tretinoin and nitrofen

Lung and pulmonary vascular maldevelopment in congenital diaphragmatic hernia (CDH) results in significant morbidity and mortality. Retinoic acid (RA) and imatinib have been shown to improve pulmonary morphology following prenatal administration in the rat nitrofen-induced CDH model. It remains unclear if these changes translate into improved function. We evaluated the effect of prenatal RA and imatinib on postnatal lung function, structure, and pulmonary artery (PA) blood flow in the rat CDH model.. Olive oil or nitrofen was administered alone or in combination with RA or imatinib to pregnant rats. Pups were assessed for PA blood flow by ultrasound and pulmonary function/morphology following delivery, intubation, and short-term ventilation.. Neither RA nor imatinib had a negative effect on lung and body growth. RA accelerated lung maturation indicated by increased alveoli number and thinner interalveolar septa and was associated with decreased PA resistance and improved oxygenation. With the exception of a decreased PA pulsatility index, no significant changes in morphology and pulmonary function were noted following imatinib.. Prenatal treatment with RA but not imatinib was associated with improved pulmonary morphology and function, and decreased pulmonary vascular resistance. This study highlights the potential of prenatal pharmacologic therapies, such as RA, for management of CDH.

Topics: Animals; Drug Administration Schedule; Female; Hernias, Diaphragmatic, Congenital; Imatinib Mesylate; Lung; Phenyl Ethers; Pregnancy; Prenatal Care; Protein Kinase Inhibitors; Pulmonary Alveoli; Pulmonary Artery; Random Allocation; Rats; Rats, Sprague-Dawley; Treatment Outcome; Tretinoin

Erythropoietin (EPO), an essential stimulator of erythropoiesis produced by the fetal liver, is important both in vascular remodeling and modulation of the endothelial response in the pulmonary vasculature. In addition, EPO guides alveolar development, along with retinoic acid (RA). EPO is a direct target of RA, and the retinoid pathway is altered in the nitrofen-induced congenital diaphragmatic hernia (CDH) model. In the present study, we tested the hypothesis that the synthesis of EPO is suppressed in a rat model of CDH.. Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D19 and D21 and divided into control and CDH groups. Immunohistochemistry and quantitative real-time polymerase chain reaction (RT-PCR) were performed to determine the expression of EPO in the fetal liver and kidney. We also estimated the expression of EPO receptor in the fetal lung.. The relative EPO mRNA expression in the liver on D19 and in the kidney on D21 were significantly lower in the CDH group than in the controls (P = 0.0008 and P = 0.0064, respectively). In addition, the results of immunohistochemistry supported the findings from the RT-PCR analysis. No significant changes were noted in the expression pattern or EPO receptor levels in the fetal lungs of the CDH group compared to the controls.. Our results reveal the suppressed EPO synthesis in the CDH fetus, which may contribute to the pathogenesis of lung hypoplasia and modification of pulmonary vasculature in the CDH rat model. Pediatr Pulmonol. 2017;52:606-615. © 2016 Wiley Periodicals, Inc.

Topics: Animals; Disease Models, Animal; Erythropoietin; Female; Gene Expression Regulation, Developmental; Hernias, Diaphragmatic, Congenital; Lung; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley; Tretinoin

Low pulmonary retinol levels and disrupted retinoid signaling pathway (RSP) have been implicated in the pathogenesis of congenital diaphragmatic hernia (CDH) and associated pulmonary hypoplasia (PH). It has been demonstrated that nitrofen disturbs the main retinol-binding protein (RBP)-dependent trophoblastic retinol transport. Several studies have demonstrated that prenatal treatment with retinoic acid (RA) can reverse PH in the nitrofen-induced CDH model. We hypothesized that maternal administration of RA can increase trophoblastic RBP-dependent retinol transport in a nitrofen model of CDH.. Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9) and sacrificed on D21. RA was given i.p. on D18, D19, and D20. Retinol and RA levels were measured using high-performance liquid chromatography. Immunohistochemistry was performed to evaluate trophoblastic expression of RBP. Expression levels of the primary RSP genes were determined using quantitative real-time PCR and immunohistochemistry.. Markedly increased trophoblastic RBP immunoreactivity was observed in CDH+RA compared to CDH. Significantly increased serum and pulmonary retinol and RA levels were detected in CDH+RA compared to CDH. Pulmonary expression of RSP genes and proteins were increased in CDH+RA compared to CDH.. Increased trophoblastic RBP expression and retinol transport after antenatal administration of RA suggest that retinol-triggered RSP activation may attenuate CDH-associated PH by elevating serum and pulmonary retinol levels.

