Spatiotemporal alterations in Sprouty-2 expression and tyrosine phosphorylation in nitrofen-induced pulmonary hypoplasia

Background/Purpose

Pulmonary hypoplasia (PH) is a life-threatening condition of newborns presenting with congenital diaphragmatic hernia (CDH). Sprouty-2 functions as a key regulator of fibroblast growth factor receptor (FGFR) signalling in developing foetal lungs. It has been reported that FGFR-mediated alveolarization is disrupted in nitrofen-induced PH. Sprouty-2 knockouts show severe defects in lung morphogenesis similar to nitrofen-induced PH. Upon FGFR stimulation, Sprouty-2 is tyrosine-phosphorylated, which is essential for its physiological function during foetal lung development. We hypothesized that Sprouty-2 expression and tyrosine phosphorylation are altered in nitrofen-induced PH.

Methods

Time-pregnant rats received either nitrofen or vehicle on gestation day 9 (D9). Foetal lungs were dissected on D18 and D21. Pulmonary Sprouty-2 gene and protein expression levels were analyzed by qRT-PCR, Western blotting and immunohistochemical staining.

Results

Relative mRNA expression of Sprouty-2 was significantly decreased in hypoplastic lungs without CDH (0.1050 ± 0.01 vs. 0.3125 ± 0.01; P < .0001) and with CDH (0.1671 ± 0.01 vs. 0.3125 ± 0.01; P < .0001) compared to controls on D18. Protein levels of Sprouty-2 were markedly decreased in hypoplastic lungs on D18 with decreased tyrosine phosphorylation levels on D18 and D21 detected at the molecular weight of Sprouty-2 consistent with Sprouty-2 tyrosine phosphorylation. Sprouty-2 immunoreactivity was markedly decreased in hypoplastic lungs on D18 and D21.

Conclusion

Spatiotemporal alterations in pulmonary Sprouty-2 expression and tyrosine phosphorylation during the late stages of foetal lung development may interfere with FGFR-mediated alveolarization in nitrofen-induced PH.     

Antenatal vitamin A administration attenuates lung hypoplasia by interfering with early instead of late determinants of lung underdevelopment in congenital diaphragmatic hernia

Background/Purpose

Early and late lung underdevelopment in congenital diaphragmatic hernia (CDH) is likely caused by nonmechanical (directly mediated by nitrofen) and mechanical (mediated by thoracic herniation) factors, respectively. The authors investigated if vitamin A enhances lung growth because of effects on both early and late determinants of lung hypoplasia.

Methods

Twenty-seven pregnant Wistar rats were exposed on embryonic day (E)9.5 to 100 mg of nitrofen or just olive oil. From nitrofen-exposed pregnant rats, 12 were treated at day 9.5 or 18.5 with 15,000 IU of vitamin A. Lungs were harvested at E18, E20, and E22, weighed, and analyzed for DNA and protein contents. Left and/or right lung hypoplasia was estimated by assessment of the ratios of lung to body weight and left to right lung weight. Fetuses were assigned to 5 experimental groups: baseline (exposed neither to nitrofen nor vitamin A), nitrofen (exposed to nitrofen without CDH), CDH (exposed to nitrofen with CDH), nitr+vitA (exposed to nitrofen without CDH and treated with vitamin A), and CDH+vitA (exposed to nitrofen with CDH and treated with vitamin A).

Results

Incidence of hernia was significantly reduced in fetuses treated with vitamin A. When vitamin A was administered at E9.5, the authors observed similar effect on lung hypoplasia measured through ratio of lung to body weight at E18 in the nitrofen and CDH groups (nitrofen 1.92% ± 0.05%, CDH 1.92% ± 0.04%), whereas lung hypoplasia was attenuated relative to baseline (2.45% ± 0.05%) in 5% and 4% in nitrofen (nitr+vitA 2.05% ± 0.03%) and CDH (CDH+vitA 2.08% ± 0.04%) groups, respectively. At E20, lung hypoplasia was increased in CDH compared with nitrofen groups (nitrofen 2.52% ± 0.1%, CDH 2.39% ± 0.05%), whereas vitamin A attenuated lung hypoplasia, in relation to baseline (3.20% ± 0.07%), 14% in both nitrofen-exposed groups (nitr+vitA 2.96% ± 0.03%, CDH+vitA 2.83% ± 0.03%). At E22, lung hypoplasia was significantly higher in CDH group than nitrofen group (nitrofen 2.13% ± 0.06%, CDH 1.48% ± 0.03%), whereas lung hypoplasia was attenuated in 9% of both nitrofen-exposed groups (nitr+vitA 2.35% ± 0.06%, CDH+vitA 1.69% ± 0.05%) in relation to baseline group (2.38% ± 0.04%). Administration of vitamin A at E18.5 produced no significant effects on lung growth.

Conclusions

The authors conclude from these results that antenatal administration of vitamin A attenuates lung hypoplasia in CDH by interfering with early determinants of lung underdevelopment. This finding may have clinical implications because prenatal diagnosis of human CDH commonly occurs after 16 weeks' gestation when late determinants of lung hypoplasia likely predominate.     

