Reductions in the incidence of nitrofen-induced diaphragmatic hernia by vitamin A and retinoic acid

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.     

Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Birth Defects Res (Part B) 89:223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Prenatal administration of retinoic acid upregulates insulin-like growth factor receptors in the nitrofen-induced hypoplastic lung

BACKGROUND : 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. METHODS : 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. RESULTS : 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. CONCLUSIONS : 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. Birth Defects Res (Part B) 92:148–151, 2011. © 2011 Wiley‐Liss, Inc.     

Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development

The teratogen nitrofen produces a congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia in rodent fetuses that closely parallel observations made in humans. We hypothesized that these changes may be due to primary pulmonary hypoplasia and not herniation of the abdominal contents. Timed-pregnant rats were given nitrofen on day 9, and fetuses were harvested on days 13 through 21. Initial evagination of lung buds on gestational day 11 was not delayed in nitrofen-treated fetuses. On gestational day 13, however, there was a significant decrease in the number of terminal end buds in the lungs of nitrofen-exposed fetuses vs. controls. Thymidine-labeled lung epithelial and mesenchymal cells were significantly decreased in nitrofen-treated lungs. Lungs from nitrofen-treated fetuses exhibited wide septae with disorganized, compacted tissue, particularly around the air spaces. Expression of surfactant protein B and C mRNAs was significantly decreased in the nitrofen litters. In situ hybridization of fetal lung tissue at all gestational ages showed no difference in the expression of vascular endothelial growth factor, Flk-1, or Flt-1 mRNAs. Because closure of the diaphragm is completed on gestational day 16 in the rat, our results suggest that lung hypoplasia in this model of CDH is due at least in part to a primary effect of nitrofen on the developing lung.     

Ligand-dependent synergy of thyroid hormone and retinoid X receptors.

The binding of thyroid hormone receptors to DNA is enhanced by heterodimerization with nuclear proteins. One such heterodimerization partner has recently been characterized as the retinoid X receptor. 9-cis-Retinoic acid has been identified as a natural ligand for retinoid X receptors, suggesting a potential receptor-mediated interaction between thyroid hormone and 9-cis-retinoic acid in the regulation of thyroid hormone-responsive genes. A transient cotransfection assay was used to test for such an interaction. When a complex thyroid hormone response element composed of both direct and inverted repeat hexamers was tested, these two ligands activated gene expression synergistically. In contrast, when the response element consisted only of directly repeated hexamers, unliganded retinoid X receptors enhanced thyroid hormone responsiveness, but 9-cis-retinoic acid induced no additional activation. The results suggest a unique mechanism to achieve differential suggest a unique mechanism to achieve differential thyroid hormone sensitivity of thyroid hormone-responsive genes within a cell. Genes with appropriate response elements will show amplification of the thyroid hormone response by 9-cis-retinoic acid in the presence of retinoid X receptors; other thyroid hormone-responsive genes will be influenced by retinoid X receptors, but not 9-cis-retinoic acid.     

Early development of the primordial mammalian diaphragm and cellular mechanisms of nitrofen‐induced congenital diaphragmatic hernia

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.     

Effects of retinoid and thyroid receptors during development of the inner ear

The sensory epithelia of the vertebrate inner ear are comprised of a complex pattern of hair cells and supporting cells. Several different families of signaling molecules have been shown to play a role in the development and maintenance of this structure. In particular, the steroid/thyroid receptor superfamily, and specifically retinoid and thyroid receptors appear to influence the determination, differentiation, maintenance, and possibly regeneration, of the sensory epithelia of the vertebrate inner ear. Clinical and experimental evidence demonstrates that changes in the relative availability of retinoic acid and thyroid hormone, the ligands for retinoid and thyroid receptors, result in perturbations in the development of hair cell sensory epithelia. Retinoic acid and retinoid receptors appear to play a role in early developmental events including cellular proliferation and determination whereas thyroid hormone and thyroid receptors appear to play a role in later events including differentiation and maintenance.     

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-β 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.     

Down-regulation of the tumor suppressor gene retinoic acid receptor beta2 through the phosphoinositide 3-kinase/Akt signaling pathway

The retinoic acid receptor beta2 (RARbeta2) is a potent, retinoid-inducible tumor suppressor gene, which is a critical molecular relay for retinoid actions in cells. Its down-regulation, or loss of expression, leads to resistance of cancer cells to retinoid treatment. Up to now, no primary mechanism underlying the repression of the RARbeta2 gene expression, hence affecting cellular retinoid sensitivity, has been identified. Here, we demonstrate that the phosphoinositide 3-kinase/Akt signaling pathway affects cellular retinoid sensitivity, by regulating corepressor recruitment to the RARbeta2 promoter. Through direct phosphorylation of the corepressor silencing mediator for retinoic and thyroid hormone receptors (SMRT), Akt stabilized RAR/SMRT interaction, leading to an increased tethering of SMRT to the RARbeta2 promoter, decreased histone acetylation, down-regulation of the RARbeta2 expression, and impaired cellular differentiation in response to retinoid. The phosphoinositide 3-kinase/Akt signaling pathway, an important modulator of cellular survival, has thus a direct impact on cellular retinoid sensitivity, and its deregulation may be the triggering event in retinoid resistance of cancer cells.     

