Decreased apelin and apelin-receptor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: The high morbidity and mortality in congenital diaphragmatic hernia (CDH) are attributed to severe pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by structural changes in pulmonary arteries, resulting in adventitial and medial thickness. These effects are triggered by abnormal apoptosis and proliferation of pulmonary vascular endothelial and smooth muscle cells (SMCs). Apelin (APLN), a target gene of bone morphogenic protein receptor 2 (BMPR2), is known to play an important and manifold role in regulating pulmonary homeostasis promoting endothelial cell (EC) survival, proliferation and migration. In addition to these autocrine effects of apelin, it displays a paracrine function attenuating the response of pulmonary SMCs to growth factors and promoting apoptosis. Apelin exerts its effect via its G-protein-coupled receptor (APLNR) and is solely expressed by pulmonary vascular EC, whereas APLNR is co-localized in pulmonary ECs and SMCs. Dysfunction of BMPR2 and downstream signalling have been shown to disturb the crucial balance of proliferation of SMCs contributing to the pathogenesis of human and experimentally induced PH. We designed this study to investigate the hypothesis that apelin and APLNR signalling are disrupted in the pulmonary vasculature of rats in nitrofen-induced CDH. METHODS: Pregnant rats were exposed to nitrofen or vehicle on D9 of gestation. Foetuses were sacrificed on D21 and divided into nitrofen and control group (n = 32). Pulmonary RNA was extracted and mRNA levels of APLN and APLNR were determined by quantitative real-time PCR. Protein expression of apelin and APLNR was investigated by western blotting. Confocal immunofluorescence double staining for apelin, APLNR and SMCs were performed. RESULTS: Relative mRNA level of APLN and APLNR were significantly decreased in the CDH group compared to control lungs. Western blotting and confocal microscopy confirmed the qRT-PCR results showing decreased pulmonary protein expression of apelin and APLNR in lungs of nitrofen-exposed foetuses compared to controls. CONCLUSION: This study provides striking evidence of markedly decreased gene and protein expression of apelin and its receptor APLNR in the pulmonary vasculature of nitrofen-induced CDH. The disruption of the apelin-APLNR signalling axis in the pulmonary vasculature may lead to extensive vascular remodelling and contribute to PPH in the nitrofen-induced CDH model.

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

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

Disruption of the bone morphogenetic protein receptor 2 pathway in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Congenital diaphragmatic hernia (CDH) remains a major therapeutic challenge despite advances in neonatal resuscitation and intensive care. The high mortality and morbidity in CDH has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). Bone morphogenetic protein receptor 2 (BMPR2) plays a key role in pulmonary vasculogenesis during the late stages of fetal lung development. BMPR2 is essential for control of endothelial and smooth muscle cell proliferation. Dysfunction of BMPR2 and downstream signaling have been shown to disturb the crucial balance of proliferation of smooth muscle cells contributing to the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that BMPR2 signaling is disrupted in nitrofen-induced CDH. METHODS: Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of BMPR2 and related proteins. RESULTS: Pulmonary Bmpr2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy revealed decreased pulmonary BMPR2 protein expression and increased activation of p38(MAPK) in CDH compared to controls. CONCLUSION: The observed disruption of the BMPR2 signaling pathway may lead to extensive vascular remodeling and contribute to PH in the nitrofen-induced CDH model. BMPR2 may therefore represent a potential target for the treatment of PH in CDH.