Topics: Animals; Biological Transport; Disease Models, Animal; Female; Fetal Weight; Hernias, Diaphragmatic, Congenital; Lung; Phenyl Ethers; Pregnancy; Rats; Retinol-Binding Proteins; Tretinoin; Trophoblasts; Vitamin A

The high mortality rate in congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH). Insulin-like growth factor 2 (IGF2) is an important regulator of fetal growth. The highest levels of IGF2 expression are found in the placenta, which are negatively regulated by decidual retinoid acid receptor alpha (RARα). It has been demonstrated that prenatal administration of retinoic acid (RA) suppresses decidual RARα expression. Previous studies have further shown that prenatal administration of RA can reverse PH in nitrofen-induced CDH model. In IGF2 knockout animals, low levels of IGF2 are associated with decreased placental growth and PH. We therefore hypothesized that nitrofen decreases trophoblastic IGF2 expression and prenatal administration of RA increases it through decidual RARα in the nitrofen-induced CDH model.. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given intraperitoneally on D18, D19 and D20. Fetuses were harvested on D21 and divided into three groups: control, CDH and nitrofen+RA. Immunohistochemistry was performed to evaluate decidual RARα and trophoblastic IGF2 expression. Protein levels of IGF2 in serum, intra-amniotic fluid and left lungs were measured by enzyme-linked immunosorbent assay.. Significant growth retardation of placenta and left lungs was observed in the CDH group compared to control and nitrofen+RA group. Markedly increased decidual RARα and decreased IGF2 immunoreactivity were found in the CDH group compared to control and nitrofen+RA group. Significantly decreased IGF2 protein levels were detected in serum, intra-amniotic fluid and left lungs in the CDH group compared to control and nitrofen+RA group.. Our findings suggest that nitrofen may disturb trophoblastic IGF2 expression through decidual RARα resulting in retarded placental growth and PH in the nitrofen-induced CDH. Prenatal administration of RA may promote lung and placental growth by increasing trophoblastic IGF2 expression.

Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation, Developmental; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Insulin-Like Growth Factor II; Lung; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin; Trophoblasts; Up-Regulation

Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH.. Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo, and Nitrofen+ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining.. Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo (0.31±0.02 vs. 0.08±0.01; P<0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen+ATRA compared to Nitrofen+Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen+ATRA compared to Nitrofen+Placebo.. Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development.

Topics: Animals; Cell Differentiation; Female; Fibroblasts; Gene Expression Regulation, Developmental; Hernias, Diaphragmatic, Congenital; Immunohistochemistry; Lung; Membrane Proteins; Organogenesis; Perilipin-2; Phenyl Ethers; Pregnancy; Pregnancy, Animal; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Up-Regulation

Lung hypoplasia can be prevented in vitro by retinoic acid (RA). Recent evidence suggests that amniotic fluid stem (AFS) cells may integrate injured lungs and influence their recovery. We tested the hypothesis that AFS cells might improve lung growth and motility by paracrine mechanisms. Pregnant rats received either nitrofen or vehicle on E9.5. In vitro E13 embryonic lungs were cultured in the presence of culture medium alone or with RA, basophils, or AFS cells. In vivo green fluorescent protein-expressing (GFP(+)) rat AFS cells were transplanted in nitrofen-exposed rats on E10.5. E13 lung explants were cultured before analysis. The surface, the number of terminal buds, and the frequency of bronchial contractions were assessed. Protein gene product 9.5 (PGP 9.5) and α-actin protein levels were measured. The lung explants transplanted with AFS cells were stained for α-actin, PGP 9.5, and TTF-1. The levels of FGF-10, VEGFα, and TGF-β1 secreted by the AFS cells in the culture medium were measured. Comparison between groups was made by ANOVA. In vitro, the surface, the number of terminal buds, and the bronchial peristalsis were increased in nitrofen+AFS cell explants in comparison with nitrofen-exposed lungs. While nitrofen+RA lungs were similar to nitrofen+AFS ones, basophils did not normalize these measurements. PGP 9.5 protein was decreased in nitrofen lungs, but after adding AFS cells, the value was similar to controls. No differences were found in the expression of α-actin. In vivo, the surface, number of terminal buds, and peristalsis were similar to control after injection of AFS cells in nitrofen-exposed rats. Colocalization with TTF-1-positive cells was found. The levels of FGF-10 and VEGFα were increased in nitrofen+AFS cell explants, while the levels of TGF-β1 were similar to controls. Lung growth, bronchial motility, and innervation were decreased in nitrofen explants and rescued by AFS cells both in vitro and in vivo, similarly to that observed before with RA. The AFS cell beneficial effect was probably related to paracrine action of growth factor secretion.

