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Prem Puri - One of the best experts on this subject based on the ideXlab platform.
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follistatin like 1 expression is decreased in the alveolar epithelium of hypoplastic rat lungs with Nitrofen induced congenital diaphragmatic hernia
2017Co-Authors: Toshiaki Takahashi, Julia Zimmer, Florian Friedmacher, Prem PuriAbstract:Abstract Background/purpose Pulmonary hypoplasia (PH), characterized by incomplete alveolar development, remains a major therapeutic challenge associated with congenital diaphragmatic hernia (CDH). Follistatin-like 1 (Fstl1) is a crucial regulator of alveolar formation and maturation, which is strongly expressed in distal airway epithelium. Fstl1 -deficient mice exhibit reduced airspaces, impaired alveolar epithelial cell differentiation, and insufficient production of surfactant proteins similar to PH in human CDH. We hypothesized that pulmonary Fstl1 expression is decreased during alveolarization in the Nitrofen-induced CDH model. Methods Timed-pregnant rats received Nitrofen or vehicle on gestational day 9 (D9). Fetal lungs were harvested on D18 and D21 and divided into control−/Nitrofen-exposed specimens. Alveolarization was assessed using morphometric analysis techniques. Pulmonary gene expression of Fstl1 was determined by qRT-PCR. Immunofluorescence-double-staining for Fstl1 and alveolar epithelial marker surfactant protein C (SP-C) was performed to evaluate protein expression/localization. Results Radial alveolar count was significantly reduced in hypoplastic lungs of Nitrofen-exposed fetuses with significant down regulation of Fstl1 mRNA expression on D18 and D21 compared to controls. Confocal-laser-scanning-microscopy revealed strikingly diminished Fstl1 immunofluorescence and SP-C expression in distal alveolar epithelium of Nitrofen-exposed fetuses with CDH-associated PH on D18 and D21 compared to controls. Conclusions Decreased expression of Fstl1 in alveolar epithelium may disrupt alveolarization and pulmonary surfactant production, thus contributing to the development of PH in the Nitrofen-induced CDH model. Level of evidence 2b (Centre for Evidence-Based Medicine, Oxford).
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Expression of Prx1 and Tcf4 is decreased in the diaphragmatic muscle connective tissue of Nitrofen-induced congenital diaphragmatic hernia
2016Co-Authors: Toshiaki Takahashi, Julia Zimmer, Florian Friedmacher, Prem PuriAbstract:Abstract Background/purpose Pleuroperitoneal folds (PPFs) are the source of the primordial diaphragm's muscle connective tissue (MCT), and developmental mutations have been shown to result in congenital diaphragmatic hernia (CDH). The protein paired-related homeobox 1 (Prx1) labels migrating PPF cells and stimulates expression of transcription factor 4 (Tcf4), a novel MCT marker that controls morphogenesis of the fetal diaphragm. We hypothesized that diaphragmatic Prx1 and Tcf4 expression is decreased in the Nitrofen-induced CDH model. Methods Time-mated rats were exposed to either Nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15, and D18, and divided into control and Nitrofen-exposed specimens. Gene expression levels of Prx1 and Tcf4 were analyzed by qRT-PCR. Immunofluorescence double staining for Prx1 and Tcf4 was performed to evaluate protein expression and localization. Results Relative mRNA expression of Prx1 and Tcf4 was significantly downregulated in PPFs (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of Nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy revealed markedly diminished Prx1 and Tcf4 expression in diaphragmatic MCT of Nitrofen-exposed fetuses on D13, D15, and D18 compared to controls. Conclusions Decreased expression of Prx1 and Tcf4 in the fetal diaphragm may cause defects in the PPF-derived MCT, leading to development of CDH in the Nitrofen model. Level of evidence Level 2c (Centre for Evidence-Based Medicine, Oxford).