Downregulated bone morphogenetic protein signaling in nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: Bone morphogenetic proteins (BMP) have been shown to play crucial roles in not only lung and heart development, but also in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH). We therefore hypothesized that BMP signaling could be altered in nitrofen-induced congenital diaphragmatic hernia (CDH) and associated PH. METHODS: Pregnant rats were exposed to either 100 mg nitrofen or vehicle on embryonic day (E) 9.5. On E17 and E21, fetuses were delivered by cesarean section, killed and checked for left-sided CDH. The tissue was then harvested for pathobiological evaluation. RESULTS: In nitrofen-induced CDH, pulmonary expressions of BMP4, BMP receptor (BMPR) type 2 and Id1 decreased on E17 and E21. On E17, pulmonary gremlin-1 expression increased, while BMP7 decreased. In the lungs, Id1 expression was correlated to BMP4 and BMPR2 and inversely correlated to gremlin-1 expression. Myocardial expressions of BMPR2, BMPR1A, BMP7 and SERCA-2A decreased, while gremlin-1 and noggin expressions increased on E17. On E21, myocardial expressions of Id1 and SERCA-2A decreased, while gremlin-1 expression increased. Moreover, BMPR2 and BMPR1A expressions were correlated to SERCA-2A expression and inversely correlated to pro-apoptotic Bax/Bcl2 ratio within the myocardium. CONCLUSION: Downregulation of BMP signaling seems to contribute to pulmonary and myocardial anomalies observed in nitrofen-induced CDH.

Increased trophoblastic apoptosis mediated by neutrophil gelatinase-associated lipocalin (NGAL) activation in the nitrofen model of congenital diaphragmatic hernia.

BACKGROUND: Retinoids play a key role in fetal lung development. It has been suggested that the maternal-fetal retinol transport is disrupted by trophoblastic apoptosis. The mechanism underlying nitrofen-induced apoptosis in placenta is not fully understood. Neutrophil gelatinase-associated lipocalin (NGAL) is expressed in the fetal part of the maternal-fetal interface. NGAL is part of the immune barrier and serves primarily as a transport protein transferring biologically hazardous molecules in a safe and controlled way. It has been shown that over-activation of NGAL induces apoptosis. We hypothesized that increased placental NGAL expression induces trophoblastic apoptosis in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Placenta harvested on D21 and divided into two groups: control and nitrofen with CDH. Immunohistochemistry was performed to evaluate trophoblasts (by cytokeratin expression), NGAL expression, and apoptotic trophoblastic cells (using TUNEL assay). Total RNA was extracted from each placenta and the relative mRNA expression levels of NGAL were analyzed using RT-PCR. RESULTS: Immunohistochemistry showed NGAL immunoreactivity both in control and CDH in the fetal part of the fetal-maternal interface of placenta. Markedly increased NGAL expression was detected in CDH group compared to controls. Relative mRNA expression levels of NGAL gene were significantly increased in the CDH group compared to control in the placenta (5.924 ± 0.93 vs. 1.895 ± 0.54, p < 0.001). Markedly increased numbers of apoptotic trophoblastic cells were seen in the maternal-fetal interface in the CDH group compared to controls. CONCLUSIONS: NGAL activation may lead to increased trophoblastic apoptosis in the maternal-fetal interface in the nitrofen model of CDH. These changes may therefore cause disturbance in maternal-fetal retinol transport affecting fetal lung morphogenesis.

Decreased pulmonary c-Cbl expression and tyrosine phosphorylation in the nitrofen-induced rat model of congenital diaphragmatic hernia.