Topics: Amniotic Fluid; Animals; Cell Movement; Female; Hyperplasia; Lung; Microscopy, Confocal; Pesticides; Phenyl Ethers; Pregnancy; Random Allocation; Rats; Rats, Sprague-Dawley; Stem Cell Transplantation; Tretinoin

Bronchial peristalsis modulates lung growth and is deficient in hypoplastic nitrofen-exposed rat lung explants. Retinoic acid (RA) rescues lung hypoplasia. This study examines whether decreased bronchial innervation contributes to this developmental deficiency and if RA is able to recover bronchial innervation and motility.. After IRB approval, pregnant rats received either 100 mg nitrofen or vehicle on gestational day 9.5 (E9.5). Embryonic lung primordia harvested on E13 were cultured for 72 h and RA was added daily to the medium when appropriate. Lung growth was assessed by counting the number of terminal buds and measuring explant surface, total DNA and protein in control, control + RA, nitrofen and nitrofen + RA groups. Peristaltic contractions were recorded for 10 min under an inverted microscope. Lung explants stained for anti-protein gene product 9.5 (PGP 9.5) and smooth muscle α-actin were examined under a confocal microscope for depicting the specific relationship between neural and smooth muscle cells. PGP 9.5 and smooth muscle α-actin levels were quantified by Western blot analysis for assessing the neural and muscle cell expressions. Comparisons between groups were made with non-parametric tests.. The number of terminal buds, the explants' surface and the DNA and protein contents were significantly decreased in nitrofen-exposed lungs in comparison with controls. In contrast, these measurements were normal in explants exposed to both nitrofen and RA. Bronchial peristalsis (contractions/min) was significantly decreased in nitrofen-exposed lungs in comparison with controls; in contrast, in nitrofen + RA lungs it was similar to controls. In all study groups, the airways were surrounded by smooth muscle and ensheathed in a plexus of nerve fibers containing ganglia. PGP 9.5 protein levels were decreased in nitrofen-exposed lungs, but they normalized when RA was added. No differences were found in α-actin protein levels. Explants exposed only to RA were similar to control.. Lung growth, bronchial innervation and peristalsis are decreased in nitrofen-exposed lung explants and are rescued by RA. If deficient airway innervation contributing to dysmotility and pulmonary hypoplasia can be pharmacologically rescued, new relatively simple prenatal interventions could be envisioned.

Topics: Actins; Animals; Female; Gene Expression Regulation, Developmental; Hernia, Diaphragmatic; Keratolytic Agents; Lung; Male; Organ Culture Techniques; Peristalsis; Pesticides; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley; Tretinoin; Ubiquitin Thiolesterase

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.. 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.. 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.. 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.

Topics: Animals; Cell Differentiation; Connexin 43; Disease Models, Animal; Drug Evaluation, Preclinical; Epithelial Cells; Female; Fetal Therapies; Gene Expression Regulation, Developmental; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Injections, Intraperitoneal; Lung; Phenyl Ethers; Pregnancy; Random Allocation; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Respiratory Mucosa; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Up-Regulation

We sought to investigate the effects of antenatal retinoic acid on the pulmonary vasculature and vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) expression in a nitrofen-induced congenital diaphragmatic hernia (CDH) model.. Rat fetuses were exposed to nitrofen at gestational day 9.5 and/or all-trans retinoic acid (ATRA) at gestational days 18.5-20.5. We assessed lung growth, airway, and vascular morphometry. VEGF, VEGFR1, and VEGFR2 expression was analyzed by Western blotting and immunohistochemistry. Continuous data were analyzed by analysis of variance and Kruskal-Wallis test.. CDH decreased lung to body weight ratio, increased mean linear intercept and mean transection length/airspace, and decreased mean airspace cord length. ATRA did not affect lung growth or morphometry. CDH increased proportional medial wall thickness of arterioles while ATRA reduced it. ATRA recovered expression of VEGF and receptors, which were reduced in CDH.. Retinoic acid and VEGF may provide pathways for preventing pulmonary hypertension in CDH.