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decreased expression of hepatocyte growth factor in the Nitrofen model of congenital diaphragmatic hernia
2016Co-Authors: Toshiaki Takahashi, Julia Zimmer, Florian Friedmacher, Prem PuriAbstract:Pleuroperitoneal folds (PPFs) are essential for normal diaphragmatic development, representing the only source of the diaphragm’s muscle connective tissue. Hepatocyte growth factor (Hgf), which is secreted in PPFs, plays a crucial role in the formation of the muscular diaphragmatic components by regulating the migration of myogenic progenitor cells into the primordial diaphragm. Hgf is also a known downstream target of Gata4 and it has been demonstrated that the expression of Hgf was significantly downregulated in PPF cells of Gata4 knockouts with congenital diaphragmatic hernia (CDH). Furthermore, mutations in PPF-derived cells have been shown to result in CDH. We hypothesized that Hgf expression is decreased in developing diaphragms of fetal rats with Nitrofen-induced CDH. Timed-pregnant rats were exposed to either Nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18. Dissected diaphragms (n = 72) were divided into control and Nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Diaphragmatic gene expression of Hgf was analyzed by qRT-PCR. Immunofluorescence double staining for Hgf and the mesenchymal marker Gata4 or muscular progenitor marker Myogenin was performed to evaluate protein expression and localization in fetal diaphragms. Relative mRNA expression of Hgf was significantly downregulated in PPFs of Nitrofen-exposed fetuses on D13 (3.08 ± 1.46 vs. 5.24 ± 1.93; p < 0.05), developing diaphragms of Nitrofen-exposed fetuses on D15 (2.01 ± 0.79 vs. 4.10 ± 1.50; p < 0.05) and fully muscularized diaphragms of Nitrofen-exposed fetuses on D18 (1.60 ± 0.78 vs. 3.21 ± 1.89; p < 0.05) compared to controls. Confocal laser scanning microscopy revealed markedly diminished diaphragmatic immunofluorescence of Hgf in Nitrofen-exposed fetuses on D13, D15 and D18 compared to controls, which was associated with disruptions in muscle connective tissue formation and reduced myogenic progenitor cell invasion. Decreased diaphragmatic expression of Hgf may disturb the formation of muscle connective tissue in PPFs and thus prevent essential migration of muscle progenitor cells into the developing diaphragm, leading to diaphragmatic defects in the Nitrofen CDH model.
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decreased expression of monocarboxylate transporter 1 and 4 in the branching airway epithelium of Nitrofen induced congenital diaphragmatic hernia
2016Co-Authors: Toshiaki Takahashi, Julia Zimmer, Florian Friedmacher, Prem PuriAbstract:Abstract Background/Purpose Monocarboxylate transporters (MCTs) are crucial for the maintenance of intracellular pH homeostasis in developing fetal lungs. MCT1/4 is strongly expressed by epithelial airway cells throughout lung branching morphogenesis. Functional inhibition of MCT1/4 in fetal rat lung explants has been shown to result in airway defects similar to pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). We hypothesized that pulmonary expression of MCT1/4 is decreased during lung branching morphogenesis in the Nitrofen model of CDH-associated PH. Methods Timed-pregnant rats received Nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on D15, D18, and D21, and divided into control and Nitrofen-exposed group. Pulmonary gene expression levels of MCT1 / 4 were analyzed by qRT-PCR. Immunofluorescence staining for MCT1/4 was combined with E-cadherin in order to evaluate protein expression in branching airway tissue. Results Relative mRNA levels of MCT1 / 4 were significantly reduced in lungs of Nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly decreased immunofluorescence of MCT1/4 in distal bronchial and primitive alveolar epithelium of Nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Conclusion Decreased expression of MCT1/4 in distal airway epithelium may disrupt lung branching morphogenesis and thus contribute to the development of PH in the Nitrofen-induced CDH model.