Abnormal platelet-derived growth factor signaling accounting for lung hypoplasia in experimental congenital diaphragmatic hernia

Purpose

The pathogenesis of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) is not fully understood. Platelet-derived growth factor A (PDGFA) and platelet-derived growth factor receptor α (PDGFRα) play a crucial role in lung development. It has been reported that PDGF induces H₂O₂-production and that oxidative stress may be an important mechanism for the impaired lung development in the nitrofen rat model. We hypothesized that pulmonary expression of PDGFA and PDGFRα is altered in the nitrofen induced CDH model.

Materials and Methods

Pregnant rats received 100 mg nitrofen or vehicle on gestational day 9 (D9) and were sacrificed on D15, D18 or D21. RNA was extracted from fetal left lungs and mRNA levels of PDGFA and PDGFRα were determined using real-time polymerase chain reaction. Immunohistochemistry for protein expression of PDGFA and PDGFRα was performed. Pulmonary H₂O₂ was measured colorimetrically.

Results

mRNA levels of PDGFRα at D15 (4.50 ± 0.87) and PDGFA at D18 (2.90 ± 1.38) were increased in the nitrofen group (P < .05). Immunohistochemistry revealed increased pulmonary expression of PDGFRα and PDGFA. H₂O₂ content was significantly higher in the nitrofen group.

Conclusions

Increased expression of PDGFA and PDGFRα suggests that pulmonary hypoplasia in the nitrofen CDH model may be owing to PDGF-induced oxidative stress during lung development.     

Upregulation of serotonin-receptor-2a and serotonin transporter expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia

Purpose

Congenital diaphragmatic hernia (CDH) is attributed to severe pulmonary hypoplasia and pulmonary hypertension (PH). PH is characterized by structural changes resulting in vascular remodeling. Serotonin, a potent vasoconstrictor, plays a central role in the development of PH. It exerts its constricting effects on the vessels via Serotonin receptor 2A (5-HT_2A) and induces pulmonary smooth muscle cell proliferation via the serotonin transporter (5-HTT). This study was designed to investigate expressions of 5-HT_2A and 5-HTT in the pulmonary vasculature of rats with nitrofen-induced CDH.

Methods

Rats were exposed to nitrofen or vehicle on D9. Fetuses were sacrificed on D21 and divided into nitrofen and control group (n = 32). Pulmonary RNA was extracted and mRNA level of 5HT_2A was determined by qRT-PCR. Protein expression of 5HT_2A and 5-HTT was investigated by western blotting. Confocal immunofluorescence double-staining for 5-HT_2A, 5-HTT, and alpha smooth muscle actin were performed.

Results

Pulmonary 5-HT_2A gene expression levels were significantly increased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy confirmed increased pulmonary protein expression in CDH lungs compared to controls.

Conclusion

Increased gene and protein expression of 5HT_2A and 5-HTT in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that 5HT_2A and 5-HTT are important mediators of PH in nitrofen-induced CDH.     

VEGF expression is downregulated in nitrofen-induced congenital diaphragmatic hernia

Background

Vascular endothelial growth factor (VEGF) is upregulated in pulmonary alveolarization. However, developmental expression of pulmonary VEGF and its possible role in the pathogenesis of CDH are not well described.

Methods

Timed-pregnant VEGF-LacZ mice, possessing a β-galactosidase reporter introduced into the 3′ region of the VEGF gene, were used to examine fetal lung gene expression in a model of nitrofen-induced CDH.

Results

VEGF gene expression increased from embryonic day 13 until its peak at embryonic day 16 and then decreased until term in all groups. This pattern was most apparent in the periphery with smaller differences noted in central lung locations. Expression of VEGF/β-gal in the lungs of nitrofen-treated mice was less than controls at all time-points (P < .0001) The type-II pneumocyte population did not significantly differ between the groups. Study concentrations of nitrofen showed no effect on vascular endothelial proliferation in vitro.

Conclusions

Nitrofen downregulates the production of VEGF during gestation and attenuates the peak seen at the onset of the canalicular stage, despite preservation of type-II pneumocytes. This effect was most pronounced in peripheral lung tissue. The authors speculate that altered VEGF expression may have a pivotal role in the pathogenesis of nitrofen-induced CDH.     

Effect of epidermal growth factor on pulmonary hypoplasia in experimental diaphragmatic hernia

Background/purpose

Currently, tracheal occlusion (TO) is a potent stimulus for fetal lung growth but also a rather invasive and high-risk procedure. The aim of this study was to investigate a new and much less invasive therapeutic strategy, namely the maternal intraperitoneal administration of epidermal growth factor (EGF) and its effect on pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) rat model, especially its effect on type II pneumocytes.

