Pulmonary vasculature development in congenital diaphragmatic hernia: a novel automated quantitative imaging analysis.

PURPOSE: Impaired fetal lung vasculature determines the degree of pulmonary hypertension in the congenital diaphragmatic hernia (CDH). This study aims to demonstrate the morphometric measurements that differ in pulmonary vessels of fetuses with CDH. METHODS: Nitrofen-induced CDH Sprague-Dawley rat fetuses were scanned with microcomputed tomography. The analysis of the pulmonary vascular tree was performed with artificial intelligence. RESULTS: The number of segments in CDH was significantly lower than that in the control group on the left (U = 2.5, p = 0.004) and right (U = 0, p = 0.001) sides for order 1(O1), whereas there was a significant difference only on the right side for O2 and O3. The pooled element numbers in the control group obeyed Horton's law (R2 = 0.996 left and R2 = 0.811 right lungs), while the CDH group broke it. Connectivity matrices showed that the average number of elements of O1 springing from elements of O1 on the left side and the number of elements of O1 springing from elements of O3 on the right side were significantly lower in CDH samples. CONCLUSION: According to these findings, CDH not only reduced the amount of small order elements, but also destroyed the fractal structure of the pulmonary arterial trees.

Dysregulation of CITED2 in abnormal lung development in the nitrofen rat model.

PURPOSE: CITED2 both modulates lung, heart and diaphragm development. The role of CITED2 in the pathogenesis of congenital diaphragmatic hernia (CDH) is unknown. We aimed to study CITED2 during abnormal lung development in the nitrofen model. METHODS: Timed-pregnant rats were given nitrofen on embryonic day (E) 9 to induce CDH. Fetal lungs were harvested on E15, 18 and 21. We performed RT-qPCR, RNAscope™ in situ hybridization and immunofluorescence staining for CITED2. RESULTS: We observed no difference in RT-qPCR (control: 1.09 ± 0.22 and nitrofen: 0.95 ± 0.18, p = 0.64) and in situ hybridization (1.03 ± 0.03; 1.04 ± 0.03, p = 0.97) for CITED2 expression in E15 nitrofen and control pups. At E18, CITED2 expression was reduced in in situ hybridization of nitrofen lungs (1.47 ± 0.05; 1.14 ± 0.07, p = 0.0006), but not altered in RT-qPCR (1.04 ± 0.16; 0.81 ± 0.13, p = 0.33). In E21 nitrofen lungs, CITED2 RNA expression was increased in RT-qPCR (1.04 ± 0.11; 1.52 ± 0.17, p = 0.03) and in situ hybridization (1.08 ± 0.07, 1.29 ± 0.04, p = 0.02). CITED2 protein abundance was higher in immunofluorescence staining of E21 nitrofen lungs (2.96 × 109 ± 0.13 × 109; 4.82 × 109 ± 0.25 × 109, p < 0.0001). CONCLUSION: Our data suggest that dysregulation of CITED2 contributes to abnormal lung development of CDH, as demonstrated by the distinct spatial-temporal distribution in nitrofen-induced lungs.

Upregulated TSP1 and CD47 expression in the lung in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). The secreted glycoprotein thrombospondin-1 (TSP1), a ligand for receptor CD47, is widely expressed on both systemic and pulmonary vascular cells. TSP1-CD47 signaling has been reported to be one of the pathogeneses of pulmonary hypertension (PH). METHODS: After creating a nitrofen-induced CDH rat model, fetuses were sacrificed on D17, D19 and D21 and divided into a control group and a CDH group. Quantitative real-time polymerase chain reaction was performed to determine the pulmonary gene expression of TSP1, CD47 and Runx3 (a regulator of TSP1). An immunofluorescence study was performed to evaluate the expression and localization of TSP1, CD47 and Runx3. RESULTS: The relative mRNA expression of pulmonary TSP1, CD47 and Runx3 on D21 was significantly increased in the CDH group (p = 0.005, p = 0.001, p = 0.046, and p = 0.002, respectively). The immunofluorescence study also confirmed the overexpression of TSP1, CD47 and Runx3 in the CDH group. CONCLUSION: Our results provide evidence that TSP1-CD47 signaling is involved in the pathogenesis of PH in a nitrofen-induced CDH model. Our data suggest that anti-CD47 antibodies can be novel therapeutic targets for the treatment of PH in CDH.