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expression of hepatic lipid droplets is decreased in the Nitrofen model of congenital diaphragmatic hernia
2016Co-Authors: Hiromizu Takahashi, Florian Friedmacher, Toshiaki Takahashi, Balazs Kutasy, Prem PuriAbstract:Prenatal mortality in newborn infants with congenital diaphragmatic hernia (CDH) has been attributed to increased amounts of liver hernia ion through the diaphragmatic defect. Antenatal studies in human and rodent fetus with CDH further demonstrated a contribution of the developing liver in the pathogenesis of CDH. The abnormal hepatic growth in experimental animal models, therefore, indicates a disruption of normal liver development in CDH. However, the underlying structural, histological and functional changes in the liver of animals with CDH remain unclear. We design this study to test the hypothesis that the morphological and cellular liver development is altered in the nitrogen-induced CDH model. Pregnant rats were exposed to either olive oil or Nitrofen on day 9 of gestation (D9). Livers and chest were harvested on D21 and divided into two groups: control (n = 8), Nitrofen with CDH (CDH, n = 8). Haematoxylin–eosin (Straub et al. Histopathology 68:617–631, 2013) staining was performed to evaluate underlying morphological changes. Apoptosis was checked by using TUNEL staining and apoptotic cell number was counted on 16–16 slides in 25 fields by two independent viewers. Hepatic lipid droplet expressions were evaluated by hepatic adipose differentiation-related protein (ARDP) expression. Compared to controls markedly increased hypertrophy was seen in CDH group. Significantly increased apoptotic cell numbers were detected in CDH group compared to controls (5.1 ± 1.5 vs 2.1 ± 0.6) (p < 0.05). The relative mRNA expression levels of ARDP were significantly reduced in CDH group compared to controls. Immunohistochemistry showed markedly decreased hepatic ADRP immunoreactivity in CDH fetuses compared to controls. Our findings provide strong evidence of hepatic hypertrophy and increased cell apoptosis in the liver of Nitrofen-induced CDH. These morphological changes may affect liver lipid droplet expression function.
Takashi Doi - One of the best experts on this subject based on the ideXlab platform.
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pax3 gene expression is not altered during diaphragmatic development in Nitrofen induced congenital diaphragmatic hernia
2012Co-Authors: Janhendrik Gosemann, Florian Friedmacher, Prem Puri, Takashi Doi, Jens Dingemann, Balazs KutasyAbstract:Abstract Background/Purpose Malformations of the pleuroperitoneal folds (PPFs) have been identified as the origin of the diaphragmatic defect in congenital diaphragmatic hernia (CDH). Pax3, expressed in muscle precursor cells (MPCs), plays a key role in regulating myogenesis and muscularization in the fetal diaphragm. Pax3 mutant mice display absence of muscular diaphragm. However, the distribution of muscle precursor cells is reported to be normal in the PPF of the Nitrofen-CDH model. We designed this study to investigate the hypothesis that Pax3 gene expression is unaltered in the PPF and developing diaphragm in the Nitrofen-induced CDH model. Methods Pregnant rats were treated with Nitrofen or vehicle on gestational day (D) 9 and sacrificed on D13, D18, and D21. Pleuroperitoneal folds (D13) and developing diaphragms (D18 and D21) were dissected, total RNA was extracted, and real-time quantitative polymerase chain reaction was performed to determine Pax3 messenger RNA levels. Confocal immunofluorescence microscopy was performed to evaluate protein expression/distribution of Pax3. Results Relative messenger RNA expression levels of Pax3 in PPFs and developing diaphragms were not significantly different in the Nitrofen group compared with controls. Intensity of Pax3 immunofluorescence was also not altered in PPFs and developing diaphragms of the Nitrofen group compared with controls. Conclusion Pax3 gene expression is not altered in the PPFs and developing diaphragm of Nitrofen-CDH model, suggesting that the diaphragmatic defect is not caused by disturbance of myogenesis and muscularization.
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prenatal retinoic acid upregulates connexin 43 cx43 gene expression in pulmonary hypoplasia in the Nitrofen induced congenital diaphragmatic hernia rat model
2012Co-Authors: Prem Puri, Takashi Doi, Elke Ruttenstock, Jens DingemannAbstract:Abstract 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.
