SFTA2 - a novel secretory peptide highly expressed in the lung - is modulated by lipopolysaccharide but not hyperoxia

Tissue-specific transcripts are likely to be of importance for the corresponding organ. While attempting to define the specific transcriptome of the human lung, we identified the transcript of a yet uncharacterized protein, SFTA2. In silico analyses, biochemical methods, fluorescence imaging and animal challenge experiments were employed to characterize SFTA2. Human SFTA2 is located on Chr. 6p21.33, a disease-susceptibility locus for diffuse panbronchiolitis. RT-PCR verified the abundance of SFTA2-specific transcripts in human and mouse lung. SFTA2 is synthesized as a hydrophilic precursor releasing a 59 amino acid mature peptide after cleavage of an N-terminal secretory signal. SFTA2 has no recognizable homology to other proteins while orthologues are present in all mammals. SFTA2 is a glycosylated protein and specifically expressed in nonciliated bronchiolar epithelium and type II pneumocytes. In accordance with other hydrophilic surfactant proteins, SFTA2 did not colocalize with lamellar bodies but colocalized with golgin97 and clathrin-labelled vesicles, suggesting a classical secretory pathway for its expression and secretion. In the mouse lung, Sfta2 was significantly downregulated after induction of an inflammatory reaction by intratracheal lipopolysaccharides paralleling surfactant proteins B and C but not D. Hyperoxia, however, did not alter SFTA2 mRNA levels. We have characterized SFTA2 and present it as a novel unique secretory peptide highly expressed in the lung

PEDIATRIC ORIGINAL Adaptive mechanical backup ventilation for preterm infants on respiratory assist modes -a pilot study

Abstract Background: Mechanical respiratory-assist modes, such as assist/control, low-rate intermittent mandatory ventilation, continuous positive airway pressure, or proportional assist ventilation (PAV), require a continuous respiratory effort. Because of the frequent occurrence of periodic breathing and/or apnea, mechanical backup ventilation must be initiated during episodes of reduced or absent respiratory drive to maintain gas exchange. The common approach to this problem is a regular conventional mechanical ventilation, which is initiated and withdrawn in an "on/off" function. Objective: To develop and evaluate a mechanical backup ventilation mode that is adaptive to the rapidly changing breathing pattern of preterm infants. Design: Prospective randomized clinical crossover trial. Setting: Neonatal intensive care unit at the University of Munich, Germany. Patients: Preterm infants undergoing PAV. Interventions: The infants were ventilated with PAV using a newly developed adaptive backup support, with and without pulse-oximetryguided operation (SpO 2 -sensitive backup). Each infant was ventilated with both modes of backup support on 2 consecutive days, with the sequence randomized. Measurements and results: The analysis on 11 preterm infants showed a statistically significant and clinically relevant reduction of the incidence (33%) and duration of oxygen desaturations (52%) when SpO 2 -sensitive adaptive backup support was used. Conclusions: SpO 2 -sensitive adaptive backup proved safe and effective in reducing the incidence and duration of oxygen desaturation in this short-term trial. This technology is potentially applicable to other assisted modalities of ventilation, such as noninvasive nasal ventilation

Markers of extracellular matrix and epithelial cell maturation after fetal tracheal occlusion in newborn rabbits with lung hypoplasia induced by diaphragmatic hernia.

