Surfactant Protein mRNA 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 of Aging-Related Emphysematous and Lymphoma-Like Lesions is Enhanced by the Lack of Secretoglobin 3A2 in Mouse Lungs.

BackgroundSecretoglobin (SCGB) 3A2 is a novel bioactive molecule with anti-inflammatory and anti-fibrotic activities. SCGB3A2 also promotes the maturation of bronchial divergence and the lungs during embryonic development. However, much remains unknown concerning the roles of SCGB3A2 in diseases associated with aging.MethodsThe lungs of Scgb3a2-knockout (KO) mice and their wild-type (WT) littermates were subjected to histological analysis, Victoria blue staining to evaluate of elastic fibers, and lung morphometric analysis during the postnatal period (birth to 8 weeks) and during aging (8 weeks to 2 years). Their spleens were also histologically evaluated. The expression of lung surfactant protein (SP) mRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction. RNA sequencing (RNAseq) analysis was performed on 3-month-old KO and WT mouse lungs.ResultsThe alveolar spaces of KO mice continuously expanded between 0.5 and 2 years of age, accompanied by increases of the mean linear intercept and destructive index. KO mouse lungs displayed inflammation associated with lymphocyte aggregate starting at 1 year of age, and the inflammation was worse than that of WT mouse lungs. A high number of lymphoma-like cells were presented in 2-year-old KO mouse lungs. White pulp fusion was detected in the spleens of both WT and KO mice older than 0.5 years; however, the fusion was more severe in KO mice than in WT mice. The expression of surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNAs in KO mouse lungs decreased with age, and after 1 year of age, the expression of most SPs was significantly lower in KO mice than in WT mice. RNAseq demonstrated that the expression of immune system-related genes was highly altered in KO mouse lungs.ConclusionSCGB3A2 may be required for maintaining homeostasis and immune activity in the lungs during aging. SCGB3A2 deficiency might increase the risk of emphysema of the lung.

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.

An internal amino-terminal FLAG-tag octapeptide alters oligomerization of expressed surfactant protein-A

Pulmonary surfactant protein-A (SP-A) is expressed by lung alveolar and bronchiolar epithelial cells and plays a critical role in innate immunity of the lung. Exposure of the lung to various environmental insults alters SP-A homeostasis. To investigate the cellular mechanisms involved in these alterations, we added the FLAG octapeptide (DYKDDDDK) to the carboxy-terminus (SP-A/C-FLAG) or near the amino-terminus (SP-A/N-FLAG) of mouse SP-A (WT-SP-A) to tag specific pools of protein. We hypothesized that addition of FLAG would have negligible effects on SP-A expression, oligomerization and secretion. Analysis of Chinese hamster ovary cells expressing these proteins indicated that tagged SP-A mRNA could be distinguished from WT-SP-A by northern analysis and RT-PCR using sequence-specific oligonucleotides. Tagged SP-A protein could be differentiated from WT-SP-A by western analysis using antibodies specific for the FLAG epitope. Subcellular fractionation and immunocytochemistry indicated the majority of each protein was present in punctuate (presumably endocytic) vesicles, and all forms of SP-A protein were secreted. These results suggest that a FLAG epitope added to the carboxy-terminus or inserted into the amino-terminus of the mature SP-A protein has little effect on its expression and cellular processing. However, disruptions of the amino-terminal end of SP-A prevents proper oligomerization, suggesting that this region of mature SP-A is critical in proper oligomeric assembly and is not useful for studies intended to define mechanisms underlying SP-A homeostasis.

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.

Fetal growth restriction is associated with an altered cardiopulmonary and cerebral hemodynamic response to surfactant therapy in preterm lambs.

Efficacy of surfactant therapy in fetal growth restricted (FGR) preterm neonates is unknown. Twin-bearing ewes underwent surgery at 105 days gestation to induce FGR in one twin by single umbilical artery ligation. At 123-127 days, catheters and flow probes were implanted in pulmonary and carotid arteries to measure flow and pressure. Lambs were delivered, intubated and mechanically ventilated. At 10 min, surfactant (100 mg kg-1) was administered. Ventilation, oxygenation, and hemodynamic responses were recorded for 1 h before euthanasia at 120 min. Lung tissue and bronchoalveolar lavage fluid was collected for analysis of surfactant protein mRNA and phosphatidylcholines (PCs). FGR preterm lambs were 26% lighter than appropriate for gestational age (AGA) lambs and had baseline differences in lung mechanics and pulmonary blood flows. Surfactant therapy reduced ventilator and oxygen requirements and improved lung mechanics in both groups, although a more rapid improvement in compliance and tidal volume was observed in AGA lambs. Surfactant administration was associated with decreased mean pulmonary and carotid blood flow in FGR but not AGA lambs. No major differences in surfactant protein mRNA or PC levels were noted. Surfactant therapy was associated with an altered pulmonary and cerebral hemodynamic response in preterm FGR lambs.