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smad1 and wif1 genes are downregulated during saccular stage of lung development in the Nitrofen rat model
2012Co-Authors: Naho Fujiwara, Florian Friedmacher, Takashi Doi, Balazs Kutasy, Janhendrik Gosemann, Prem PuriAbstract:The exact pathogenesis of pulmonary hypoplasia in the Nitrofen-induced congenital diaphragmatic hernia (CDH) still remains unclear. Smad1, one of the bone morphogenesis protein (BMP) receptor downstream signaling proteins, plays a key role in organogenesis including lung development and maturation. Smad1 knockout mice display reduced sacculation, an important feature of pulmonary hypoplasia. Wnt inhibitor factor 1 (Wif1) is a target gene of Smad1 in the developing lung epithelial cells (LECs). Smad1 directly regulates Wif1 gene expression and blockade of Smad1 function in fetal LECs is reported to downregulate Wif1 gene expression. We designed this study to test the hypothesis that pulmonary Smad1 and Wif1 gene expression is downregulated during saccular stage of lung development in the Nitrofen CDH model. Pregnant rats were exposed to either olive oil or Nitrofen on day 9 of gestation (D9). Fetuses were harvested on D18, and D21. Fetal lungs were dissected and divided into 2 groups: control and Nitrofen (n = 9 at each time point, respectively). Pulmonary gene expression of Smad1 and Wif1 were analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate protein expression/distribution of Smad1 and Wif1. The relative mRNA expression levels of Smad1 and Wif1 were significantly downregulated in the Nitrofen group compared to controls on D18 and D21 (*p < 0.01, **p < 0.05). Immunoreactivity of Smad1 and Wif1 was also markedly decreased in Nitrofen lungs compared to controls on D18 and D21. We provide evidence, for the first time, that the pulmonary gene expression of Smad1 and Wif1 is downregulated on D18 and D21 (saccular stage of lung development) in the Nitrofen-induced hypoplastic lung. These findings suggest that the downregulation of Smad1/Wif1 gene expression may contribute to pulmonary hypoplasia in the Nitrofen CDH model by retardation of lung development during saccular stage.
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upregulation of fibroblast growth factor receptor 2 and 3 in the late stages of fetal lung development in the Nitrofen rat model
2012Co-Authors: Florian Friedmacher, Prem Puri, Takashi Doi, Balazs Kutasy, Naho Fujiwara, Janhendrik GosemannAbstract:Nitrofen model of congenital diaphragmatic hernia (CDH) has been widely used to investigate the pathogenesis of pulmonary hypoplasia (PH). Fibroblast growth factor (FGF) signaling pathway plays a fundamental role in fetal lung development. FGF7 and FGF10, which are critical for lung morphogenesis, have been reported to be downregulated in Nitrofen-induced PH. FGF signaling is mediated by a family of four single transmembrane receptors, FGFR1–4. FGFR2 and FGFR3 have been shown to be expressed predominantly in the late stages of developing lungs. In addition, the upregulation of FGFR2 gene expression has been associated with severe defects in lung development and resulted in arrested alveologenesis similar to PH seen in the Nitrofen model. Furthermore, FGFR3−/−FGFR4−/− double mutants showed thinner mesenchyme and larger air spaces. We designed this study to test the hypothesis that FGFR gene expression is upregulated in the late stages of lung development in the Nitrofen CDH model. Pregnant rats were exposed to either olive oil or Nitrofen on day 9 of gestation (D9). Cesarean section was performed and fetuses were harvested on D18 and D21. Fetal lungs were divided into three groups: control, Nitrofen without CDH [CDH(−)], and Nitrofen with CDH [CDH(+)] (n = 24 at each time-point). Pulmonary gene expression levels of FGFR1–4 were analyzed by real-time RT-PCR. Immunohistochemistry was also performed to evaluate protein expression/distribution at each time-point. The relative messenger RNA expression levels of pulmonary FGFR2 and FGFR3 on D21 were significantly increased in CDH(−) (6.38 ± 1.93 and 7.84 ± 2.86, respectively) and CDH(+) (7.09 ± 2.50 and 7.25 ± 3.43, respectively) compared to controls (P < 0.05 and P < 0.01, respectively), whereas no significant alteration was observed on D18. There were no differences in FGFR1 and FGFR4 expression at both time-points. Increased immunoreactivity of FGFR2 and FGFR3, mainly in the distal epithelium and mesenchyme, was observed in the Nitrofen-induced hypoplastic lungs on D21 compared to controls. Upregulation of FGFR2 and FGFR3 pulmonary gene expression in the late stages of fetal lung development may disrupt FGFR-mediated alveologenesis resulting in PH in the CDH model.
