Decreased expression of GATA4 in the diaphragm of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: The molecular mechanisms underlying the diaphragmatic defect in congenital diaphragmatic hernia (CDH) are still poorly understood. The transcription factor GATA4 is essential for normal development of the diaphragm. Recently, mutations in the GATA4 gene have been linked to human and rodent CDH. We hypothesized that diaphragmatic GATA4 expression is downregulated in the nitrofen CDH model. METHODS: Pregnant rats received Nitrofen or vehicle on day 9 of gestation (D9). Fetuses were sacrificed on D13, D18, or D21. Pleuroperitoneal folds (n=20) and fetal diaphragms (n=40) were (micro) dissected and divided into CDH group and controls. RNA and protein were extracted. GATA4 mRNA levels were determined by real-time PCR. Protein levels were determined by ELISA and Immunohistochemistry. RESULTS: mRNA levels and Protein levels were significantly decreased in the CDH group compared to controls on D13 (mRNA 15.96±6.99 vs. 38.10±5.01, p<0.05), D18 (mRNA 10.45±1.84 vs. 17.68±2.11, Protein 2.59±0.06 vs. 4.58±0.35 p<0.05) and D21 (mRNA 4.31±0.83 vs. 6.87±0.88, Protein 0.16±0.08 vs. 1.26±0.49, p<0.05). Immunoreactivity of GATA4 was markedly decreased in CDH-diaphragms on D13, D18, and D21. CONCLUSIONS: We provide evidence for the first time that diaphragmatic expression of GATA4 is downregulated in the nitrofen model, suggesting that decreased expression of GATA4 may impair diaphragmatic development in nitrofen-induced CDH.

Prenatal retinoic acid upregulates connexin 43 (Cx43) gene expression in pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia rat model.

PURPOSE: Connexin 43 (Cx43), a major gap junction protein, is necessary for alveologenesis and plays an important role in the differentiation of type II to type I alveolar epithelial cells. Knockout mice of Cx43 display severe pulmonary hypoplasia (PH). Prenatal administration of retinoic acid (RA) is known to stimulate alveologenesis in nitrofen-induced PH. Recent studies revealed that retinoids upregulate Cx43 expression. We hypothesized that gene expression of Cx43 is downregulated during alveologenesis and that administration of RA upregulates Cx43 expression in the nitrofen-induced PH. METHODS: Pregnant rats were exposed to olive oil or nitrofen on day 9 (D9) of gestation. Retinoic acid was given intraperitoneally on D18, D19, and D20. Fetal lungs were harvested on D18 and D21 and divided into control, nitrofen, control+RA (D21), and nitrofen+RA (D21). The Cx43 expression levels were determined using reverse transcription polymerase chain reaction and immunohistochemistry. RESULTS: On D18 and D21, Cx43 relative messenger RNA expression levels were significantly downregulated in nitrofen compared with those in the control group. On D21, expression levels of Cx43 were significantly upregulated in nitrofen+RA and control+RA compared with those in nitrofen group. Immunohistochemical studies confirmed these results. CONCLUSION: Downregulation of Cx43 expression may interfere with normal alveologenesis. Upregulation of Cx43 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in nitrofen-induced PH.

Prenatal administration of retinoic acid upregulates connective tissue growth factor in the nitrofen CDH model.

PURPOSE: Recent studies have suggested that retinoids may be involved in the molecular mechanisms of pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). Connective tissue growth factor (CTGF) plays a key role in foetal lung development and remodelling during later gestation. CTGF knockout mice exhibit PH with similar characteristics to the human and nitrofen-induced PH. Prenatal administration of retinoic acid (RA) has been shown to stimulate alveologenesis in nitrofen-induced PH. In vitro studies have revealed that RA can induce CTGF gene expression. We hypothesized that pulmonary gene expression of CTGF is downregulated during the later stages of lung development, and that prenatal administration of RA upregulates CTGF in the nitrofen CDH model. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on D18, D19 and D20. Foetuses were harvested on D21 and divided into control, CDH, control + RA and CDH + RA group. Pulmonary CTGF gene and protein expression levels were determined using RT-PCR and immunohistochemistry. RESULTS: On D21, CTGF relative mRNA expression levels were significantly downregulated in CDH group compared to controls. After RA treatment, expression levels of CTGF were significantly upregulated in CDH + RA and control + RA compared to the CDH group. Immunohistochemical studies confirmed these results. CONCLUSION: Downregulation of pulmonary CTGF gene and protein expression during later stages of lung development may interfere with normal alveologenesis in the nitrofen CDH model. Upregulation of CTGF pulmonary gene expression after prenatal RA treatment may promote lung growth by promoting alveologenesis in the nitrofen-induced CDH model.

Prenatal retinoic acid treatment upregulates late gestation lung protein 1 in the nitrofen-induced hypoplastic lung in late gestation.

PURPOSE: Pulmonary hypoplasia (PH), the leading cause of mortality in congenital diaphragmatic hernia (CDH), is associated with arrested alveolarization. Late gestation lung protein 1 (LGL1) plays a crucial role in the regulation of alveolarization. Inhibition of LGL1 impairs alveolar maturation in fetal rat lungs. LGL1 heterozygotus knockout mice display delayed lung maturation. It is well known that prenatal administration of retinoic acid (RA) stimulates alveologenesis in nitrofen-induced PH. In vitro studies have reported that RA is a key modulator of LGL1 during alveologenesis. We hypothesized, that pulmonary gene expression of LGL1 is downregulated in the late stage of lung development, and that prenatal administration of RA upregulates pulmonary LGL1 expression in the nitrofen CDH model. METHODS: Pregnant rats were exposed to nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on D18, D19 and D20. Fetal lungs were dissected on D21 and divided into control, control + RA, CDH and CDH + RA group. Expression levels of LGL1 were determined using RT-PCR and immunohistochemistry. RESULTS: On D21, LGL1 relative mRNA expression levels were significantly downregulated in CDH group compared to controls. After RA treatment, gene expression levels of LGL1 were significantly upregulated in CDH + RA and control + RA compared to CDH group. Immunohistochemical studies confirmed these results. CONCLUSION: Downregulation of pulmonary LGL1 gene expression in the late stage of lung development may interfere with normal alveologenesis. Upregulation of LGL1 pulmonary gene expression after RA treatment may promote lung growth by stimulating alveologenesis in the nitrofen CDH model.