Effects of budesonide and surfactant in preterm fetal sheep.

Mechanical ventilation causes lung injury and systemic inflammatory responses in preterm sheep and is associated with bronchopulmonary dysplasia (BPD) in preterm infants. Budesonide added to surfactant decreased BPD by 20% in infants. We wanted to determine the effects of budesonide and surfactant on injury from high tidal volume (VT) ventilation in preterm lambs. Ewes at 125 ± 1 days gestational age had fetal surgery to expose fetal head and chest with placental circulation intact. Lambs were randomized to 1) mechanical ventilation with escalating VT to target 15 ml/kg by 15 min or 2) continuous positive airway pressure (CPAP) of 5 cmH2O. After the 15-min intervention, lambs were given surfactant 100 mg/kg with saline, budesonide 0.25 mg/kg, or budesonide 1 mg/kg. The fetuses were returned to the uterus for 24 h and then delivered and ventilated for 30 min to assess lung function. Budesonide levels were low in lung and plasma. CPAP groups had improved oxygenation, ventilation, and decreased injury markers compared with fetal VT lambs. Budesonide improved ventilation in CPAP lambs. Budesonide decreased lung weights and lung liquid and increased lung compliance and surfactant protein mRNA. Budesonide decreased proinflammatory and acute-phase responses in lung. Airway thickness increased in animals not receiving budesonide. Systemically, budesonide decreased monocyte chemoattractant protein-1 mRNA and preserved glycogen in liver. Results with 0.25 and 1 mg/kg budesonide were similar. We concluded that budesonide with surfactant matured the preterm lung and decreased the liver responses but did not improve lung function after high VT injury in fetal sheep.

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.

Lipopolysaccharide Stimulates Surfactant Protein-A in Human Renal Epithelial HK-2 Cells through Upregulating Toll-like Receptor 4 Dependent MEK1/2-ERK1/2-NF-κB Pathway.

Background:Surfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.Methods:Tetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.Results:HK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.Conclusions:The present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

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.

The protective effect of different airway humidification liquids to lung after tracheotomy in traumatic brain injury: The role of pulmonary surfactant protein-A (SP-A)

The purpose of this study was to establish a rat model of a brain injury with tracheotomy and compared the wetting effects of different airway humidification liquids, afterward, the best airway humidification liquid was selected for the clinical trial, thus providing a theoretical basis for selecting a proper airway humidification liquid in a clinical setting. Rats were divided into a sham group, group A (0.9% NaCl), group B (0.45% NaCl), group C (0.9% NaCl+ambroxol) and group D (0.9% NaCl+Pulmicort). An established rat model of traumatic brain injury with tracheotomy was used. Brain tissue samples were taken to determine water content, while lung tissue samples were taken to determine wet/dry weight ratio (W/D), histological changes and expression levels of SP-A mRNA and SP-A protein. 30 patients with brain injury and tracheotomy were selected and divided into two groups based on the airway humidification liquid instilled in the trachea tube, 0.45% NaCl and 0.9% NaCl+ambroxol. Blood was then extracted from the patients to measure the levels of SP-A, interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α). The difference between group C and other groups in lung W/D and expression levels of SP-A mRNA and SP-A protein was significant (P<0.05). In comparison, the histological changes showed that the lung tissue damage was smallest in group C compared to the three other groups. Aspect of patients, 0.45% NaCl group and 0.9% NaCl+ambroxol group were significantly different in the levels of SP-A, IL-6, IL-8 and TNF-α (P<0.01). In the present study, 0.9% NaCl+ambroxol promote the synthesis and secretion of pulmonary surfactant, and has anti-inflammatory and antioxidant effects, which inhibit the release of inflammatory factors and cytokines, making it an ideal airway humidification liquid.

Keratinocyte growth factor-2 inhibits bacterial infection with Pseudomonas aeruginosa pneumonia in a mouse model.