PURPOSE: The high morbidity of newborn infants with congenital diaphragmatic hernia (CDH) is attributed to pulmonary hypoplasia (PH), which is characterized by a failure of alveolar development. The nitrofen-induced CDH model has been widely used to investigate the pathogenesis of PH in CDH. It has previously been shown that the fibroblast growth factor receptor (FGFR) pathway, which is essential for a proper lung development, is disrupted during late gestation of nitrofen-induced CDH. Casitas B-lineage lymphoma (c-Cbl) proteins are known regulators of signal transduction through FGFRs, indicating their important role during alveolarization in developing lungs. Furthermore, it has been demonstrated that tyrosine phosphorylation of c-Cbl proteins has a pivotal role for their physiological function and activity during fetal lung development. We designed this study to test the hypothesis that pulmonary c-Cbl expression and tyrosine phosphorylation status are decreased in the nitrofen-induced CDH model. METHODS: Timed-pregnant rats received either 100 mg nitrofen or vehicle on gestation day 9 (D9). Fetuses were harvested on D18 and D21, and lungs were divided into two groups: control and hypoplastic lungs with CDH (CDH(+)) (n = 10 at each time-point, respectively). Pulmonary gene expression levels of c-Cbl were analyzed by quantitative real-time polymerase chain reaction. Western blotting combined with densitometry analysis was used for semi-quantification of protein levels of pulmonary c-Cbl and tyrosine phosphorylation status. Confocal-immunofluorescence staining was performed to evaluate c-Cbl protein expression and distribution. RESULTS: Relative mRNA expression levels of pulmonary c-Cbl were significantly decreased in CDH(+) on D18 and D21 compared to controls. Western blotting showed markedly decreased protein levels of pulmonary c-Cbl and tyrosine phosphorylation status in CDH(+) on D18 and D21. Confocal-immunofluorescence analysis confirmed decreased c-Cbl expression in CDH(+) on D18 and D21 mainly in the distal alveolar epithelium compared to controls. CONCLUSION: Decreased pulmonary c-Cbl gene and protein expression accompanied by a decreased tyrosine phosphorylation status during the late stages of fetal lung development may result in reduced c-Cbl activity, and thus interfere with the FGFR-mediated alveolarization in the nitrofen-induced CDH model.

Asystole in 2 Pediatric Patients During Dexmedetomidine Infusion.

INTRODUCTION: Bradycardia is a known side effect of dexmedetomidine. Reports of sinus pauses or asystole, however, are rare. We present 2 cases of pediatric patients who developed asystole on a dexmedetomidine infusion. SUMMARY OF CASES: An 8-week-old male with RSV bronchiolitis and acute hypoxemic respiratory failure was started on dexmedetomidine for sedation at 0.2 mcg/kg/h with a maximum dose of 0.7mcg/kg/h. On Hospital day (HD) 4, on dexmedetomidine at 0.7 mcg/kg/h, he developed intermittent episodes of bradycardia with heart rates in the 60 s. Echocardiogram on HD 6 showed normal function. On HD 7, he began having periods of asystole lasting up to 6 seconds. Dexmedetomidine was discontinued, with the resolution of episodes of asystole after 6 hours. A 27-month-old male with a congenital left diaphragmatic hernia and pulmonary hypertension who had been weaned off sildenafil 6 months earlier underwent re-repair of left diaphragmatic hernia. Postoperatively he remained intubated and paralyzed. Dexmedetomidine was started at 0.3 mcg/kg/h for sedation, with a maximum dose of 1.2 mcg/kg/h. An echocardiogram on HD 3 showed good function with mild to moderate pulmonary hypertension. That evening, with dexmedetomidine at 1.1 mcg/kg/h, he developed a 15 second period of asystole requiring CPR. Dexmedetomidine was discontinued, and he was started on a midazolam infusion with no further episodes. DISCUSSION: Both cases occurred in patients without cardiac conduction defects or on negative chronotropic or sympatholytic medications that have been associated with dexmedetomidine-induced asystole. We hypothesize that both episodes of asystole were due to increased patient-related vagal tone exacerbated by dexmedetomidine.

Antenatal sildenafil treatment attenuates pulmonary hypertension in experimental congenital diaphragmatic hernia.

