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The aim of this study was to investigate the expression and distribution pattern of connective tissue growth factor (CTGF) in lung development during different stages, and to compare with a model of stimulated lung growth after tracheal ligation (TL) and with the teratogen model of induced congenital diaphragmatic hernia (CDH) and lung hypoplasia after nitrofen. Sprague-Dawley rat fetuses were obtained on gestational days 14, 17, and 21 (E14, E17, E21). For the experimental CDH group, pregnant rats were given 100 mg nitrofen on gestational day 9.5 (E9.5), and delivered E21. In another group, Sprague-Dawley rat fetuses were subjected to intrauterine tracheal ligation (TL) on gestational day 19 (E19), and delivered on day 21 (E21). All fetuses were delivered by cesarean section and lungs harvested. Lungs from 1-day-old newborn healthy, nonoperated rats were also obtained. Immunohistochemical (IHC) analysis for CTGF was performed on the different lung sections. CTGF mRNA expression levels in hyperplastic lungs after TL, hypoplastic lungs and CDH after nitrofen administration, and fetal controls at E21 were analyzed with real-time polymerase chain reaction (PCR). Immunohistochemical staining for CTGF at E14 showed that it was merely localized to the epithelium of terminal bronchiole, increasing during gestation, being more abundant at E17 and at E21. In the CDH group, lungs had an immature appearance and CTGF protein expression was decreased in the epithelium of the distal airways compared to the control group at E21, and was mainly observed in the lung mesenchyme. In the TL group, CTGF expression was more abundant compared to the control group at E21, especially in the epithelium of the terminal bronchioles, with a decreasing expression pattern distally. In the newborn lungs, CTGF had a pattern of expression in the epithelium of terminal bronchiole similar to TL lungs. At the mRNA level, CTGF expression was increased after TL, and decreased in the teratogen model of CDH and lung hypoplasia after nitrofen administration. This is, to the authors’ knowledge, the first report of CTGF expression pattern during lung development, and of an impaired expression in CDH lungs after nitrofen. CTGF is suggested to enhance alveologenesis and microvascular development at late stages of lung development, and a decreased expression could lead to the impaired alveologenesis and abnormal microvascular pulmonary bed observed in CDH lungs. Increased understanding of the molecular mechanisms that control lung growth could provide a key to develop novel therapeutic techniques to stimulate pre- and/or postnatal lung growth in infants with impaired lung growth and development, such in congenital diaphragmatic hernia.

Previously, the authors have shown that tracheal occlusion (TO) partially reverses the onset of congenital diaphragmatic hernia (CDH)-induced pulmonary hypertension (PH) and abnormal pulmonary vascular development whereas release of the occlusion (TR) abolishes these clinical benefits. As a consequence of their mitogenic and vasoactive properties, the authors hypothesize that the expression of endothelin (ET)-1 and ET receptor (ETA) genes is increased in lungs of CDH lambs, and that this increase is abolished partially in CDH + TO but not in CDH + TO + TR. A surgical left-sided CDH was created in fetal lambs at 80 days of gestation (gd), followed by TO at 108 gd, and by TR at 129 gd. Four groups were compared: CDH, CDH + TO, CDH + TO + TR, and nonoperated controls (C). Assessment of mRNA expression by Northern blot showed significantly lower ET-1 and ETA levels in the CDH group than in the CDH + TO +/- TR groups (P < .05). Endothelin protein expression levels were lower in CDH +/- TO +/- TR groups when compared with controls for airways and vessels (P < .05) with the exception of endothelial cells. In contrast, ETA protein expression levels were higher in CDH +/- TO +/- TR groups compared with controls for airways and blood vessels smooth muscles (P < .05). These results suggest that involvement of the endothelin system in the pulmonary hypertension associated with CDH is limited. However, the endothelin system appears to be modulated during development.

The aim of the present study was to compare the architecture and alveolar epithelial cell composition of the pulmonary acinus in hypoplastic and normal fetal rat lungs. For this purpose, a rat model of pulmonary hypoplasia in association with congenital diaphragmatic hernia (CDH) induced by Nitrofen (100 mg on day 10 of pregnancy) was studied. Sections (5 microns) from lungs of control and Nitrofen-exposed fetal Sprague Dawley rats with or without CDH aged 18-22 days (vaginal plug on day 1, birth on day 23) were stained with hematoxylin and eosin. To identify developing alveolar epithelial cells, sections were incubated with anti-surfactant protein A (SP-A; rabbit anti-mouse) or preimmunization serum (indirect immunofluorescence). On days 18 and 19, control lungs and exposed lungs from fetuses with and without CDH looked similar (pseudoglandular stage of lung development). The prospective pulmonary acinus consisted of acinar tubules with small round lumens, lined by cuboid, fluorescent type II cells. Morphometric analysis on day 19 showed significantly smaller lung volumes and lung tissue volumes after Nitrofen exposure. On day 20 (canalicular stage), some tubules were slightly dilated and lined by cuboid and thinner fluorescent cells; these dilated tubules were less numerous in lungs from exposed fetuses with CDH. On days 21 and 22 (saccular stage), the saccular lining consisted of cuboid to thin fluorescent cells in exposed lungs from fetuses with and without CDH, and fluorescent (low) cuboid cells interspersed with dark zones (type I cell areas) in control lungs. In the exposed lungs from fetuses with CDH, the lumens of all airspaces were frequently slit-like, and the septa were thicker. These phenomena gave the lungs a primitive, compact aspect. Morphometric analysis on day 22 showed smaller lung volumes and lung tissue volumes, smaller airspace/tissue ratios, smaller epithelial surface areas, and more type II cells per surface area in Nitrofen-exposed lungs than in normal control lungs. The results suggest that Nitrofen-exposed, and thus hypoplastic, fetal rat lungs are retarded with respect to the differentiation of cuboid type II cells into squamous type I cells whether or not CDH is present, and with respect to the development of the future airspaces between days 20 and 22 if CDH is present.