To determine protective effects of concurrent administration of Keratinocyte growth factor-2 (KGF-2) with Pseudomonas aeruginosa (P. aeruginosa) inoculation on the induced pneumonia. KGF-2 (5 mg/kg) was concurrently administered into the left lobe of 55 mice with P. aeruginosa PAO1 (5 × 10(6) CFU, half-lethal dose); 55 mice in the control group were concurrently administered PBS with the PAO1. We detected and analyzed: body temperature; amount of P. aeruginosa in homogenates; count of total number of nucleated cells and of mononuclear macrophages; protein concentration in bronchoalveolar lavage fluid (BALF); lung wet-to-dry weight ratio; cytokines in BALF and blood; and lung morphology. To study survival rate, concurrent administration of KGF-2 (experimental group) versus PBS (control) with a lethal dose of PAO1 (1 × 10(7) CFU was performed, and survivorship was documented for 7 days post-inoculation. The bacterial CFU in lung homogenates was significantly decreased in the KGF-2 group compared to the control group. There were significantly more mononuclear macrophages in the BALF from the KGF-2 group than from the control group (p < 0.05). KGF-2 increased the surfactant protein and GM-CSF mRNA in lung at 6 h and 72 h after inoculation. Significant reduction of lung injury scores, protein concentrations, lung wet-to-dry weight ratio, and IL-6 and TNF-α levels was noted in the KGF-2 treated rats at 72 h after inoculation (p < 0.05). The 7-day survival rate of the KGF-2 group was significantly higher than that of the control group (p < 0.05). Concurrent administration of KGF-2 facilitates the clearance of P. aeruginosa from the lungs, attenuates P. aeruginosa-induced lung injury, and extends the 7-day survival rate in mice model with P. aeruginosa pneumonia.

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.

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.

Knockdown of Drosha in human alveolar type II cells alters expression of SP-A in culture: A pilot study

ABSTRACTHuman surfactant protein A (SP-A) plays an important role in surfactant metabolism and lung innate immunity. SP-A is synthesized and secreted by alveolar type II (ATII) cells, one of the two cell types of the distal lung epithelium (ATII and ATI). We have shown that miRNA interactions with sequence polymorphisms on the SP-A mRNA 3'UTRs mediate differential expression of SP-A1 and SP-A2 gene variants in vitro. In the present study, we describe a physiologically relevant model to study miRNA regulation of SP-A in human ATII. For these studies, we purified and cultured human ATII on an air-liquid interface matrix (A/L) or plastic wells without matrix (P). Gene expression analyses confirmed that cells cultured in A/L maintained the ATII phenotype for over 5 days, whereas P-cultured cells differentiated to ATI. When we transfected ATII with siRNAs to inhibit the expression of Drosha, a critical effector of miRNA maturation, the levels of SP-A mRNA and protein increased in a time dependent manner. We next characterized cultured ATII and ATI by studying expression of 1,066 human miRNAs using miRNA PCR arrays. We detected expression of >300 miRNAs with 24 miRNAs differentially expressed in ATII versus ATI, 12 of which predicted to bind SP-A 3'UTRs, indicating that these may be implicated in SP-A downregulation in ATI. Thus, miRNAs not only affect SP-A expression, but also may contribute to the maintenance of the ATII cell phenotype and/or the trans-differentiation of ATII to ATI cells, and may represent new molecular markers that distinguish ATII and ATI.

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.

An 11-nt sequence polymorphism at the 3'UTR of human SFTPA1 and SFTPA2 gene variants differentially affect gene expression levels and miRNA regulation in cell culture.

Surfactant protein A (SP-A) plays a vital role in maintaining normal lung function and in host defense. Two genes encode SP-A in humans (SFTPA1, SFTPA2), and several gene variants have been identified for these. We have previously shown that sequence elements of SFTPA1 and SFTPA2 3' untranslated regions (UTRs) differentially affect translation efficiency in vitro. Polymorphisms at the 3'UTRs of mRNA variants may account for differential binding of miRNAs, a class of small noncoding RNAs that regulate gene expression. In this work, we generated 3'UTR reporter constructs of the SFTPA1 and SFTPA2 variants most frequently found in the population, as well as mutants of a previously described 11-nt indel element (refSNP rs368700152). Reporter constructs were transfected in NCI-H441 cells in the presence or absence of miRNA mimics, and reporter gene expression was analyzed. We found that human miRNA mir-767 negatively affected expression of constructs containing SFTPA1 and SFTPA2 variants, whereas mir-4507 affected only constructs with 3'UTRs of SFTPA1 variants 6A, 6A(3), and 6A(4) (not containing the 11-nt element). Three miRNAs (mir-183, mir-449b, and mir-612) inhibited expression of recombinants of SFTPA2 variants and the SFTPA1 variant 6A(2), all containing the 11-nt element. Similar results were obtained for SP-A expression when these miRNAs were transfected in Chinese hamster ovary cells expressing SFTPA1 or SFTPA2 variants or in NCI-H441 cells (genotype 1A(5)/1A(5)-6A(4)/6A(4)). Moreover, transfection with a specific antagomir (antagomir-183) reversed the effects of mir-183 on SP-A mRNA levels. Our results indicate that sequence variability at the 3'UTR of SP-A variants differentially affects miRNA regulation of gene expression.