Alveolarization Genes Modulated by Fetal Tracheal Occlusion in the Rabbit Model for Congenital Diaphragmatic Hernia: A Randomized Study

Abstract

BackgroundThe 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.MethodsFetal 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.ResultsRegarding 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.ConclusionsExperimental 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.

Increased TGF-β: a drawback of tracheal occlusion in human and experimental congenital diaphragmatic hernia?

“…All experiments were conducted in agreement with the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. Anesthesia and surgical procedures for the creation of CDH and TO have been detailed elsewhere (22,62). Briefly, rabbit fetuses were randomly operated at day 23 (pseudoglandular stage of lung development) for partial diaphragm excision (DH) or thoracotomy (sham DH), and at day 28 (early saccular stage) for tracheal ligation (TO) or tracheal dissection without ligation (sham TO).…”

“…Due to high fetal losses and the small size of neonatal rabbit lungs, left and right lungs from each pup were differently allocated to postmortem analyses. We previously showed that biological and histological features of lung hypoplasia were also present in the right lung, i.e., contralateral to the hernia (62,63). The right upper lobe was gently expanded with 4% paraformaldehyde under vacuum and stored for 24 h at 4°C in fixative solution.…”

“…Negative and positive controls were included to ensure qualitative measurements. Data transformed following the efficiencycorrected model were normalized against the geometric mean of validated reference genes (61,62), which were identified by the genorm software (ATP5B, RPLP0, and PGK1 in humans; SDHA and TOP1 in rabbits).…”

“…Protein extraction, gel electrophoresis, transfer and immunoblotting were performed using human and rabbit whole lung homogenates as previously reported (62). After the blocking step, membranes were blotted with one of the following primary antibodies: goat polyclonal anti-phospho-Smad2/3 (p-Smad2/3, Ser 423/425; 1:200; Santa Cruz, Heidelberg, Germany), rabbit monoclonal anti-Smad2/3 (1:100; Cell Signaling Technology, Leiden, The Netherlands), mouse monoclonal anti-ROCK1 (1:100; R&D Systems, Abingdon, UK), goat polyclonal anti-ROCK2 (1:100; R&D Systems), and mouse monoclonal anti-␣-smooth muscle actin (␣-SMA; 1:1,000; Dako, Heverlee, Belgium).…”

“…Membranes were stripped and reprobed with GAPDH (1:5,000; Sigma-Aldrich, Bornem, Belgium) as loading control. Signals were quantified by densitometry using the NIH ImageJ gel analysis tool as previously reported (62). For rabbit analyses, four samples per group were included, which were run in two different gels processed in parallel.…”