Sildenafil during the 2nd and 3rd Trimester of Pregnancy: Trials and Tribulations.

Sildenafil, a phosphodiesterase 5 inhibitor with a vasodilatory and anti-remodeling effect, has been investigated concerning various conditions during pregnancy. Per indication, we herein review the rationale and the most relevant experimental and clinical studies, including systematic reviews and meta-analyses, when available. Indications for using sildenafil during the second and third trimester of pregnancy include maternal pulmonary hypertension, preeclampsia, preterm labor, fetal growth restriction, oligohydramnios, fetal distress, and congenital diaphragmatic hernia. For most indications, the rationale for administering prenatal sildenafil is based on limited, equivocal data from in vitro studies and rodent disease models. Clinical studies report mild maternal side effects and suggest good fetal tolerance and safety depending on the underlying pathology.

Low maternal vitamin A intake increases the incidence of teratogen induced congenital diaphragmatic hernia in mice.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a severe birth defect associated with high perinatal mortality and long-term morbidity. The etiology of CDH is poorly understood although abnormal retinoid signaling has been proposed to contribute to abnormal diaphragm development. Existing epidemiological data suggest that inadequate dietary vitamin A intake is a risk factor for developing CDH. METHODS: Using a mouse model of teratogen-induced CDH, the objective of this study was to test the hypothesis that low maternal vitamin A intake contributes to abnormal diaphragm development. To test this hypothesis, we optimized a model of altered maternal dietary vitamin A intake and a teratogenic model of CDH in mice that recapitulates the hallmark features of posterolateral diaphragmatic hernia in humans. RESULTS: Our data uniquely show that low maternal dietary vitamin A intake and marginal vitamin A status increases the incidence of teratogen-induced CDH in mice. CONCLUSION: Low dietary vitamin A intake and marginal vitamin A status lead to an increased incidence of teratogen-induced CDH in mice, highlighting the importance of adequate dietary vitamin A intake and CDH risk. IMPACT: This study describes and validates a mouse model of altered maternal and fetal vitamin A status. This study links existing epidemiological data with a mouse model of teratogen-induced congenital diaphragmatic hernia, highlighting the importance of low maternal vitamin A intake as a risk factor for the development of congenital diaphragmatic hernia. This study supports the Retinoid Hypothesis, which posits that the etiology of congenital diaphragmatic hernia is linked to abnormal retinoid signaling in the developing diaphragm.

Vitamin D improves pulmonary function in a rat model for congenital diaphragmatic hernia.

A congenital diaphragmatic hernia (CDH) is an anomaly caused by defects in the diaphragm; the resulting limited thorax cavity in turn restricts lung growth (pulmonary hypoplasia). This condition is related to pulmonary hypertension. Despite advances in neonatal CDH therapy, the mortality for severe pulmonary hypoplasia remains high. Therefore, it is essential to establish prenatal therapeutic interventions. Vitamin D was reported to have beneficial effects on adult pulmonary hypertension. This study aims to evaluate the efficacy of prenatal vitamin D administration for CDH. First, serum 25-hydroxyvitamin D [25(OH)D] levels in umbilical cord blood were evaluated among CDH newborns. Second, Sprague Dawley rat CDH models were exposed to nitrofen on embryo day 9 (E9). Randomly selected rats in the nitrofen-treated group were infused with calcitriol from E9 to E21. Samples from CDH pups diagnosed after birth were used for lung weight measurements, blood gas analysis, and immunohistochemical analysis. Third, microarray analysis was performed to examine the effect of vitamin D on gene expression profiles in CDH pulmonary arterial tissues. Serum 25(OH)D levels in the umbilical cord blood of newborns who did not survive were significantly lower than those who were successfully discharged. Prenatal vitamin D showed no significant effect on CDH incidence or lung weight but attenuated alveolarization and pulmonary artery remodeling accompanied the improved blood gas parameters. Vitamin D inhibited several gene expression pathways in the pulmonary arteries of CDH rats. Our results suggest that prenatal vitamin D administration attenuates pulmonary vascular remodeling by influencing several gene pathways in CDH.

ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND: Characterized by abnormal lung growth or maturation, congenital diaphragmatic hernia (CDH) affects 1:3000 live births. Cellular studies report proximal (SOX2+) and distal (SOX9+) progenitor cells as key modulators of branching morphogenesis and epithelial differentiation, whereas transcriptome studies demonstrate ROBO/SLIT as potential therapeutic targets for diaphragm defect repair in CDH. In this study, we tested the hypothesis that (a) experimental-CDH could changes the expression profile of ROBO1, ROBO2, SOX2 and SOX9; and (b) ROBO1 or ROBO2 receptors are regulators of branching morphogenesis and SOX2/SOX9 balance. METHODS: The expression profile for receptors and epithelial progenitor markers were assessed by Western blot and immunohistochemistry in a nitrofen-induced CDH rat model. Immunohistochemistry signals by pulmonary structure were also quantified from embryonic-to-saccular stages in normal and hypoplastic lungs. Ex vivo lung explant cultures were harvested at E13.5, cultures during 4 days and treated with increasing doses of recombinant rat ROBO1 or human ROBO2 Fc Chimera proteins for ROBO1 and ROBO2 inhibition, respectively. The lung explants were analyzed morphometrically and ROBO1, ROBO2, SOX2, SOX9, BMP4, and ß-Catenin were quantified by Western blot. RESULTS: Experimental-CDH induces distinct expression profiles by pulmonary structure and developmental stage for both receptors (ROBO1 and ROBO2) and epithelial progenitor markers (SOX2 and SOX9) that provide evidence of the impairment of proximodistal patterning in experimental-CDH. Ex vivo functional studies showed unchanged branching morphogenesis after ROBO1 inhibition; increased fetal lung growth after ROBO2 inhibition in a mechanism-dependent on SOX2 depletion and overexpression of SOX9, non-phospho ß-Catenin, and BMP4. CONCLUSIONS: These studies provided evidence of receptors and epithelial progenitor cells which are severely affected by CDH-induction from embryonic-to-saccular stages and established the ROBO2 inhibition as promoter of branching morphogenesis through SOX2/SOX9 balance.

Extracellular vesicles influence the pulmonary arterial extracellular matrix in congenital diaphragmatic hernia.

OBJECTIVE: Abnormal pulmonary vasculature directly affects the development and progression of congenital diaphragmatic hernia (CDH)-associated pulmonary hypertension (PH). Though overarching structural and cellular changes in CDH-affected pulmonary arteries have been documented, the precise role of the extracellular matrix (ECM) in the pulmonary artery (PA) pathophysiology remains undefined. Here, we quantify the structural, compositional, and mechanical CDH-induced changes in the main and distal PA ECM and investigate the efficacy of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) as a therapy to ameliorate pathological vascular ECM changes. METHODS: Pregnant Sprague-Dawley rodents were administered nitrofen to induce CDH-affected pulmonary vasculature in the offspring. A portion of CDH-affected pups was treated with intravenous infusion of MSC-EVs (1 × 1010 /mL) upon birth. A suite of histological, mechanical, and transmission electron microscopic analyses were utilized to characterize the PA ECM. RESULTS: The CDH model main PA presented significantly altered characteristics-including greater vessel thickness, greater lysyl oxidase (LOX) expression, and a relatively lower ultimate tensile strength of 13.6 MPa compared to control tissue (25.1 MPa), suggesting that CDH incurs ECM structural disorganization. MSC-EV treatment demonstrated the potential to reverse CDH-related changes, particularly through rapid inhibition of ECM remodeling enzymes (LOX and MMP-9). Additionally, MSC-EV treatment bolstered structural aspects of the PA ECM and mitigated pathological disorganization as exhibited by increased medial wall thickness and stiffness that, while not significantly altered, trends away from CDH-affected tissue. CONCLUSIONS: These data demonstrate notable ECM remodeling in the CDH pulmonary vasculature, along with the capacity of MSC-EVs to attenuate pathological ECM remodeling, identifying MSC-EVs as a potentially efficacious therapeutic for CDH-associated pulmonary hypertension.