Background and aims: Tracheal occlusion (TO), a prenatal treatment promoting lung growth, improves defective elastogenesis in congenital diaphragmatic hernia (CDH). Because the transforming growth factor-ß (TGF-ß) regulates extracellular matrix (ECM) during late lung development, we hypothesized that the effects of TO on ECM would be mediated by the TGF-ß/Smad2 pathway. We therefore investigated the effects of TO on lung connective tissue composition and function, and on TGF-ß signaling expression in an experimental model of CDH. Methods: Rabbit fetuses were randomized twice during gestation: at day 23 (pseudoglandular phase), for a DH or a sham thoracotomy, and five days later (canalicular phase) for a tracheal ligation or a sham operation. Non-operated littermates served as controls. At term (day 31), live fetuses underwent an evaluation of global and peripheral lung mechanics, followed by pulmonary tissue sampling for morphometric and biological studies. Results: TO normalized the lung to body weight ratio and morphometry of airways in DH fetuses compared to controls. TO did not improve dynamic resistance and compliance in DH fetuses, and even resulted in worsening of lung function in sham DH fetuses. TO had no effect on peripheral lung mechanics assessed by forced oscillation technique. In DH fetuses, gene expressions for tropoelastin and lysyl-oxidase were downregulated (by 50%) and restored by TO. Besides, TO stimulates gene expressions for fibulin-5, types I and III procollagens, integrins (respectively by 700%, 180% and 200%), and the density of elastic fibers was increased. These effects on ECM composition co-occurred with an increase in gene and protein expressions for TGF-ß, and a rise of the phosphorylation of Smad2 (by 100%). Conclusions: In surgically induced CDH, sustained TO restores elastogenesis, but also enhances the synthesis of many ECM components, coinciding with an activation of the TGF-ß/Smad2 signaling pathway. These biological effects, together with decreased level of surfactant after TO, may contribute to the impairment of neonatal lung mechanics.info:eu-repo/semantics/nonPublishe

Alveolarization genes modulated by fetal tracheal occlusion in the rabbit model for congenital diaphragmatic hernia: a randomized study.

BACKGROUND: The mechanisms by which tracheal occlusion (TO) improves alveolarization in congenital diaphragmatic hernia (CDH) are incompletely understood. Therefore transcriptional and histological effects of TO on alveolarization were studied in the rabbit model for CDH. The question of the best normalization strategy for gene expression analysis was also addressed. METHODS: Fetal rabbits were randomized for CDH or sham operation on gestational day 23/31 and for TO or sham operation on day 28/31 resulting in four study groups. Untouched littermates were added. At term and before lung harvest, fetuses were subjected to mechanical ventilation or not. Quantitative real-time PCR was performed on lungs from 4-5 fetuses of each group with and without previous ventilation. Stability of ten housekeeping genes (HKGs) and optimal number of HKGs for normalization were determined, followed by assessment of HKG expression levels. Expression levels of eleven target genes were studied in ventilated lungs, including genes regulating elastogenesis, cell-environment interactions, and thinning of alveolar walls. Elastic staining, immunohistochemistry and Western blotting completed gene analysis. RESULTS: Regarding HKG expression, TO increased β-actin and β-subunit of ATP synthase. Mechanical ventilation increased β-actin and β2-microglobulin. Flavoprotein subunit of succinate dehydrogenase and DNA topoisomerase were the most stable HKGs. CDH lungs showed disorganized elastin deposition with lower levels for tropoelastin, fibulin-5, tenascin-C, and α6-integrin. After TO, CDH lungs displayed a normal pattern of elastin distribution with increased levels for tropoelastin, fibulin-5, tenascin-C, α6-integrin, ß1-integrin, lysyl oxidase, and drebrin. TO increased transcription and immunoreactivity of tissue inhibitor of metalloproteinase-1. CONCLUSIONS: Experimental TO might improve alveolarization through the mechanoregulation of crucial genes for late lung development. However part of the transcriptional changes involved genes that were not affected in CDH, raising the question of TO-induced disturbances of alveolar remodeling. Attention should also be paid to selection of HKGs for studies on mechanotransduction-mediated gene expressions.info:eu-repo/semantics/publishedJournal articl

Antenatal BAY 41-2272 reduces pulmonary hypertension in the rabbit model of congenital diaphragmatic hernia