BACKGROUND: Lung hypoplasia and persistent pulmonary hypertension of the newborn limit survival in congenital diaphragmatic hernia (CDH). Unlike other diseases resulting in persistent pulmonary hypertension of the newborn, infants with CDH are refractory to inhaled nitric oxide (NO). Nitric oxide mediates pulmonary vasodilatation at birth in part via cyclic GMP production. Phosphodiesterase type 5 (PDE5) limits the effects of NO by inactivation of cyclic GMP. Because of the limited success in postnatal management of CDH, we hypothesized that antenatal PDE5 inhibition would attenuate pulmonary artery remodeling in experimental nitrofen-induced CDH. METHODS AND RESULTS: Nitrofen administered at embryonic day 9.5 to pregnant rats resulted in a 60% incidence of CDH in the offspring and recapitulated features seen in human CDH, including structural abnormalities (lung hypoplasia, decreased pulmonary vascular density, pulmonary artery remodeling, right ventricular hypertrophy), and functional abnormalities (decreased pulmonary artery relaxation in response to the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide). Antenatal sildenafil administered to the pregnant rat from embryonic day 11.5 to embryonic day 20.5 crossed the placenta, increased fetal lung cyclic GMP and decreased active PDE5 expression. Antenatal sildenafil improved lung structure, increased pulmonary vessel density, reduced right ventricular hypertrophy, and improved postnatal NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide-induced pulmonary artery relaxation. This was associated with increased lung endothelial NO synthase and vascular endothelial growth factor protein expression. Antenatal sildenafil had no adverse effect on retinal structure/function and brain development. CONCLUSIONS: Antenatal sildenafil improves pathological features of persistent pulmonary hypertension of the newborn in experimental CDH and does not alter the development of other PDE5-expressing organs. Given the high mortality/morbidity of CDH, the potential benefit of prenatal PDE5 inhibition in improving the outcome for infants with CDH warrants further studies.

Defective pulmonary innervation and autonomic imbalance in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is associated with significant mortality due to lung hypoplasia and pulmonary hypertension. The role of embryonic pulmonary innervation in normal lung development and lung maldevelopment in CDH has not been defined. We hypothesize that developmental defects of intrapulmonary innervation, in particular autonomic innervation, occur in CDH. This abnormal embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH. To define patterns of pulmonary innervation in CDH, human CDH and control lung autopsy specimens were stained with the pan-neural marker S-100. To further characterize patterns of overall and autonomic pulmonary innervation during lung development in CDH, the murine nitrofen model of CDH was utilized. Immunostaining for protein gene product 9.5 (a pan-neuronal marker), tyrosine hydroxylase (a sympathetic marker), vesicular acetylcholine transporter (a parasympathetic marker), or VIP (a parasympathetic marker) was performed on lung whole mounts and analyzed via confocal microscopy and three-dimensional reconstruction. Peribronchial and perivascular neuronal staining pattern is less complex in human CDH than control lung. In mice, protein gene product 9.5 staining reveals less complex neuronal branching and decreased neural tissue in nitrofen-treated lungs from embryonic day 12.5 to 16.5 compared with controls. Furthermore, nitrofen-treated embryonic lungs exhibited altered autonomic innervation, with a relative increase in sympathetic nerve staining and a decrease in parasympathetic nerve staining compared with controls. These results suggest a primary defect in pulmonary neural developmental in CDH, resulting in less complex neural innervation and autonomic imbalance. Defective embryonic pulmonary innervation may contribute to lung developmental defects and postnatal physiological derangement in CDH.

Antenatal imatinib treatment reduces pulmonary vascular remodeling in a rat model of congenital diaphragmatic hernia.

The pathophysiology of congenital diaphragmatic hernia (CDH) is constituted by pulmonary hypoplasia and pulmonary hypertension (PH). We previously reported successful treatment with imatinib of a patient with CDH. This study examines the effect of antenatal imatinib administration on the pulmonary vasculature in a rat model of CDH. Pregnant rats were given nitrofen to induce CDH. Controls were given olive oil. Half of the CDH fetuses and half of the controls were treated with imatinib antenatally E17-E21, rendering four groups: Control, Control+Imatinib, CDH, and CDH+Imatinib. Lung sections were obtained for morphometry and immunohistochemistry, and protein was purified for Western blot. Effects of nitrofen and imatinib on Ki-67, caspase-3, PDGF-B, and PDGF receptors were analyzed. Imatinib significantly reduced medial wall thickness in pulmonary arteries of rats with CDH. It also normalized lumen area and reduced the proportion of fully muscularized arteries. Imatinib also caused medial thinning in the control group. Cell proliferation was increased in CDH, and this proliferation was significantly reduced by imatinib. PDGF-B and PDGFR-ß were upregulated in CDH, and imatinib treatment resulted in a downregulation. PDGFR-α remained unchanged in CDH but was significantly downregulated by imatinib. Antenatal imatinib treatment reduces development of medial wall thickness and restores lumen area in pulmonary arteries in nitrofen-induced CDH. The mechanism is reduced cell proliferation. Imatinib is an interesting candidate for antenatal therapy for PH in CDH, but potential side effects need to be investigated and more specific targeting of PDGF signaling is needed.