Overexpression of miR-455-5p affects retinol (vitamin A) absorption by downregulating STRA6 in a nitrofen-induced CDH with lung hypoplasia rat model.

Lung hypoplasia is the main cause of congenital diaphragmatic hernia (CDH)-associated death but pathogenesis remains unclear. MiR-455-5p is involved in lung hypoplasia. We hypothesized that nitrofen causes abnormal miR-455-5p expression during lung development and designed this study to determine the relationship between miR-455-5p, stimulated by retinoic acid 6 (STRA6), and retinol in a nitrofen-induced CDH with lung hypoplasia rat model. Nitrofen or olive oil was administered to Sprague-Dawley rats by gavage on day 9.5 of gestation, and the rats were divided into a nitrofen group and a control group (n = 6). The left lung of fetuses was dissected on day 15.5. The expression of miR-455-5p or STRA6 messenger RNA (mRNA) was determined by quantitative real-time polymerase chain reaction. Average integrated optical density (IOD) of STRA6 protein was determined by immunofluorescence histochemistry. The average retinol level was detected by enzyme-linked immunosorbent assay (n = 6 lungs, respectively). Compared with the control group, the nitrofen group exhibited significantly increased miR-455-5p expression levels (29.450 ± 9.253 vs 5.955 ± 2.330; P = .00045) and significantly decreased STRA6 mRNA levels (0.197 ± 0.097 vs 0.588 ± 0.184; P = .0047). In addition, the average IOD of the STRA6 protein was significantly lower in the nitrofen group (805.643 ± 291.182 vs 1616.391 ± 572.308, P = .015), and the average retinol level was significantly reduced (4.013 ± 0.195 vs 5.317 ± 0.337 µg/L, P = .000). In summary, the overexpression of miR-455-5p affected retinol absorption by downregulating STRA6 in the nitrofen-induced CDH with lung hypoplasia rat model, and this downregulation may be one cause of CDH with lung hypoplasia.

MiR-130a-5p/Foxa2 axis modulates fetal lung development in congenital diaphragmatic hernia by activating the Shh/Gli1 signaling pathway.

AIMS: Congenital diaphragmatic hernia (CDH) is a lethal birth defect characterized by congenital lung malformation, and the severity of pulmonary hypoplasia directly affects the prognosis of infants with CDH. Using a nitrofen-induced CDH rat model, we previously reported that Foxa2 expression was downregulated in CDH lungs by proteomics analysis. Here, we investigate the role of miR-130a-5p/Foxa2 axis in lung development of the nitrofen-induced CDH and evaluate its potential role in vivo prenatal therapy. MAIN METHODS: Nitrofen was orally administrated on embryonic day (E) 8.5 to establish a rat CDH model, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5 and E21.5. The binding sites of miR-130a-5p on Foxa2 mRNA were identified using bioinformatics prediction software and were validated via luciferase assay. The expression levels of miR-130a-5p and Foxa2 were detected using qRT-PCR, ISH, IHC and western blotting. The role of miR-130a-5p/Foxa2 axis in CDH-associated lung development was investigated in ex vivo lung explants. KEY FINDINGS: We found that Foxa2 was downregulated in CDH lung tissues, and Foxa2 upregulating improved CDH branching morphogenesis in ex vivo lung explants. Meanwhile, we also showed that miR-130a-5p was significantly upregulated in CDH lungs and thus inversely correlated with Foxa2. Increasing miR-130a-5p abundance with mimics decreases Foxa2-driven Shh/Gli1 signaling and inhibits branching morphogenesis in ex vivo lung explants. SIGNIFICANCE: This study was the first to show that the miR-130a-5p/Foxa2 axis played a crucial role in CDH-associated pulmonary hypoplasia. These findings may provide relevant insights into the prenatal diagnosis and prenatal therapy of CDH.

