Lung growth and lung hypoplasia in congenital diaphragmatic hernia

Abstract: Pulmonary hypoplasia and persistent pulmonary hypertension are the main causes of mortality and morbidity in newborns with congenital diaphragmatic hernia (CDH). Prenatal tracheal occlusion or ligation (TL) is known to accelerate lung growth, but the mechanism of this is poorly understood. Nitrofen is well known to induce CDH and lung hypoplasia in a rat model of CDH, but its mechanism of action remains unknown. The aim of the present study is to increase the understanding of the biological mechanisms underlying growth and development after prenatal TL in the fetal lung and the underlying pathogenesis of CDH, in order to improve outcomes for neonates with CDH. The experiments were conducted in an animal model of stimulated lung growth after prenatal TL, and in the teratogen model of induced CDH and lung hypoplasia after nitrofen administration. In order to be able to establish the accuracy of the experimental model for further molecular examinations, the histological structure of these fetal lungs was first evaluated, and global gene expression analysis using the Affymetrix Platform and the RAE 230 set arrays was then performed. For validation of the microarray data quantitative real-time PCR of the most significantly up or down-regulated genes was performed, combined with immunohistochemical (IHC) analysis of lung sections, or Western Blot analysis, for validation at a protein level. Fetal lungs after TL showed evidence of growth stimulation with increased volume density of alveolar air space (Vva) and increased radial alveolar count (RAC), comparable to findings in normal neonatal lungs. After nitrofen administration lungs had a more immature appearance, both in the ipsi and contralateral side of the diaphragm defect, with decreased Vva and RAC. In the TL-group, several transcripts with growth factor activity had an increased expression, including connective tissue growth factor (CTGF), insulin-like growth factor-I (IGF-I) and fibroblast growth factor 18 (FGF-18). Some of the genes with a decreased expression after TL are involved in surfactant synthesis and metabolism, such as surfactant protein A (SP-A), apolipoprotein E (Apo-E) and phospholipase group II A2 (plg2a2). These results were confirmed with real-time PCR and IHC studies. Genes with a decreased expression in nitrofen induced CDH included several growth factors, including CTGF, and growth factors receptors involved in lung development, transcription factors, water and ion channels, genes involved in angiogenesis and extracellular matrix. These results were confirmed with real-time PCR and Western Blot. CTGF expression on gestational day 14 (E14) was localized to the epithelium of distal airways, increasing during gestation in days 17 (E17) and 21 (E21). CTGF was increased after TL, both at the mRNA and protein level, and decreased in nitrofen induced CDH, compared to controls (E21). The expression pattern for CTGF in TL-lungs was mostly located to the epithelium of the terminal bronchiole, with decreasing expression pattern distally, whereas in the CDH group, CTGF protein expression seemed to be located mostly to the lung mesenchyma. Summarizing, prenatal TL has been proven to accelerate lung growth, with increased expression of genes and proteins with growth factor activity, such as CTGF. We further describe the CTGF expression pattern during lung development. The pathogenesis of lung hypoplasia and congenital diaphragmatic hernia in the nitrofen model includes alteration at a molecular level of several pathways involved in lung growth and development, fluid balance and vascular development. The complexity of the nitrofen mechanism of action reminds of human CDH, and the picture is consistent with lung hypoplasia and vascular disease, both important contributors to the high mortality and morbidity in CDH. Increased understanding of the molecular mechanisms that control lung growth may be the key to develop novel therapeutic techniques in order to stimulate pre and postnatal lung growth.