Prenatal retinoic acid improves lung vascularization and VEGF expression in CDH rat.

OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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. CONCLUSION: Retinoic acid and VEGF may provide pathways for preventing pulmonary hypertension in CDH.

A longer tracheal occlusion period results in increased lung growth in the nitrofen rat model.

OBJECTIVE: Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with severe congenital diaphragmatic hernia. Limited data are available regarding the effect of duration of TO on lung development. Our objective was to evaluate the effects of long (2 and 2.5 days) versus short (1 day) TO on lung development in rats with nitrofen-induced diaphragmatic hernia. METHOD: Nitrofen was administered on embryonic day (ED) 9 and fetal TO performed either on ED18.5, 19 or 20 (term = 22 days). Sham-operated and untouched littermates served as controls. On ED21, lungs were harvested and only fetuses with a left-sided diaphragmatic defect were included in further analyses. RESULTS: Lung-body-weight ratio incrementally increased with the duration of TO. Increased proliferation following long TO was confirmed by immunohistochemistry and qRT-PCR for the proliferation marker Ki-67. Irrespective of duration, TO induced more complex airway architecture. Medial wall thickness of pulmonary arteries was thinner after long rather than short TO. CONCLUSION: In the nitrofen rat model of congenital diaphragmatic hernia, a longer period of TO leads to enhanced lung growth and less muscularized pulmonary arteries.

Smad1 and WIF1 genes are downregulated during saccular stage of lung development in the nitrofen rat model.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Upregulation of fibroblast growth factor receptor 2 and 3 in the late stages of fetal lung development in the nitrofen rat model.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Nitrofen interferes with trophoblastic expression of retinol-binding protein and transthyretin during lung morphogenesis in the nitrofen-induced congenital diaphragmatic hernia model.

BACKGROUND: Retinoids play a key role in lung development. Retinoid signaling pathway has been shown to be disrupted in the nitrofen model of congenital diaphragmatic hernia (CDH) but the exact mechanism is not clearly understood. Retinol-binding protein (RBP) and transthyretin (TTR) are transport proteins for delivery of retinol to the tissues via circulation. Previous studies have shown that pulmonary retinol levels are decreased during lung morphogenesis in the nitrofen CDH model. In human newborns with CDH, both retinol and RBP levels are decreased. It has been reported that maternal RBP does not cross the placenta and the fetus produces its own RBP by trophoblast. RBP and TTR synthesized in the fetus are essential for retinol transport to the developing organs including lung morphogenesis. We hypothesized that nitrofen interferes with the trophoblastic expression of RBP and TTR during lung morphogenesis and designed this study to examine the trophoblastic expression of RBP and TTR, and the total level of RBP and TTR in the lung in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs and placenta harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Total lung RBP and TTR levels using protein extraction were compared with enzyme linked immunoassay (ELISA). Immunohistochemistry was performed to evaluate trophoblastic RBP and TTR expression. RESULTS: Total protein levels of lung RBP and TTR were significantly lower in CDH (0.26 ± 0.003 and 6.4 ± 0.5 µg/mL) compared with controls (0.4 ± 0.001 and 9.9 ± 1.6 µg/mL, p < 0.05). In the control group, immunohistochemical staining showed strong immunoreactivity of RBP and TTR in the trophoblast compared to CDH group. CONCLUSIONS: Decreased trophoblast expression of retinol transport proteins suggest that nitrofen may interfere with the fetal retinol transport resulting in reduced pulmonary RBP and TTR levels and causing pulmonary hypoplasia in CDH.

Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

BACKGROUND: Heparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures. RESULTS: The pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme.We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2. CONCLUSIONS: The observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.