Methods

CDH was induced by maternal administration of a single oral dose (100 mg) of nitrofen on day 8.5 of pregnancy. Four groups of pregnant rats were designed on day 18.5: normal control (n = 4), CDH (n = 4), CDH plus Dex (n = 4), CDH plus EGF (n = 8). All fetuses were delivered by cesarean section on day 21. Accordingly, there were 4 groups of fetuses: normal controls (n = 33), nitrofen-induced CDH (n = 19), CDH plus Dex treatment (n = 15), and CDH plus EGF treatment (n = 24). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic and morphometric evaluations were performed, and image analysis was combined after lung processing. Transmission electron microscopy was used for ultrastructural observation, especially type II pneumocytes.

Results

CDH was observed in 58 of the 94 rat fetuses (61.7%). Lw/Bw of CDH group was significantly lower than those of Dex and EGF (P < .05). The lungs of CDH fetuses showed marked hypoplasia, in contrast to improved mesenchymal differentiation in that of Dex and EGF fetuses. Statistical differences of these morphologic parameters (RAC, MTBD, interstitial%, and alveoli%) were found (P < .05). As to ultrastructural features, type II cells of CDH lungs had few if any lamellar bodies and cytoplasmic organelles, and showed evidence of abundant glycogen granules. The sparse type II cells also showed cytoplasmic degenerative changes. By contrast, type II cells of EGF lungs showed numerous mitochondria, abundant lamellar bodies (surfactant) and deficiency of glycogen granules, and displayed prominent microvillous projections and pitlike depressions. The density of type II pneumocyte were 65 ± 4.5, 31 ± 3.1, and 8 ± 1.5 for EGF, Dex, and CDH, respectively (EGF v Dex, P < .05; EGF v CDH, P < 0.01).

Conclusions

Compared with TO, prenatal EGF administration as a much less-invasive therapeutic strategy had shown marked improvement in pulmonary hypoplasia and promotion of type II pneumocyte differentiation in the nitrofen-induced CDH rat model. Thus, EGF could improve the prognosis of CDH by means of promoting pulmonary hypoplasia and improving the surfactant deficiency, which suggested a potential role in the clinical treatment of CDH.     

Kidney development in the nitrofen-induced pulmonary hypoplasia and congenital diaphragmatic hernia in rats

Background/Purpose

The relationship of the developing lung and kidney is not completely understood. Renal enlargement has been reported in association with pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Recent studies suggest that retinoids may be involved in the pathogenesis of CDH. The aims of this study were to investigate the effects of pulmonary hypoplasia on renal development and to evaluate retinoids status of kidneys in the nitrofen model of CDH.

Methods

Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 of gestation. Fetuses were recovered at term and divided into 3 groups: 1, control (n = 69); 2, nitrofen without CDH (n = 25); and 3, nitrofen with CDH (n = 40). Kidneys were dissected, weighed, and processed for biochemical measurements of DNA, proteins, total retinol content, and for immunohistochemical staining of proliferating cells.

Results

Kidneys were smaller in nitrofen-exposed animals vs control animals (group 3, 0.65 ± 0.08; group 2, 0.62 ± 0.09 vs group 1, 0.73 ± 0.09% of body weight, P < .001), and there were no differences between right and left kidney weight in all the 3 groups. Regression of total kidney weight on body weight showed a linear direct correlation between them in all the groups. Total amount of DNA was significantly reduced in nitrofen-exposed animals vs controls (group 3, 80.58 ± 35.65; group 2, 64.71 ± 20.28 vs group 1, 110.34 ± 42.15 μg, P < .01), but the DNA concentration remained the same in the 3 groups (group 3, 3.59 ± 1.26; group 2, 3.06 ± 1.19; group 1, 3.43 ± 1.05 μg DNA/mg kidney). Total protein content (group 3, 1145.59 ± 500.36; group 2, 993.2 ± 276.62; group 1, 1287.48 ± 312.52 μg), protein concentration (group 3, 49.76 ± 11.12; group 2, 43.95 ± 6.79; group 1, 47.38 ± 6.93 μg protein/mg kidney), and protein-to-DNA ratio (group 3, 15.12 ± 5.98; group 2, 16.22 ± 6.85; group I, 16.16 ± 7.02 μg/μg) were similar in all groups. Retinol concentration was significantly reduced in both nitrofen-exposed groups compared with the control group (group 3, 1.35 ± 0.24; group 2, 1.28 ± 0.11; group 1, 2.53±0.61 μg retinol/g kidney). Proliferation index was similar in all 3 groups (group 3, 50.43 ± 8.81; group 2, 47.96 ± 6.01; group 1, 47.64 ± 5.76% of proliferating cells).

Conclusions

Our data clearly show that renal enlargement in association with pulmonary hypoplasia is not seen in the nitrofen-induced CDH. These results rule out any possible relationship between lung and kidney development. Moreover, kidneys are hypoplastic in both nitrofen-exposed groups and have reduced retinol content, suggesting that a retinoid pathway disruption could be the common mechanism in the pathogenesis of lung and kidney hypoplasia in the nitrofen model of CDH.