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs.

BACKGROUND: Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs. OBJECTIVE: We used the nitrofen/bisdiamine mouse model to induce congenital diaphragmatic hernia and accurately assess the status of alveolar epithelial cell differentiation in relation to the common bipotent progenitors. STUDY DESIGN: Pregnant Swiss mice were gavage-fed with nitrofen/bisdiamine or vehicle at embryonic day 8.5. The administered dose was optimized by assessing the survival, congenital diaphragmatic hernia and facial abnormality rates of the exposed mouse pups. NanoCT was performed on embryonic day 11.5 and 16.5 to assess the embryonic and early canalicular stages of lung development. At embryonic day 17.5 corresponding to late canalicular stage, congenital diaphragmatic hernia lungs were characterized by measuring the lung weight/body weight ratio, morphometry, epithelial cell marker gene expression levels and alveolar cell type quantification. RESULTS: Nitrofen/bisdiamine associated congenital diaphragmatic hernia lungs showed delayed development, hypoplasia with morphologic immaturity and thickened alveolar walls. Expression levels of distal epithelial progenitor marker Id2 increased, alveolar type 1 cell markers Pdpn and Hopx decreased, while type 2 cell markers pro-SPC and Muc1 remained constant during the canalicular stage. The number of Pdpn+ type 1 alveolar cells also decreased in congenital diaphragmatic hernia lungs. CONCLUSION: The mouse nitrofen/bisdiamine model is a potential model allowing the study of congenital diaphragmatic hernia lung development from early stages using a wide array of methods. Based on this model, the alveolar epithelium showed a decrease in the number of alveolar type 1 cell in congenital diaphragmatic hernia lungs while type 2 cell population remains unchanged.

MIF inhibition enhances pulmonary angiogenesis and lung development in congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is a complex birth anomaly with significant mortality and morbidity. Lung hypoplasia and persistent pulmonary hypertension (PPHN) limit survival in CDH. Macrophage migration inhibitory factor (MIF), a key regulator of innate immunity, is involved in hypoxia-induced vascular remodeling and PPHN. We hypothesized that antenatal inhibition of MIF in CDH fetuses, would reduce vascular remodeling, and improve angiogenesis and lung development. METHODS: Pregnant rats were randomized into three groups: Control, nitrofen, and nitrofen + ISO-92. Lung volumes of pups were measured by CT scanning. Right ventricular systolic pressure (RVSP) and vascular wall thickness (VWT) were measured together with MIF concentration, angiogenesis markers, lung morphometry, and histology. RESULTS: Prenatal treatment with ISO-92, an MIF inhibitor, improved normalization of static lung volume, lung volume-to-body weight ratio, decreased alveolar septal thickness, RVSP and VWT and improved radial alveolar count as compared to the non-treated group. Expression of MIF was unaffected by ISO-92; however, ISO-92 increased p-eNOS and VEGF activities and reduced arginase 1, 2 and Sflt-1. CONCLUSION: Prenatal inhibition of MIF activity in CDH rat model improves angiogenesis and lung development. This selective intervention may be a future therapeutic strategy to reduce the morbidity and mortality of this devastating condition.

Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia.