Topics: Analysis of Variance; Angiogenesis Inducing Agents; Animals; Arterioles; Biomarkers; Blotting, Western; Disease Models, Animal; Female; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Lung; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley; Teratogens; Treatment Outcome; Tretinoin; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs (HL) in the nitrofen model of congenital diaphragmatic hernia (CDH). Parathyroid hormone-related protein (PTHrP) promotes alveolar maturation by stimulating surfactant production, regulated by PTHrP receptor (PTHrP-R). PTHrP knockout and PTHrP-R null mice both exhibit pulmonary hypoplasia. We have recently reported that nitrofen inhibits PTHrP signaling in the nitrofen-induced HL. Because both PTHrP and PTHrP-R genes have RA-inducible element, we hypothesized that prenatal administration of RA upregulates pulmonary gene expression of PTHrP and PTHrP-R in the nitrofen-induced HL.. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). RA was given on days D18, D19 and D20. Fetal lungs were obtained on D21 and divided into four groups: control, control + RA, nitrofen, nitrofen + RA. RT-PCR and Immunohistochemistry were performed to investigate the pulmonary PTHrP and PTHrP-R gene and protein expression in each group, respectively.. The pulmonary gene expression levels of PTHrP and PTHrP-R were significantly increased in nitrofen + RA group compared to nitrofen group (p < 0.05). Immunoreactivity of PTHrP and PTHrP-R was also remarkably increased in nitrofen + RA group compared to nitrofen group.. Upregulation of PTHrP and PTHrP-R genes after prenatal treatment with RA in the nitrofen-induced HL suggests that RA may have a therapeutic potential in reverting lung hypoplasia in CDH, by stimulating surfactant production and alveolar maturation.

Topics: Analysis of Variance; Animals; Disease Models, Animal; Female; Lung; Lung Diseases; Olive Oil; Parathyroid Hormone-Related Protein; Phenyl Ethers; Plant Oils; Pregnancy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tretinoin; Up-Regulation

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.. 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.. 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.. 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.

Topics: Animals; Female; Gene Expression Regulation, Developmental; Gestational Age; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Immunohistochemistry; Lung; Lung Diseases; Phenyl Ethers; Pregnancy; Pregnancy, Animal; Proteins; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin

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.. 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.. 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.. 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.

Topics: Animals; Connective Tissue Growth Factor; Disease Models, Animal; Female; Gene Expression Regulation, Developmental; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Immunohistochemistry; Phenyl Ethers; Pregnancy; Pregnancy, Animal; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Up-Regulation

Pulmonary hypoplasia (PH) is the main cause of mortality in newborns with congenital diaphragmatic hernia (CDH). Prenatal administration of retinoic acid (RA) stimulates alveologenesis in the nitrofen-induced pulmonary hypoplasia. Insulin-like growth factor receptors (IGFRs) play a crucial role in alveologenesis during lung development. We recently demonstrated that IGFRs were downregulated in later stages of lung development in the nitrofen CDH model. Several studies suggest the ability of RA to regulate insulin-like growth factor signaling. We hypothesized that IGFRs pulmonary gene expression is upregulated after the administration of RA in the nitrofen-induced CDH model.. Pregnant rats were exposed to either olive oil or nitrofen on day 9 (D9) of gestation. RA was given intraperitoneally on days D18, D19, and D20. Fetal lungs were dissected on D21 and divided into control, control + RA, CDH, and CDH + RA group. IGFRs gene and protein expression were determined using RT-PCR and immunohistochemistry.. mRNA expression levels of IGFRs were significantly increased in control + RA and CDH + RA compared with CDH group. Immunoreactivity of IGFRs was markedly increased in control + RA and CDH + RA compared with CDH lungs.. Upregulation of pulmonary gene and protein expression of IGFRs after prenatal RA treatment in the nitrofen model suggests that RA may promote lung growth by stimulating IGFRs mediated alveologenesis.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Down-Regulation; Female; Gene Expression Regulation, Developmental; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Immunohistochemistry; Lung; Lung Diseases; Phenyl Ethers; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Receptor, IGF Type 1; Receptor, IGF Type 2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tretinoin; Up-Regulation

Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling.. To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response-element (RARE) was transfected into HEK-293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace-cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets.. Nitrofen-induced dose-dependent declines in RARE-reporter gene expression. However, similar reductions were observed in control-reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen-induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes.. The observed declines in nitrofen-associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Animals; Apoptosis; Cell Line; Epithelium; Gene Expression; Genes, Reporter; Humans; Isoenzymes; Lipid Metabolism; Lung; Mice; Phenyl Ethers; Rats; Response Elements; Retinal Dehydrogenase; Staining and Labeling; Tretinoin