Late administration of antenatal vitamin A promotes pulmonary structural maturation and improves ventilation in the lamb model of congenital diaphragmatic hernia.

PURPOSE: The lungs in congenital diaphragmatic hernia (CDH) are hypoplastic and immature making respiratory support one of the most challenging aspects of caring for these neonates. Vitamin A is essential for normal lung growth and development. It also promotes alveolarization. The aim of this study is to investigate the effects of antenatal vitamin A on lung growth and alveolarization and ventilation in the lamb model of CDH. METHODS: This study was approved by the Animal Care Committee of the State University of New York at Buffalo, and conforms to the National Institute of Health guidelines. Diaphragmatic defects were created at 79-81 days gestation. Group 1 lambs (CDH, n = 5) were untreated. In group 2 (CDH + vitamin A, n = 6) and group 3 lambs (control + vitamin A, n = 3) right jugular venous catheters were inserted at 118-120 days and retinyl palmitate (vitamin A) was administered until 135 days. The control group (n = 5) consisted of twin littermates. Lambs were delivered at 136-139 days and ventilated for 2 h according to a set protocol. The left lungs were harvested and fixed for histology. RESULTS: Lung compliance was significantly higher in CDH + vitamin A (median 0.27, range 0.1-0.48 ml/cmH(2)O/kg) versus CDH lambs (median 0.07, range 0.07-0.18 ml/cmH(2)O/kg), P < 0.05. At 1 h CDH + vitamin A lambs experienced significantly lower PaCO(2) (median 115, range 35-194 mmHg vs. median 192, range 168-234 mmHg) and higher arterial pH (median 7.0, range 6.74-7.35 vs. median 6.73, range 6.5-6.81) than CDH lambs, P < 0.05. The lung weight to body weight ratio of CDH + vitamin A lambs was significantly less than that of CDH lambs (P < 0.05). Histology showed small thick walled air-spaces and no true alveoli in CDH lambs. In contrast, true alveoli and thinning of the inter-alveolar septums were seen in CDH + vitamin A lambs. CONCLUSION: This is the first study to demonstrate an improvement in lung function and structural maturation when antenatal vitamin A is given in a surgical model of CDH.

Expression of the Wilm's tumor gene WT1 during diaphragmatic development in the nitrofen model for congenital diaphragmatic hernia.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Downregulation of diaphragmatic WT1 gene expression may impair diaphragmatic development in the nitrofen CDH model.

The role of primary myogenic regulatory factors in the developing diaphragmatic muscle in the nitrofen-induced diaphragmatic hernia.

PURPOSE: The nitrofen model of congenital diaphragmatic hernia (CDH) is widely used to investigate the pathogenesis of CDH. However, the exact pathomechanism of the diaphragmatic defect is still unclear. Diaphragmatic muscularization represents the last stage of diaphragmatic development. Myogenic differentiation 1 (MyoD) and myogenic factor 5 (Myf5) play a crucial role in muscularization. MyoD(-/-) : Myf5(+/-) mutant mice show reduced diaphragmatic size, whereas MyoD(+/-) : Myf5(-/-) mutants have normal diaphragms. We designed this study to investigate diaphragmatic gene expression of MyoD and Myf5 in the nitrofen CDH model. METHODS: Pregnant rats received nitrofen or vehicle on day 9 of gestation (D9), followed by cesarean section on D18 and D21. Fetal diaphragms (n = 40) were micro-dissected and divided into CDH group and controls. MyoD and Myf5 mRNA-expression were determined using Real-time PCR. Immunohistochemistry was performed to evaluate protein expression of MyoD and Myf5. RESULTS: Relative diaphragmatic mRNA expression levels and immunoreactivity of MyoD were decreased in the CDH group on D18 and D21. Myf 5 mRNA and protein expression were not altered in the CDH group. CONCLUSION: This is the first study showing that MyoD expression is selectively decreased in the diaphragm muscle in the nitrofen model of CDH.

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

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