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.

The tumor stage is the most powerful prognostic tool for predicting the survival rates of lung carcinoma patients. However, prognosis of individual patients is difficult in part because of the marked clinical heterogeneity among such patients. Galectins are involved in cell growth, apoptosis and cell migration features, and their diagnostic and prognostic values have already been demonstrated in various types of cancers.In the present paper we analyze the potential prognostic value of immunohistochemical galectin-3 expression in lung adenocarcinomas and squamous cell carcinomas. In all, 165 squamous cell carcinomas and 121 adenocarcinomas were immunostained for galectin-3. In each case the immunohistochemical analyses consisted of an evaluation of the percentage of tumor cells stained and the intensity of staining. An IP score (ie Intensity  Percentage) was thus determined for each lung carcinoma. A large majority of cases displayed galectin-3 expression. While the cytoplasmic staining in the squamous cell carcinomas was focal and moderately intense, the staining in the adenocarcinomas was diffuse and intense. The IP scores were significantly (P ¼ 0.0001) higher in the adenocarcinomas than in the squamous cell carcinomas. The difference in nuclear expression profiles between the two cancer types was statistically significant (P ¼ 0.0005). Cox multivariate analysis carried out on the patients' genders, the TNM classification and the galectin-3-related variables showed that of the galectin-3-related variables, only the nuclear location of galectin-3 was identified as a prognostic indicator of recurrence independent of the clinicopathological features characterizing the patients (P ¼ 0.02). The prognostic contribution of this latter variable was enhanced when the patients with relapse-free follow-ups longer than 8 months were considered (P ¼ 0.005). Galectin-3 immunohistochemical expression differs between squamous cell carcinomas and adenocarcinomas, but the nuclear expression of galectin-3 behaves as a significant prognostic predictor for all the cases as a group.

Summary. Rationale and objectives: Little is known about molecular changes in lungs of fetal rabbits with surgically induced diaphragmatic hernia (DH). Therefore, we examined in this model gene expressions of pivotal molecules for the developing lung. Methods: At day 23 of gestation, DH was created in 12 fetuses from 4 does. Both lungs from six live DH fetuses and from six unoperated controls were harvested and weighed at term. Transcription of 15 genes involved in alveolarization, angiogenesis, regulation of vascular tone, or epithelial maturation was investigated by real-time quantitative polymerase chain reaction. Main results: DH decreased lung-to-body weight ratio (P < 0.001). A bilateral downregulation was seen for genes encoding for tropoelastin (P < 0.01), lysyl oxidase (P < 0.05), fibulin 5 (P < 0.05), and cGMP specific phosphodiesterase 5 (P < 0.05). Lower mRNA levels for endothelial nitric oxide synthase occurred in the ipsilateral lung (P < 0.05). Conclusions: Experimental DH in fetal rabbits disrupted transcription of genes implicated in lung growth and function. Similarities with the human disease make this model appropriate for investigation of new prenatal therapies. Pediatr Pulmonol. ß

A large number of infants with severe CDH surviving after FETO have a degree of tracheomegaly that is associated with the severity of CDH as assessed by pre-FETO LHR. This tracheomegaly does not constitute an obvious clinical problem.

Survivors of severe congenital diaphragmatic hernia (CDH) present significant respiratory morbidity despite lung growth induced by fetal tracheal occlusion (TO). We hypothesized that the underlying mechanisms would involve changes in lung extracellular matrix and dysregulated transforming growth factor (TGF)-β pathway, a key player in lung development and repair. Pulmonary expression of TGF-β signaling components, downstream effectors, and extracellular matrix targets were evaluated in CDH neonates who died between birth and the first few weeks of life after prenatal conservative management or TO, and in rabbit pups that were prenatally randomized for surgical CDH and TO vs. sham operation. Before tissue harvesting, lung tissue mechanics in rabbits was measured using the constant-phase model during the first 30 min of life. Human CDH and control fetal lungs were also collected from midterm onwards. Human and experimental CDH did not affect TGF-β/Smad2/3 expression and activity. In human and rabbit CDH lungs, TO upregulated TGF-β transcripts. Analysis of downstream pathways indicated increased Rho-associated kinases to the detriment of Smad2/3 activation. After TO, subtle accumulation of collagen and α-smooth muscle actin within alveolar walls was detected in rabbit pups and human CDH lungs with short-term mechanical ventilation. Despite TO-induced lung growth, mediocre lung tissue mechanics in the rabbit model was associated with increased transcription of extracellular matrix components. These results suggest that prenatal TO increases TGF-β/Rho kinase pathway, myofibroblast differentiation, and matrix deposition in neonatal rabbit and human CDH lungs. Whether this might influence postnatal development of sustainably ventilated lungs remains to be determined.