The precise mechanism of pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) still remains unclear. Recently, prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs in the nitrofen model of CDH. The serine/threonine protein kinase B (AKT) plays a key role in lung morphogenesis through epithelial-mesenchymal interaction in phosphatidylinositide 3-kinase (PI3K)-dependent manner. It has been reported that the lung morphogenesis in explants in mice is interfered by inhibitors of PI3K-AKT signaling pathway. Furthermore, we have recently shown that nitrofen inhibits PI3K-AKT signaling during mid-to-late lung morphogenesis in the nitrofen-induced hypoplastic lung. We hypothesized that prenatal administration of RA upregulates pulmonary gene expression of PI3K and AKT in the nitrofen-induced hypoplastic lung.. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). 5 mg/kg of RA was given on D18, D19 and D20. The fetuses were harvested on D21, and fetal lungs were obtained and divided into four groups: control, control + RA, nitrofen, nitrofen + RA. The mRNA expression levels of PI3K and AKT were analyzed in each lung by real-time RT-PCR and statistically analyzed. Immunohistochemistry was also performed to evaluate protein expression of PI3K and AKT in the fetal lungs at D21.. The pulmonary gene expression levels of PI3K and AKT were significantly upregulated in nitrofen + RA group compared to nitrofen group and control + RA group (p < 0.05), whereas there were no significant differences between controls and control + RA group. Immunoreactivity of PI3K and AKT was markedly increased in nitrofen + RA lungs compared to nitrofen-induced hypoplastic lungs.. Upregulation of PI3K and AKT genes after prenatal treatment with RA in the nitrofen-induced hypoplastic lung suggests that RA may have a therapeutic potential in modulating lung alveologenesis by stimulating epithelial-mesenchymal interaction via PI3K-AKT signaling.

Topics: Animals; Disease Models, Animal; Female; Fetal Diseases; Gene Expression Regulation, Developmental; Gestational Age; Hernia, Diaphragmatic; Immunohistochemistry; Lung; Morphogenesis; Phenyl Ethers; Pregnancy; Pregnancy, Animal; Prenatal Exposure Delayed Effects; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tretinoin; Up-Regulation

Retinoids play an important role in lung development. Recently, prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia (CDH). Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is a transcription factor in the steroid/thyroid hormone receptor superfamily, and targeted ablation of COUP-TFII causes CDH and associated lung hypoplasia in mice. Friend of GATA 2 (FOG2) is a zinc finger-containing protein that modulates the transcriptional activity of GATA proteins. GATA4 is a member of a family of DNA-binding proteins, which is found in the promoter regions of many genes. The COUP-TFII, FOG2, and GATA4 genes, regulated by the retinoid signaling pathway, are located on chromosomes 15q26, 8q23, and 8p23.1 respectively, regions reported to be deleted in individuals with CDH. The aim of this study was to examine the pulmonary gene expression of COUP-TFII, FOG2, and GATA4 in the nitrofen model of CDH.. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation (D9). 5 mg/kg of RA was given intraperitoneally on days D18, D19, and D20. The fetuses were recovered by caesarean section on D21, and the diaphragm was carefully examined for the presence of a hernia under a microscope. Left lungs were obtained from CDH fetuses and controls and divided into four groups: control (n = 9), control + RA (n = 9), CDH (n = 9), and CDH + RA (n = 9). The relative mRNA expression levels of COUP-TFII, FOG2, and GATA4 were analyzed in each lung by real-time reverse transcriptase-polymerase chain reaction from cDNA generated by mRNA from pulmonary total RNA.. The relative mRNA expression levels of COUP-TFII, FOG2, and GATA4 were significantly increased in CDH + RA lungs compared to control, control + RA, and CDH (P < .05).. Up-regulation of pulmonary gene expression of COUP-TFII, FOG2, and GATA4 after prenatal treatment with retinoic acid in the nitrofen model of CDH suggests that RA may have a therapeutic potential in modulating lung growth. Furthermore, these results support the concept that these proteins work together to regulate downstream target genes that play an important role in the development of lung.

Topics: Animals; COUP Transcription Factor II; Disease Models, Animal; DNA-Binding Proteins; Female; GATA4 Transcription Factor; Gene Expression Regulation, Developmental; Hernia, Diaphragmatic; Humans; Lung; Mice; Phenyl Ethers; Pregnancy; Prenatal Exposure Delayed Effects; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transcription Factors; Tretinoin; Up-Regulation