Infants with congenital diaphragmatic hernia (CDH) fail to adapt at birth because of persistent pulmonary hypertension (PH), a condition characterized by excessive muscularization and abnormal vasoreactivity of pulmonary vessels. Activation of soluble guanylate cyclase by BAY 41-2272 prevents pulmonary vascular remodeling in neonatal rats with hypoxia-induced PH. By analogy, we hypothesized that prenatal administration of BAY 41-2272 would improve features of PH in the rabbit CDH model. Rabbit fetuses with surgically induced CDH at day 23 of gestation were randomized at day 28 for an intratracheal injection of BAY 41-2272 or vehicle. After term delivery (day 31), lung mechanics, right ventricular pressure, and serum NH2-terminal-pro-brain natriuretic peptide (NT-proBNP) levels were measured. After euthanasia, lungs were processed for biological or histological analyses. Compared with untouched fetuses, the surgical creation of CDH reduced the lung-to-body weight ratio, increased mean terminal bronchial density, and impaired lung mechanics. Typical characteristics of PH were found in the hypoplastic lungs, including increased right ventricular pressure, higher serum NT-proBNP levels, thickened adventitial and medial layers of pulmonary arteries, reduced capillary density, and lower levels of endothelial nitric oxide synthase. A single antenatal instillation of BAY 41-2272 reduced mean right ventricular pressure and medial thickness of small resistive arteries in CDH fetuses. Capillary density, endothelial cell proliferation, and transcripts of endothelial nitric oxide synthase increased, whereas airway morphometry, lung growth, and mechanics remained unchanged. These results suggest that pharmacological activation of soluble guanylate cyclase may provide a new approach to the prenatal treatment of PH associated with CDH.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Effects of ventilation on normalized expressions of the ten candidate housekeeping genes.

<p>Mean Cq values were converted into linear values according to the efficiency corrected model, and normalized to the geometric mean of SDHA and TOP1 as calculated by <i>genorm</i>. Error bars indicate the standard error of the mean, with 4 to 5 animals per study group (blue square = unventilated lungs; orange square = ventilated lungs). <b>A</b>. Effect of ventilation in all groups combined (all unventilated lungs, <i>n</i> = 21; all ventilated lungs, <i>n</i> = 22). <b>B</b>. Effect of ventilation in non-operated fetuses. <b>C</b>. Effect of ventilation in sham-operated fetuses (SHAM). <b>D</b>. Effect of ventilation in diaphragmatic hernia fetuses (DH). <b>E</b>. Effect of ventilation in DH fetuses with tracheal occlusion (DH+ TO). <b>F</b>. Effect of ventilation in sham DH fetuses with TO (TO). Unpaired Student’s t-test was used. ^a<i>P</i> < 0.001; ^b<i>P</i> < 0.01; ^c<i>P</i> <0.05. A.U. = arbitrary unit.</p

Normalized expression of eleven alveolarization genes in the fetal rabbit model for CDH undergoing TO.

<p>Mean Cq values were converted into linear values according to the efficiency corrected model, and normalized to the geometric mean of SDHA and TOP1 as calculated by <i>genorm</i>. Error bars illustrate the standard error of the mean, with 4 to 5 animals per study group. One-way ANOVA with Bonferroni correction was used for comparisons between surgical groups (SHAM, DH, DH+ TO, and TO) and unpaired Student’s t-test for comparisons between non-operated and SHAM fetuses. SHAM, sham-operated fetuses; DH, diaphragmatic hernia fetuses; DH+ TO, DH fetuses with tracheal occlusion; TO, sham DH fetuses with TO. ^a<i>P</i> < 0.05 vs SHAM; ^b<i>P</i> < 0.05 vs DH; ^c<i>P</i> < 0.05 vs non-operated. A.U. = arbitrary unit.</p

Localization of MMPs and TIMPs in alveolar septa in the fetal rabbit model for CDH undergoing TO.