OBJECTIVE: We aimed to evaluate the use of miR-200b as a prenatal transplacental therapy in the nitrofen rat model of abnormal lung development and congenital diaphragmatic hernia (CDH). BACKGROUND: Pulmonary hypoplasia (PH) and pulmonary hypertension determine mortality and morbidity in CDH babies. There is no safe medical prenatal treatment available. We previously discovered that higher miR-200b is associated with better survival in CDH babies. Here, we investigate the role of miR-200b in the nitrofen rat model of PH and CDH and evaluate its use as an in vivo prenatal therapy. METHODS: We profiled miR-200b expression during nitrofen-induced PH using RT-qPCR and in situ hybridization in the nitrofen rat model of PH and CDH. The effects of nitrofen on downstream miR-200b targets were studied in bronchial lung epithelial cells using a SMAD luciferase assay, Western blotting and Immunohistochemistry. We evaluated miR-200b as a lung growth promoting therapy ex vivo and in vivo using lung explant culture and transplacental prenatal therapy in the nitrofen rat model. RESULTS: We show that late lung hypoplasia in CDH is associated with (compensatory) upregulation of miR-200b in less hypoplastic lungs. Increasing miR-200b abundance with mimics early after nitrofen treatment decreases SMAD-driven TGF-ß signaling and rescues lung hypoplasia both in vitro and in vivo. Also, prenatal miR-200b therapy decreases the observed incidence of CDH. CONCLUSIONS: Our data indicate that miR-200b improves PH and decreases the incidence of CDH. Future studies will further exploit this newly discovered prenatal therapy for lung hypoplasia and CDH.

Effects of Simvastatin on Fetal Cardiac Impairment in the Diaphragmatic Experimental Hernia Model.

BACKGROUND: Statins and sildenafil have been shown to exert beneficial effects in cardiac injury. We hypothesized that antenatal maternal administration of simvastatin and/or sildenafil might also promote benefits in cardiac remodeling of congenital diaphragmatic hernia (CDH). Therefore, we performed micro-CT image analysis and histology of the heart after antennal treatment in experimental nitrofen-induced CDH. METHODS: At 9.5 days post conception (dpc), pregnant rats were exposed to nitrofen. At 16 and 20 dpc fetuses were treated with simvastatin and/or sildenafil. At 21 dpc postmortem micro-CT and autopsy were performed. RESULTS: All nitrofen-treated fetuses had a lower birth weight compared to controls; in the simvastatin-treated group, a significant improvement in CDH was noted. Impairment of the lung and liver was also noted in CDH. Compared to controls, CDH rats showed lower ventricular mass, with greater left ventricular thickness; simvastatin decreased the ventricular mass and improved wall thickness. CDH rats exhibited myocardial hypotrophy, severe vascular depression in the left ventricle, and intense interstitial edema compared to controls and nitrofen-exposed animals without CDH. In CDH, the cardiac morphology appeared deformed with left ventricular wall verticalization. Simvastatin improved cardiac myocyte appearance and heart morphology. CONCLUSION: The potential to treat CDH with antenatal simvastatin may improve the management of this malformation.

Assessment of the nitrofen model of congenital diaphragmatic hernia and of the dysregulated factors involved in pulmonary hypoplasia.

PURPOSE: To study pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH), investigators have been employing a fetal rat model based on nitrofen administration to dams. Herein, we aimed to: (1) investigate the validity of the model, and (2) synthesize the main biological pathways implicated in the development of PH associated with CDH. METHODS: Using a defined strategy, we conducted a systematic review of the literature searching for studies reporting the incidence of CDH or factors involved in PH development. We also searched for PH factor interactions, relevance to lung development and to human PH. RESULTS: Of 335 full-text articles, 116 reported the incidence of CDH after nitrofen exposure or dysregulated factors in the lungs of nitrofen-exposed rat fetuses. CDH incidence: 54% (27-85%) fetuses developed a diaphragmatic defect, whereas the whole litter had PH in varying degrees. Downregulated signaling pathways included FGF/FGFR, BMP/BMPR, Sonic Hedgehog and retinoid acid signaling pathway, resulting in a delay in early epithelial differentiation, immature distal epithelium and dysfunctional mesenchyme. CONCLUSIONS: The nitrofen model effectively reproduces PH as it disrupts pathways that are critical for lung branching morphogenesis and alveolar differentiation. The low CDH rate confirms that PH is an associated phenomenon rather than the result of mechanical compression alone.

Ephrin-B1, -B2, and -B4 Expression is Decreased in Developing Diaphragms and Lungs of Fetal Rats with Nitrofen-Induced Congenital Diaphragmatic Hernia.