Retinoids play an important role in lung development. A recent study has demonstrated that prenatal treatment with retinoic acid (RA) stimulates alveologenesis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia (CDH). Furthermore, it has also been demonstrated that the differentiation from alveolar epithelial cells type II (AECs-II) into alveolar epithelial cells type I (AECs-I), which is the key process in lung development, is disturbed in this model. We hypothesized that retinoids promote alveologenesis by stimulating differentiation of AECs-II to AECs-I at the end of gestation; and therefore, we investigated the effect of RA on the pulmonary expression of intercellular adhesion molecule 1 (ICAM-1), a marker for AECs-I, and thyroid transcription factor 1 (Ttf-1), a marker for AECs-II, in nitrofen-induced hypoplastic lungs.. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day of gestation (D) 9. Five milligrams per kilogram of RA was given intraperitoneally on D18, D19, and D20; and fetuses were recovered on D21. We had 4 study groups: control (n = 7), control + RA (n = 7), CDH (n = 6), and CDH + RA (n = 6). The expression of ICAM-1 and Ttf-1 was analysed in each lung by real-time reverse transcription polymerase chain reaction and immunohistochemistry. One-way analysis of variance test was used for statistical analysis.. Expression levels of ICAM-1 were significantly reduced in CDH lungs compared with normal controls, whereas levels increased significantly in CDH group after the addition of RA (P < .05). Expression levels of Ttf-1 were significantly decreased in lungs from RA-treated CDH animals compared with CDH without RA (P < .05). The ICAM-1 and Ttf-1 immunoreactivity demonstrated similar pattern of expression in various groups.. Our results demonstrate that prenatal treatment with RA accelerates AEC-I proliferation in the hypoplastic lung in CDH.

Topics: Animals; Cell Proliferation; Disease Models, Animal; Drug Administration Schedule; Epithelial Cells; Female; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Immunohistochemistry; Lung; Phenyl Ethers; Pregnancy; Pregnancy, Animal; Prenatal Care; Probability; Pulmonary Alveoli; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Respiratory System Abnormalities; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sensitivity and Specificity; Tretinoin

Severe pulmonary hypoplasia remains the main cause of the high mortality in newborn infants with congenital diaphragmatic hernia (CDH). Retinoids are a family of molecules derived from vitamin A, which play an important role in lung development. We hypothesized that retinoids promote alveologenesis at the end of gestation and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs in CDH.. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation. Retinoic acid 5 mg/kg was given intraperitoneally on days 18, 19, and 20 of gestation and fetuses were recovered on day 21. We had 4 study groups: control (n = 24), control + retinoic acid (n = 22), CDH (n = 24), and CDH + retinoic acid (n = 19). Lungs from the 4 study groups were fixed, and the following stereological measurements were performed on vertical random sections: total lung volume, volume density of airspaces, volume density of air walls, gas exchange surface area, alveolar volume, and total number of alveoli per lung. Total DNA content of each lung was measured using a spectrophotometer.. Total lung volume increased in CDH lungs after the addition of retinoic acid but remained the same in the control group. Gas exchange surface area was larger in CDH lungs after the addition of retinoic acid but remained unchanged in the control group. The total number of alveoli per lung was higher after the addition of retinoic acid. Total DNA content as well as total DNA content-lung weight ratio of the left lung increased significantly in the CDH group after the addition of retinoic acid compared with CDH without retinoic acid.. Our results demonstrate that prenatal treatment with retinoic acid stimulates alveologenesis in hypoplastic lungs in CDH.

Topics: Animals; Disease Models, Animal; Female; Fetal Organ Maturity; Hernias, Diaphragmatic, Congenital; Injections, Intraperitoneal; Lung; Phenyl Ethers; Pregnancy; Pregnancy, Animal; Prenatal Care; Probability; Pulmonary Alveoli; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Respiratory System Abnormalities; Sensitivity and Specificity; Tretinoin