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expression of the wilm s tumor gene wt1 during diaphragmatic development in the Nitrofen model for congenital diaphragmatic hernia
2011Co-Authors: Jens Dingemann, Elke Ruttenstock, Takashi Doi, Prem PuriAbstract:The Nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm’s tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1−/− mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the Nitrofen CDH model. Pregnant rats received vehicle or Nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05. Diaphragmatic mRNA expression of WT1 was significantly decreased in the Nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21. Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the Nitrofen CDH model.
John J Greer - One of the best experts on this subject based on the ideXlab platform.
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early development of the primordial mammalian diaphragm and cellular mechanisms of Nitrofen induced congenital diaphragmatic hernia
2009Co-Authors: Robin D Clugston, Wei Zhang, John J GreerAbstract:Congenital diaphragmatic hernia (CDH) is a frequently occurring cause of neonatal respiratory distress and is associated with high mortality and long-term morbidity. Evidence from animal models suggests that CDH has its origins in the malformation of the pleuroperitoneal fold (PPF), a key structure in embryonic diaphragm formation. The aims of this study were to characterize the embryogenesis of the PPF in rats and humans, and to determine the potential mechanism that leads to abnormal PPF development in the Nitrofen model of CDH. Analysis of rat embryos, and archived human embryo sections, allowed the timeframe of PPF formation to be determined for both species, thus delineating a critical period of diaphragm development in relation to CDH. Experiments on Nitrofen-exposed NIH 3T3 cells in vitro led us to hypothesize that Nitrofen might cause diaphragmatic hernia in vivo by two possible mechanisms: through decreased cell proliferation or by inducing apoptosis. Data from Nitrofen-exposed rat embryos indicates that the primary mechanism of Nitrofen teratogenesis in the PPF is through decreased cell proliferation. This study provides novel insight into the embryogenesis of the PPF in rats and humans, and it indicates that impaired cell proliferation might contribute to abnormal diaphragm development in the Nitrofen model of CDH. Birth Defects Research (Part A) 2010. © 2009 Wiley-Liss, Inc.
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mechanisms of action of the congenital diaphragmatic hernia inducing teratogen Nitrofen
2007Co-Authors: Rhiannon B Noble, Randal P Babiuk, Robin D Clugston, Michael T Underhill, Hui Sun, Riki Kawaguchi, Paul G Walfish, Rune Blomhoff, Thomas E Gundersen, John J GreerAbstract:Congenital diaphragmatic hernia (CDH) is a developmental anomaly that results in significant mortality and morbidity. The underlying etiology is poorly understood. Insights will arise from an understanding of the mechanisms by which the teratogen Nitrofen induces CDH in rodent models. In this study, we use in vitro cell assays in conjunction with whole animal rodent studies to test hypotheses regarding Nitrofen's mechanism of action. The first component examined the interaction of Nitrofen with various aspects of the retinoid signaling pathway including uptake proteins, binding proteins, receptors, conversion, and degradation enzymes. The second component examined the interactions of Nitrofen and vitamins A, C, and E to test the hypothesis that Nitrofen was functioning as an antioxidant to interfere with retinoid signaling. Third, we performed a series of experiments examining the interaction of Nitrofen and thyroid signaling. Collectively, the data suggest that the primary aspect of retinoid signaling affected by Nitrofen is via inhibition of the rate-limiting enzymes controlling retinoic acid synthesis. Retinoid signaling perturbations do not appear to involve oxidative effects of Nitrofen. Any substantial roles of Nitrofen-induced perturbations of thyroid hormone signaling or receptor function are not supported.