INTRODUCTION: Congenital diaphragmatic hernia (CDH) is assumed to originate from a malformation of the amuscular mesenchymal component of the primordial diaphragm. Mutations in ephrin-B1, a membrane protein that is expressed by mesenchymal cells, have been found in newborn infants with CDH and associated pulmonary hypoplasia (PH), highlighting its important role during diaphragmatic and airway development. Ephrin-B1, -B2, and -B4 are expressed in fetal rat lungs and have been identified as key players during lung branching morphogenesis. We hypothesized that diaphragmatic and pulmonary expression of ephrin-B1, -B2, and -B4 is decreased in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated rats received nitrofen or vehicle on day 9 (D9). Fetal diaphragms (n = 72) and lungs (n = 72) were harvested on D13, D15, and D18, and divided into control and nitrofen-exposed specimens. Ephrin-B1, -B2, and -B4 gene expression was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for ephrin-B1, -B2, and -B4 was combined with mesenchymal and epithelial markers (Gata-4/Fgf-10 and calcitonin gene-related peptide) to evaluate protein expression/localization. RESULTS: Ephrin-B1, -B2, and -B4 gene expression was significantly reduced in pleuroperitoneal folds/primordial lungs (D13), developing diaphragms/lungs (D15), and fully muscularized diaphragms/differentiated lungs (D18) of nitrofen-exposed fetuses compared with controls. Confocal laser scanning microscopy demonstrated markedly diminished ephrin-B1 immunofluorescence in diaphragmatic and pulmonary mesenchyme of nitrofen-exposed fetuses on D13, D15, and D18 compared with controls, whereas ephrin-B2 and -B4 expression was mainly decreased in distal airway epithelium. CONCLUSION: Decreased ephrin-B1, -B2, and -B4 expression may disrupt diaphragmatic development and lung branching morphogenesis by interfering with epithelial-mesenchymal interactions, thus causing diaphragmatic defects and PH.

Expression of Nitric Oxide Synthase Interacting Protein (NOSIP) is Decreased in the Pulmonary Vasculature of Nitrofen-Induced Congenital Diaphragmatic Hernia.

INTRODUCTION: Persistent pulmonary hypertension (PPH) is a major cause of morbidity and mortality in newborns with congenital diaphragmatic hernia (CDH). PPH is characterized by increased vascular resistance and smooth muscle cell (SMC) proliferation, leading to obstructive changes in the pulmonary vasculature. Nitric oxide (NO), generated by endothelial NO synthase (eNOS), is an important regulator of vascular tone and plays a key role in pulmonary vasodilatation. NO synthase interacting protein (NOSIP), which is strongly expressed by pulmonary SMCs, has recently been identified to reduce the endogenous NO production by interacting with eNOS. We designed this study to investigate the pulmonary vascular expression of NOSIP in the nitrofen-induced CDH model. MATERIALS AND METHODS: Time-mated Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and lung specimens divided into CDH and control (n = 6 for each group). Quantitative real-time polymerase chain reaction and Western blotting were performed to analyze pulmonary gene and protein expression of NOSIP. Immunofluorescence double staining for NOSIP was combined with a specific SMC marker to evaluate protein expression in the pulmonary vasculature. RESULTS: Relative messenger ribonucleic acid and protein expression of NOSIP was significantly decreased in nitrofen-exposed CDH lungs compared with controls. Confocal laser scanning microscopy revealed markedly diminished NOSIP immunofluorescence in nitrofen-exposed CDH lungs compared with controls, mainly in the muscular and endothelial components of the pulmonary vasculature. CONCLUSION: This study demonstrates for the first time decreased NOSIP expression in the pulmonary vasculature of the nitrofen-induced CDH. These findings suggest that NOSIP underexpression may interfere with NO production, contributing to abnormal vascular remodeling and PPH.

Intra-amniotic Sildenafil Treatment Modulates Vascular Smooth Muscle Cell Phenotype in the Nitrofen Model of Congenital Diaphragmatic Hernia.