Retinoids are a group of molecules derived from vitamin A, which play an important role in lung development. Within the cell, retinol can either be oxidized to retinal or esterified to retinyl esters by lecithin : retinol acyltransferase (LRAT) for storage. Retinal is then oxidized to an active metabolite of vitamin A, retinoic acid (RA) by retinal dehydrogenase (RALDH). RA is the active metabolite of vitamin A. Cyp26 (a1,b1, and c1), which is a member of the cytochrome P450 family, acts by reducing the activity of RA. Cyp26 type b1 is the predominant subtype expressed in the murine lung. Several studies have suggested that nitrofen may interfere with the retinoid pathway resulting in congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia. Recently, it was reported that nitrofen may act by inhibiting RALDH2. The aim of this study was to examine the pulmonary expression of Cyp26b1, LRAT, and RALDH2, the key enzymes involved in the synthesis of RA, in order to understand the mechanisms underlying pulmonary hypoplasia in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9 of gestation (D9). Fetal lungs were harvested at D15, D17, D19, and D21. D17, D19, and D21 lungs were divided into three groups: control, nitrofen without CDH and nitrofen with CDH, whereas D15 lungs were divided into only two groups; control and nitrofen as the diaphragm is not fully formed yet at this stage. Real- time PCR was performed to evaluate the relative level of Cyp26b1, LRAT, and RALDH2 expression in the lung. Relative levels of Cyp26b1 mRNA were significantly decreased in the lungs of nitrofen with CDH (D17;0.19 +/- 0.09, D19;0.70 +/- 0.20, D21;0.40 +/- 0.36) and nitrofen without CDH (D17;0.14 +/- 0.06, D19;0.54 +/- 0.42, D21;0.51 +/- 0.56) compared to controls (D17;0.35 +/- 0.16, D19;1.15 +/- 0.48, D21;1.28 +/- 0.78) (P < 0.05). LRAT expression was also significantly decreased in nitrofen with CDH (D17; 19.3 +/- 7.8, D19; 4.3 +/- 1.1, D21; 3.3 +/- 1.6) and nitrofen without CDH (D17; 21.2 +/- 11.1, D19; 4.5 +/- 3.6, D21; 4.1 +/- 1.6) compared to controls (D17; 153.7 +/- 29.8, D19; 26.8 +/- 16.8 D21; 10.1 +/- 3.8) (P < 0.05). There was no significant difference in the relative levels of Cyp26b1 and LRAT between nitrofen with CDH and nitrofen without CDH. There were no significant differences in RALDH2 expression among the groups at any stages. Down-regulation of Cryp26b1 and LRAT demonstrates that RA content is decreased i

Topics: Acyltransferases; Animals; Cytochrome P-450 Enzyme System; Disease Models, Animal; Down-Regulation; Female; Gene Expression Regulation, Developmental; Herbicides; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; Lung; Lung Diseases; Olive Oil; Phenyl Ethers; Plant Oils; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Congenital diaphragmatic hernia (CDH) is a congenital malformation that occurs with a frequency of 0.08 to 0.45 per 1,000 births. Children with CDH are born with the abdominal contents herniated through the diaphragm and exhibit an associated pulmonary hypoplasia which is frequently accompanied by severe morbidity and mortality. Although the etiology of CDH is largely unknown, considerable progress has been made in understanding its molecular mechanisms through the usage of genetic, teratogenic, and surgical models. The following review focuses on the teratogenic and surgical models of CDH and the possible molecular mechanisms of nitrofen (a diphenyl ether, formerly used as an herbicide) in both induction of CDH and pulmonary hypoplasia. In addition, the mechanisms of other compounds including several anti-inflammatory agents that have been linked to CDH will be discussed. Furthermore, this review will also explore the importance of vitamin A in lung and diaphragm development and the possible mechanisms of teratogen interference in vitamin A homeostasis. Continued exploration of these models will bring forth a clearer understanding of CDH and its molecular underpinnings, which will ultimately facilitate development of therapeutic strategies.

Topics: Animals; Diaphragm; Disease Models, Animal; Dogs; Hernia, Diaphragmatic; Lung; Models, Animal; Phenyl Ethers; Rabbits; Rats; Sheep; Signal Transduction; Species Specificity; Teratogens; Tretinoin; Vitamin A; Vitamin A Deficiency

Nitrofen is a diphenyl ether that induces a spectrum of birth defects subsequent to administration to pregnant rodents, in which the molecular etiology of these defects are poorly characterized. Because previous reports showed that nitrofen induced apoptosis in undifferentiated P19 teratocarcinoma cells, we hypothesized that undifferentiated fetal cells have greater susceptibility to nitrofen-induced apoptosis than their differentiated derivatives.. To investigate this hypothesis, cell lines including P19 and F9 were differentiated with retinoic acid into neuronal and endodermal derivatives respectively. Apoptosis was characterized by caspase-3 cleavage and Terminal transferase dUTP nick end labeling (TUNEL) assays.. Both differentiated cell-types had reduced nitrofen-induced caspase-3 cleavage and DNA fragmentation compared with the naive controls, strongly suggesting that differentiation of these cells protects against nitrofen-induced apoptosis. In addition, resistance to apoptotic induction was proportional to the expression levels of the differentiation marker, p27 (kip1) while direct proportionality was not observed for the antiapoptotic protein Bcl-2.. These studies show that nitrofen may induce its associated birth defects via a mechanism involving apoptosis of undifferentiated fetal cells.