<p>Representative images of immunohistochemistry for MMP2 (<b>A</b>–<b>E</b>), MMP14 (<b>F</b>–<b>J</b>), TIMP1 (<b>K</b>–<b>O</b>), and TIMP2 (<b>P</b>–<b>T</b>) in non-operated (<b>A</b>, <b>F</b>, <b>Q</b>, <b>P</b>), SHAM (<b>B</b>, <b>G</b>, <b>L</b>, <b>K</b>), DH (<b>C</b>, <b>H</b>, <b>M</b>, <b>R</b>), DH+ TO (<b>D</b>, <b>I</b>, <b>N</b>, <b>S</b>), and TO lungs (<b>E</b>, <b>J</b>, <b>O</b>, <b>T</b>). MMP2, MMP14, and TIMP2 were expressed in alveolar epithelial cells in non-operated, SHAM, DH+ TO, and TO lungs (high-magnification inserts). MMP2, MMP14, and TIMP2 were also shown in mesenchymal cells in the thickened septa of DH lungs (diffuse staining in-high magnification inserts). TIMP1 was localized in endothelial cells of capillaries in all study groups (solid arrowheads), and in some alveolar epithelial cells (open arrowheads) in non-operated, SHAM, and DH lungs (<b>K</b>, <b>L</b>, <b>M</b>). DH+ TO (<b>N</b>) and TO lungs (<b>O</b>) showed a much more diffuse immune reaction for TIMP1 in alveolar epithelial cells (open arrowheads) as compared to non-operated and SHAM lungs. Scale bar = 50 µm in A through T.</p

Gene expression ratios between MMPs and TIMPs in the fetal rabbit model for CDH undergoing TO.

<p>Mean Cq values for each gene were converted into linear values according to the efficiency corrected model, and normalized to the geometric mean of SDHA and TOP1 as calculated by <i>genorm</i>. Normalized values for MMP2 and MMP14 were then expressed relative to normalized values for TIMP1 and TIMP2. Error bars illustrate the standard error of the mean, with 4 to 5 animals per study group. One-way ANOVA with Bonferroni correction was used for comparisons between surgical groups (SHAM, DH, DH+ TO, and TO) and unpaired Student’s t-test for comparisons between non-operated and SHAM fetuses. SHAM, sham-operated fetuses; DH, diaphragmatic hernia fetuses; DH+ TO, DH fetuses with tracheal occlusion; TO, sham DH fetuses with TO. ^a<i>P</i> < 0.05 vs SHAM; ^b<i>P</i> < 0.05 vs DH; ^c<i>P</i> < 0.05 vs non-operated. A.U. = arbitrary unit.</p

Pulmonary elastin pattern in the fetal rabbit model for CDH undergoing TO.

<p>Miller’s elastic stain colored elastic fibers in black, and smooth muscle and cytoplasm in yellow. High-magnification insets show strong accumulation of elastic foci at the tips of secondary crests in the lungs of non-operated (<b>A</b>), SHAM (<b>B</b>), DH+ TO (<b>D</b>), and TO fetuses (<b>E</b>). DH lungs showed a disorganized pattern with predominant thick elastic fibers within the alveolar walls (<b>C</b>). Solid arrowheads indicate elastin-containing secondary crests. Open arrowheads indicate elastic fibers in the alveolar walls. <b>F</b>. The mean percentage of elastin-containing crests is decreased in DH lungs and restored by TO. <b>G</b>. The mean percentage of elastic fibers in the alveolar walls was increased in DH lungs and returned to SHAM values in DH+ TO lungs. Error bars represent the standard error of the mean, with 4 to 5 animals per study group. One-way ANOVA with Bonferroni correction was used for comparisons between surgical groups (SHAM, DH, DH+ TO, and TO) and unpaired Student’s t-test for comparisons between non-operated and SHAM fetuses. SHAM, sham-operated fetuses; DH, diaphragmatic hernia fetuses; DH+ TO, DH fetuses with tracheal occlusion; TO, sham DH fetuses with TO. ^a<i>P</i> < 0.05 vs SHAM; ^b<i>P</i> < 0.05 vs DH. Scale bars = 50 µm.</p