The etiology of pulmonary vascular abnormalities in CDH is incompletely understood. Studies have demonstrated improvement in pulmonary vasculature with prenatal therapy in animal models. We hypothesize that prenatal sildenafil may attenuate defective pulmonary vascular development via modulation of vSMC phenotype from undifferentiated, proliferative phenotype to differentiated, contractile phenotype. We utilized the nitrofen model of CDH to examine the effect of IA sildenafil on pulmonary vSMC phenotype during lung development. Timed-pregnant CD-1 mice were gavage fed 25 mg nitrofen or olive oil (control) at E8.5 of gestation. Single IA injections of Sildenafil (Revatio; 10 µL of 4 mg/4 ml solution) or dextrose control were performed at E12.5. Mice were sacrificed on various gestational days for embryonic lung harvest. Markers of vSMC development of undifferentiated and differentiated phenotypes were analyzed by immunostaining and western blot. Across all time points in gestation, nitrofen-treated embryonic lungs demonstrated increased vSMC expression of NOTCH3, Hes-5, PDGFR-ß, desmin and α-SMA and decreased expression of calponin and SMMHC, compared to oil controls. IA dextrose treatment had no effect on expression levels. However, IA Sildenafil treatment resulted in down-regulation of NOTCH3, Hes-5, PDGFR-ß, desmin and α-SMA and upregulation of calponin and SMMHC, comparable to oil controls. In the nitrofen model, vSMC express markers consistent with more undifferentiated proliferative phenotype, resulting in hypermuscularization of intrapulmonary arterioles in CDH. A single dose of IA Sildenafil treatment early in gestation, results in sustained normalization of vSMC phenotype. Pharmacologic modulation of the vSMC phenotype at key gestational points may have therapeutic potential.

Upregulated EFNB2 and EPHB4 promotes lung development in a nitrofen-induced congenital diaphragmatic hernia rat model.

Congenital diaphragmatic hernia (CDH) is a common congenital malformation associated with high mortality rates, mainly due to pulmonary hypoplasia and persistent pulmonary hypertension following birth. The present study aimed to investigate abnormal lung development in a rat CDH model, and examine temporal and spatial changes in the expression of ephrin type­B receptor 4 (EPHB4) and ephrin­B2 (EFNB2) during fetal lung development, to elucidate the role of these factors during lung morphogenesis. Pregnant rats received nitrofen on embryonic day (E) 8.5 to induce CDH, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5, and E21.5. The mean linear intercept (MLI) and mean alveolar number (MAN) were observed in fetal lung tissue at E21.5 following hematoxylin and eosin staining. E13.5 fetal lungs were cultured for 96 h in serum­free medium and branch development was observed under a microscope. The gene and protein expression levels of EPHB4 and EFNB2 were assessed by reverse transcription­quantitative polymerase chain reaction analysis, and immunoblotting and immunohistochemistry, respectively. The fetal rat lungs were treated with EFNB2 and the activity of key signaling pathways was assessed. The lung index (lung weight/body weight) at E21.5 was significantly lower in the CDH rats, compared with that in the control fetal rats. The MLI and MAN were also lower in the CDH group. The number of lung terminal buds at E13.5 (embryonic stage), and the lung­explant perimeter and surface were all smaller in the CDH group rats than in the control group at the same age. Pulmonary hypoplasia was observed following 96 h of in vitro culture. No significant differences were found in the expression levels of EFNB2 and EPHB4 between the CDH and control groups at E13.5 (embryonic stage) or E15.5 (pseudoglandular stage), however, EFNB2 and EPHB4 were significantly upregulated at E17.5 (canalicular stage), and at E19.5 and E21.5 (saccular/alveolar stages). EFNB2 stimulated pulmonary branching and EFNB2 supplementation decreased the activity of p38, c­Jun NH2­terminal kinase, extracellular signal­regulated kinase, and signal transducer and activator of transcription. The CDH fetal rats developed pulmonary dysplasia at an early stage of fetal pulmonary development. Upregulated expression of EFNB2 and EPHB4 was observed in the rat lung of nitrofen­induced CDH, and the increased expression of EFNB2 promoted rat lung development in the nitrofen­induced CDH model.

Lung function and pulmonary artery blood flow following prenatal maternal retinoic acid and imatinib in the nitrofen model of congenital diaphragmatic hernia.

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