Topics: Abnormalities, Drug-Induced; Animals; Apoptosis; Caspase 3; Cell Differentiation; Cell Line; Herbicides; Mice; Phenyl Ethers; Proto-Oncogene Proteins c-bcl-2; Tretinoin

Congenital diaphragmatic hernia (CDH) is a serious medical condition in which the developing diaphragm forms incompletely, leaving a hole through which the abdominal contents can enter the thoracic space and interfere with lung growth. A perturbation of the retinoid system has been linked to the etiology of CDH. This includes findings that nitrofen, which induces CDH in rodents, inhibits the key enzyme for retinoic acid (RA) production, retinaldehyde dehydrogenase-2 (RALDH2) in vitro. Published studies indicate that antenatal vitamin A administration on gestational day (D) 12 in the nitrofen model of CDH reduced the severity and incidence of right-sided defects and lung hypoplasia. In this study, we administered nitrofen on D8, to include the induction of clinically more prevalent left-sided defects, and examined the efficacy of several vitamin A administration paradigms to gain insights into the developmental stage of susceptibility. Furthermore, we tested the hypothesis that administration of RA, the product of RALDH2 activity, is more potent than administering the substrate, vitamin A, in reducing the incidence of CDH. The incidence of CDH was reduced from approximately 54% (nitrofen alone) to approximately 32% with vitamin A treatment. The efficacy of RA treatment was very marked, with a reduction in the incidence of CDH to approximately 15%. Administration of vitamin A or RA on approximately D10 was most effective. These data lend further support for the potential involvement of retinoid signaling pathways and the etiology of CDH and support data from in vitro studies demonstrating a nitrofen-induced suppression of RALDH2.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Female; Hernia, Diaphragmatic; Incidence; Pesticides; Phenyl Ethers; Pregnancy; Rats; Rats, Sprague-Dawley; Tretinoin; Vitamin A

Defects very similar to those seen in infants born with congenital diaphragmatic hernias can be induced in rodents by the administration of the teratogen nitrofen. There is an interest in understanding the biochemical mechanisms of nitrofen's actions in hopes of gaining insights into the etiology of congenital diaphragmatic hernia. In this study, we test the hypothesis that nitrofen is acting to perturb the retinoid signaling pathway by utilizing genetically engineered mice that have the lacZ reporter gene linked to a retinoic acid response element (RARE). We demonstrate a pronounced suppression of RARE-lacZ expression by nitrofen in vitro (by approximately 64%) and in vivo (by approximately 43%).

Topics: Animals; beta-Galactosidase; Disease Models, Animal; Drug Interactions; Gene Expression; Herbicides; Hernia, Diaphragmatic; Hernias, Diaphragmatic, Congenital; In Vitro Techniques; Lac Operon; Mice; Phenyl Ethers; Response Elements; Teratogens; Tretinoin

We have shown that dexamethasone (Dex) accelerates maturation and differentiation of cultured fetal murine lungs (Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Surg Forum 47: 692-695, 1996). We now demonstrate that although Dex inhibits thinning of acinar walls and secondary septa formation, it does, however, promote lung growth. CD-1 murine fetal lungs were cultured for 7 days in the presence and absence of 10 nM Dex. Dex-modulated genes were investigated and identified by differential display of mRNAs performed with specific anchor primer H-T(11)G and 24 arbitrary primers. Thirty-five differentially expressed cDNAs were isolated, subcloned, sequenced, and identified through BLAST searches. One of these cDNAs, termed Dex2, with enhanced expression in Dex-treated lungs, had 100% similarity with ras-recision gene (rrg), also known as the lysyl oxidase (LOX) gene that encodes lysyl oxidase. LOX gene is very highly conserved, with significant sequence similarity among mouse, rat, and human. Two other cDNAs, termed Dex1 and Dex4, were also identified as rrg, with 92 and 97% sequence similarity with the existing data bank sequence of rrg. LOX enzyme is known to downregulate p21(ras) protein and play a central role in the maturation of collagen and elastin in the extracellular matrix as well as modulate the cytoskeletal elements. Thus LOX may be important in lung developmental processes involving epithelial-mesenchymal interactions.

Topics: Animals; Culture Techniques; Dexamethasone; DNA, Complementary; Down-Regulation; Extracellular Matrix Proteins; Gene Expression; Gene Expression Profiling; Immunoblotting; Lung; Mice; Oncogene Protein p21(ras); Phenyl Ethers; Protein-Lysine 6-Oxidase; Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 3; Receptors, Fibroblast Growth Factor; RNA, Messenger; Sequence Analysis, DNA; Sequence Homology, Nucleic Acid; Tretinoin; Up-Regulation