-A and SP-B mRNA levels; suggesting a role of a component of infant formula derived from cow milk. These results indicate that infant formula has a direct, dose-dependent effect to reduce surfactant protein gene expression. Ultimately, milk aspiration may potentially result in a reduced capacity of the lung to defend against environmental insults.

Effects of prenatal ethanol exposure on the lungs of postnatal lambs

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.

Early gestational intrauterine infection induces postnatal lung inflammation and arrests lung development in a rat model

Objective. In order to investigate the early gestational inflammation effect on the prenatal and postnatal lung development, identification of the proinflammatory cytokines (IL-1β and TNF-α), genes implicated in angiogenesis (Vascular endothelial growth factor [VEGF], fms-like tyrosine kinase-1 [Flt-1], fetal liver kinase-1 [Flk-1]), and surfactant proteins (SPs) were observed.Methods. Escherichia coli (E. coli) was inoculated into uterine cervix of pregnant rats at embryonic day 15 (E15) during pseudoglandular period of lung development and the control group was inoculated with normal saline. IL-1β, TNF-α, VEGF, Flt-1, Flk-1, SP-A, and SP-B mRNA in pup's lung at E17, 19, 21 and postnatal day (P) 1, 3, 7, 14 were quantified by real-time RT-PCR. Western blot or immunohistochemistry analysis was also performed for the evaluation of VEGF, Flk-1, Flt-1, and SP-A expression in pup's lung.Results. Compared with the control group, the fetal lung of the E. coli-treated group was more immature, the postnatal lung development was impaired marked by less alveoli, fewer secondary septa, and thicker alveolar wall. The lung weight and lung/body weight ratio were lower in the E. coli-treated group pups. IL-1β and TNF-α mRNA were increased significantly in E. coli-treated pup's lung after birth, but no significant difference of IL-1β and TNF-α mRNA levels in fetal lung were found between the two groups. SP-A expression was depressed at E17, E19, and E21 after intrauterine E. coli treated, accompanied with lower SP-B mRNA level at E19 and E21. Furthermore, intrauterine E. coli treated reduced the VEGF mRNA and protein levels in the fetal lung at E17 and E19, while the expression of Flt-1 and Flk-1 were higher at P7, P14 and P1, P7, P14, respectively, compared to the controls.Conclusions. These results suggested early gestational intrauterine E. coli infection could induce a postnatal pulmonary inflammation and might arrest the alveolarization in developing lung which was involved with the VEGF signaling. However, intrauterine E. coli infection could not induce the increase of proinflammatory cytokines in fetal lung and might fail to accelerate the maturation of fetal lung.

ErbB4 regulates the timely progression of late fetal lung development

The ErbB4 receptor has an important function in fetal lung maturation. Deletion of ErbB4 leads to alveolar hypoplasia and hyperreactive airways similar to the changes in bronchopulmonary dysplasia (BPD). BPD is a chronic pulmonary disorder affecting premature infants as a consequence of lung immaturity, lung damage, and abnormal repair. We hypothesized that proper ErbB4 function is needed for the timely progression of fetal lung development. An ErbB4 transgenic cardiac rescue mouse model was used to study the effect of ErbB4 deletion on fetal lung structure, surfactant protein (SP) expression, and synthesis, and inflammation. Morphometric analyses revealed a delayed structural development with a significant decrease in saccular size at E18 and more pronounced changes at E17, keeping these lungs in the canalicular stage. SP-B mRNA expression was significantly down regulated at E17 with a subsequent decrease in SP-B protein expression at E18. SP-D protein expression was significantly decreased at E18. Surfactant phospholipid synthesis was significantly decreased on both days, and secretion was down regulated at E18. We conclude that pulmonary ErbB4 deletion results in a structural and functional delay in fetal lung development, indicating a crucial regulatory role of ErbB4 in the timely progression of fetal lung development.

Gender-related effects of prenatal administration of estrogen and progesterone receptor antagonists on VEGF and surfactant-proteins and on alveolarisation in the developing piglet lung

Background Vascular endothelial growth factor (VEGF) is essential for embryonic lung development and has been shown to be regulated by estradiol (E2) and progesterone (P). Aim To investigate the effects of prenatal E2 and P withdrawal by specific receptor antagonists on the mRNA expression of VEGF, surfactant proteins (SP-B and SP-C) and on alveolarisation in lung tissue of male and female pig fetuses. Methods Fetuses from 10 sows were randomized to receive either both an intramuscular injection of the E2 receptor blocker ICI 182.780 and the P receptor blocker RTI 3021-022 (ICI + RTI, n = 5) or a placebo injection ( n = 5) at 90 days of gestation (DOG, 115 = term). After delivery by cesarean section on 114 DOG, tissue of the left lingula of the piglet's lung (28 placebo, 26 ICI + RTI) was obtained to determine the mRNA expression of VEGF, SP-B and SP-C. Lungs from 15 placebo and 14 ICI + RTI group piglets were removed and alveolar counts performed. Results The ICI + RTI group showed significantly lower SP-C mRNA expression and alveolar counts compared to the placebo group ( p = 0.04 and 0.03, respectively). Diminished alveolarisation in the ICI + RTI group was mainly due to the reduction of alveolar counts in male piglets ( p = 0.02). Within the placebo group VEGF and SP-B mRNA expression in male piglets were significantly lower compared to female piglets ( p = 0.01 and 0.004, respectively). ICI + RTI treatment abolished this gender-related difference. Conclusion Estradiol and P antagonism affected gender-related differences of key proteins for pulmonary function and development and especially in males was associated with diminished alveolarisation.

Repeated ethanol exposure during late gestation alters the maturation and innate immune status of the ovine fetal lung

Little is known about the effects of fetal ethanol exposure on lung development. Our aim was to determine the effects of repeated ethanol exposure during late gestation on fetal lung growth, maturation, and inflammatory status. Pregnant ewes were chronically catheterized at 91 days of gestational age (DGA; term approximately 147 days). From 95-133 DGA, ewes were given a 1-h daily infusion of either 0.75 g ethanol/kg (n = 9) or saline (n = 8), with tissue collection at 134 DGA. Fetal lungs were examined for changes in tissue growth, structure, maturation, inflammation, and oxidative stress. Between treatment groups, there were no differences in lung weight, DNA and protein contents, percent proliferating and apoptotic cells, tissue and air-space fractions, alveolar number and mean linear intercept, septal thickness, type-II cell number and elastin content. Ethanol exposure caused a 75% increase in pulmonary collagen I alpha1 mRNA levels (P < 0.05) and a significant increase in collagen deposition. Surfactant protein (SP)-A and SP-B mRNA levels were approximately one third of control levels following ethanol exposure (P < 0.05). The mRNA levels of the proinflammatory cytokines interleukin (IL)-1beta and IL-8 were also lower (P < 0.05) in ethanol-exposed fetuses compared with controls. Pulmonary malondialdehyde levels tended to be increased (P = 0.07) in ethanol-exposed fetuses. Daily exposure of the fetus to ethanol during the last third of gestation alters extracellular matrix deposition and surfactant protein gene expression, which could increase the risk of respiratory distress syndrome after birth. Changes to the innate immune status of the fetus could increase the susceptibility of the neonatal lungs to infection.