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Airway Smooth Muscle Changes in the Nitrofen-Induced Congenital Diaphragmatic Hernia Rat Model
2003Co-Authors: Jaques Belik, Jingyi Pan, Sandra T Davidge, Wei Zhang, John J GreerAbstract:In the fetal rat, Nitrofen induces congenital diaphragmatic hernia (CDH) and pulmonary vascular remodeling similar to what is observed in the human condition. Airway hyperactivity is common in infants with CDH and attributed to the ventilator-induced airway damage. The purpose of this study was to test the hypothesis that airway smooth muscle mechanical properties are altered in the Nitrofen-induced CDH rat model. Lungs from Nitrofen-exposed fetuses with hernias (CDH) or intact diaphragm (Nitrofen) and untreated fetuses (control) were studied on gestation d 21. The left intrapulmonary artery and bronchi were removed and mounted on a wire myograph, and lung expression, content, and immunolocalization of cyclooxygenases COX-1 and COX-2 were evaluated. Pulmonary artery muscle in the CDH group had significantly ( p < 0.01) lower force generation compared with control and Nitrofen groups. In contrast, the same generation bronchial smooth muscle of the CDH and Nitrofen groups developed higher force compared with control. Whereas no differences were found in endothelium-dependent pulmonary vascular muscle tone, the epithelium-dependent airway muscle relaxation was significantly decreased ( p < 0.01) in the CDH and Nitrofen groups. The lung mRNA levels of COX-1 and COX-2 were increased in the CDH and Nitrofen groups. COX-1 vascular and airway immunostaining, as well as COX-1 and COX-2 lung protein content, were increased in the CDH group. This is the first report of airway smooth muscle abnormalities in the Nitrofen-induced fetal rat model of CDH. We speculate that congenital airway muscle changes may be present in the human form of this disease.
Elke Ruttenstock - One of the best experts on this subject based on the ideXlab platform.
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prenatal retinoic acid upregulates connexin 43 cx43 gene expression in pulmonary hypoplasia in the Nitrofen induced congenital diaphragmatic hernia rat model
2012Co-Authors: Prem Puri, Takashi Doi, Elke Ruttenstock, Jens DingemannAbstract:Abstract 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.
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expression of the wilm s tumor gene wt1 during diaphragmatic development in the Nitrofen model for congenital diaphragmatic hernia
2011Co-Authors: Jens Dingemann, Elke Ruttenstock, Takashi Doi, Prem PuriAbstract:The Nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm’s tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1−/− mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the Nitrofen CDH model. Pregnant rats received vehicle or Nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05. Diaphragmatic mRNA expression of WT1 was significantly decreased in the Nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21. Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the Nitrofen CDH model.
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prenatal administration of retinoic acid upregulates connective tissue growth factor in the Nitrofen cdh model
2011Co-Authors: Elke Ruttenstock, Prem Puri, Takashi Doi, Jens DingemannAbstract: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.
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prenatal treatment with retinoic acid activates parathyroid hormone related protein signaling in the Nitrofen induced hypoplastic lung
2011Co-Authors: Prem Puri, Takashi Doi, Kaoru Sugimoto, Elke Ruttenstock, Jens DingemannAbstract: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.
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expression of iroquois genes is up regulated during early lung development in the Nitrofen induced pulmonary hypoplasia
2011Co-Authors: Takashi Doi, Prem Puri, Elke Ruttenstock, Jens Dingemann, Ausra LukosiūtėAbstract:Abstract Background/Purpose Iroquois homeobox ( Irx ) genes have been implicated in the early lung morphogenesis of vertebrates. Irx1-3 and Irx5 gene expression is seen in fetal lung in rodents up to day (D) 18.5 of gestation. Fetal lung in Irx knockdown mice shows loss of mesenchyme and dilated airspaces, whereas Nitrofen-induced hypoplastic lung displays thickened mesenchyme and diminished airspaces. We hypothesized that the Irx genes are up-regulated during early lung morphogenesis in the Nitrofen-induced hypoplastic lung. Methods Pregnant rats were exposed either to olive oil or Nitrofen on D9. Fetal lungs harvested on D15 were divided into control and Nitrofen groups; and the lungs harvested on D18 were divided into control, Nitrofen without congenital diaphragmatic hernia (CDH[−]), and Nitrofen with CDH (CDH[+]). Irx gene expression levels were analyzed by reverse transcriptase polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression of Irx family. Results Pulmonary Irx1-3 and Irx5 messenger RNA expression levels were significantly up-regulated in Nitrofen group compared with controls at D15. On D15, Irx immunoreactivity was increased in Nitrofen-induced hypoplastic lung compared with controls. Conclusion Overexpression of Irx genes in the early lung development may cause pulmonary hypoplasia in the Nitrofen CDH model by inducing lung dysmorphogenesis with thickened mesenchyme and diminished airspaces.
Jens Dingemann - One of the best experts on this subject based on the ideXlab platform.