Structure and function of cytoplasmic retinoid binding proteins

We examined the ligand protein interactions of two highly homologous cellular retinol binding proteins, CRBP and CRBP-II, and two highly homologous cellular retinoic acid binding proteins, CRABP-I and CRABP-II. While the crystal structures of all four have been determined, nuclear magnetic resonance studies provide a means for observing dynamic aspects of ligand protein interactions of these proteins in solution. The cellular functions of these proteins are less well understood. We have modeled retinoid flux between cytoplasmic retinoid proteins and model membranes and with nuclear receptors. Based on our in vitro studies, we propose that certain retinoids may indirectly influence retinoid signaling by displacing endogenous retinoids from the cytoplasmic proteins to the nuclear receptors.     

Improvement of pulmonary hypoplasia associated with congenital diaphragmatic hernia by in utero CFTR gene therapy

Congenital diaphragmatic hernia (CDH) may be an ideal candidate disease for in utero gene therapy as disrupted fetal lung growth plays a significant role in disease outcome. We previously demonstrated that transient in utero overexpression of CFTR during fetal development resulted in lung epithelial proliferation and differentiation. We hypothesized that gene therapy with CFTR would improve the pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH). CDH was induced by the herbicide 2,4-dichlorophenyl-4-nitrophyl ether (nitrofen) following maternal ingestion at either 10 or 13 days gestation. In utero gene transfer of the CFTR gene was subsequently performed at 16 days gestation. Examination of the fetuses at 22 days gestation revealed little improvement in the CFTR-treated lungs following induction of hernias with nitrofen at 10 days gestation. However, the CFTR gene treatment significantly improved internal surface area, saccular density, overall saccular number, and amount of saccular air space in the lungs that were treated with nitrofen at 13 days gestation. RT-PCR demonstrated that gene transfer occurred following treatment at 13 days gestation but not in the lungs treated with nitrofen at 10 days gestation, despite gene transfer at the same gestational age (16 days) in both groups. As disruption of lung development correlates with the gestational stage at which nitrofen exposure occurs, these results confirmed previous findings that in utero gene transfer efficiency depends on the stage of lung development. Lung development may be significantly delayed in human CDH to allow for successful gene transfer later in gestation, providing a substantial therapeutic window.     

Retinoic acid regulates postnatal neurogenesis in the murine subventricular zone-olfactory bulb pathway

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ)-olfactory bulb pathway. The molecular regulation of this neurogenic circuit is poorly understood. Because the components for retinoid signaling are present in this pathway, we examined the influence of retinoic acid (RA) on postnatal SVZ-olfactory bulb neurogenesis. Using both SVZ neurosphere stem cell and parasagittal brain slice cultures derived from postnatal mouse, we found that RA exposure increased neurogenesis by enhancing the proliferation and neuronal differentiation of forebrain SVZ neuroblasts. The RA precursor retinol had a similar effect, which was reversed by treating cultures with the RA synthesis inhibitor disulfiram. Electroporation of dominant-negative retinoid receptors into the SVZ of slice cultures also blocked neuroblast migration to the olfactory bulb and altered the morphology of the progenitors. Moreover, the administration of disulfiram to neonatal mice decreased in vivo cell proliferation in the striatal SVZ. These results indicate that RA is a potent mitogen for SVZ neuroblasts and is required for their migration to the olfactory bulb. The regulation of multiple steps in the SVZ-olfactory bulb neurogenic pathway by RA suggests that manipulation of retinoid signaling is a potential therapeutic strategy to augment neurogenesis after brain injury.     

TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1

BACKGROUND: Chromosome segregation and mitotic exit depend on activation of the anaphase-promoting complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. RESULTS: We have biochemically characterized subcomplexes of the human APC. One subcomplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subcomplex contains all known APC subunits except APC2/11. This subcomplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. CONCLUSIONS: APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. In analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10.     

Linking animal models to human congenital diaphragmatic hernia

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a major life-threatening malformation, occurring in approximately 1 in 3,000 live births. Over the years, different animal models have been used to gain insight into the etiology of this complex congenital anomaly and to develop treatment strategies. However, to date the pathogenic mechanism is still not understood, and treatment remains difficult because of the associated pulmonary hypoplasia and pulmonary hypertension. METHODS: In this review, data available from several animal models will be discussed. The retinoic acid signaling pathway (RA pathway, retinoid pathway) will be addressed as a developmental pathway that is potentially disrupted in the pathogenesis of CDH. Furthermore, genetic factors involved in diaphragm and lung development will be discussed. CONCLUSIONS: With this review article, we aim to provide a concise overview of the current most important experimental genetic data available in the field of CDH.