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pax3 gene expression is not altered during diaphragmatic development in Nitrofen induced congenital diaphragmatic hernia
2012Co-Authors: Janhendrik Gosemann, Florian Friedmacher, Prem Puri, Takashi Doi, Jens Dingemann, Balazs KutasyAbstract:Abstract Background/Purpose Malformations of the pleuroperitoneal folds (PPFs) have been identified as the origin of the diaphragmatic defect in congenital diaphragmatic hernia (CDH). Pax3, expressed in muscle precursor cells (MPCs), plays a key role in regulating myogenesis and muscularization in the fetal diaphragm. Pax3 mutant mice display absence of muscular diaphragm. However, the distribution of muscle precursor cells is reported to be normal in the PPF of the Nitrofen-CDH model. We designed this study to investigate the hypothesis that Pax3 gene expression is unaltered in the PPF and developing diaphragm in the Nitrofen-induced CDH model. Methods Pregnant rats were treated with Nitrofen or vehicle on gestational day (D) 9 and sacrificed on D13, D18, and D21. Pleuroperitoneal folds (D13) and developing diaphragms (D18 and D21) were dissected, total RNA was extracted, and real-time quantitative polymerase chain reaction was performed to determine Pax3 messenger RNA levels. Confocal immunofluorescence microscopy was performed to evaluate protein expression/distribution of Pax3. Results Relative messenger RNA expression levels of Pax3 in PPFs and developing diaphragms were not significantly different in the Nitrofen group compared with controls. Intensity of Pax3 immunofluorescence was also not altered in PPFs and developing diaphragms of the Nitrofen group compared with controls. Conclusion Pax3 gene expression is not altered in the PPFs and developing diaphragm of Nitrofen-CDH model, suggesting that the diaphragmatic defect is not caused by disturbance of myogenesis and muscularization.
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prenatal retinoic acid upregulates connexin 43 cx43 gene expression in pulmonary hypoplasia in the Nitrofen induced congenital diaphragmatic hernia rat model
2012Co-Authors: Prem Puri, Takashi Doi, Elke Ruttenstock, Jens DingemannAbstract:Abstract 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.
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expression of the wilm s tumor gene wt1 during diaphragmatic development in the Nitrofen model for congenital diaphragmatic hernia
2011Co-Authors: Jens Dingemann, Elke Ruttenstock, Takashi Doi, Prem PuriAbstract:The Nitrofen model of congenital diaphragmatic hernia (CDH) reproduces a typical diaphragmatic defect. However, the exact pathomechanism of CDH is still unknown. The Wilm’s tumor 1 gene (WT1) is crucial for diaphragmatic development. Mutations in WT1 associated with CDH have been described in humans. Additionally, WT1−/− mice display CDH. Furthermore, WT1 is involved in the retinoid signaling pathway, a candidate pathway for CDH. We hypothesized that diaphragmatic WT1 gene expression is downregulated during diaphragmatic development in the Nitrofen CDH model. Pregnant rats received vehicle or Nitrofen on gestational day 9 (D9). Embryos were delivered on D13, D18 and D21. The pleuroperitoneal folds (PPFs) were dissected using laser capture microdissection (D13). Diaphragms of D18 and D21 were manually dissected. RNA was extracted and relative mRNA expression of WT1 was determined using real-time PCR. Immunofluorescence was performed to evaluate protein expression of WT1. Statistical significance was considered p < 0.05. Diaphragmatic mRNA expression of WT1 was significantly decreased in the Nitrofen group on D13, D18 and D21. Intensity of immunofluorescencence of WT1 was markedly decreased in the CDH diaphragms on D13, D18 and D21. Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the Nitrofen CDH model.
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prenatal administration of retinoic acid upregulates connective tissue growth factor in the Nitrofen cdh model
2011Co-Authors: Elke Ruttenstock, Prem Puri, Takashi Doi, Jens DingemannAbstract: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.
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prenatal treatment with retinoic acid activates parathyroid hormone related protein signaling in the Nitrofen induced hypoplastic lung
2011Co-Authors: Prem Puri, Takashi Doi, Kaoru Sugimoto, Elke Ruttenstock, Jens DingemannAbstract: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.