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 antenatal tetrandrine administration on endothelin-1 and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia

Purpose

The aim of this study was to evaluate the effect of the traditional Chinese medicine tetrandrine (Tet) and to determine its possible mechanism on expression of endothelin-1 (ET-1) and epidermal growth factor (EGF) in the lung of a rat model of nitrofen-induced congenital diaphragmatic hernia (CDH).

Methods

A single oral dose (115 mg/kg) of nitrofen on day 9.5 of pregnancy was maternally administered to induce CDH. Pregnant rats were divided into 4 groups on day 18.5: control (n = 5), CDH (n = 5), CDH+dexamethasone (Dex) (n = 5), and CDH+Tet (n = 5). All fetuses were delivered by cesarean delivery on day 21.5. Accordingly, there were 4 groups of fetuses: control (n = 38), CDH (n = 25), CDH+Dex (n = 21), and CDH+Tet (n = 22). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic evaluations and ET-1 and EGF immunohistochemistry staining were performed, and image analysis was performed after lung processing.

Results

Five female rats in the control group produced 38 fetuses without CDH. CDH was observed in 68 of the 128 rat fetuses (53.1%) among the other 3 groups. The LW/BW ratio of the CDH group was significantly lower than those of the Dex and EGF groups (P < .05). The lungs of fetuses with CDH showed marked abnormal structure such as pulmonary hypoplasia and vascular remodeling, in contrast to improved pulmonary structure in lungs of fetuses in the CDH+Dex and CDH+Tet groups. Statistical differences in morphologic parameters (radial alveolar counts, percentage of alveoli, percentage of medial wall thickness, and vascular volume) were found (P < .05). The immunoreactivity of EGF and ET-1 in the CDH group was markedly stronger than that in the control, CDH+Dex, and CDH+Tet groups (P < .01). In addition, EGF and ET-1 expression in the CDH+Dex and CDH+Tet groups was stronger than that in the control group (P < .05). There was no difference in lung EGF and ET-1 immunoreactivity between CDH+Dex and CDH+Tet groups (P > .05).

Conclusion

Antenatal treatment with Tet may improve lung growth and vascular remodeling, and its mechanism seems to be involved in decreasing EGF and ET-1 expression. Tet administered maternally may be a hopeful new therapeutic option in the treatment of CDH and may be effective in helping to avoid the side effects of Dex.     

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.     

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.     

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.     

Effect of prenatal tetrandrine administration on transforming growth factor-β1 level in the lung of nitrofen-induced congenital diaphragmatic hernia rat model

Purpose

Tetrandrine (Tet) is a bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra, which has been used in traditional Chinese medicine to treat patients with silicosis, asthma, and pulmonary hypertension, and others and can be used as a pulmonary therapeutic agent. We hypothesized that it can also improve the lung growth in congenital diaphragmatic hernia (CDH) for its multiple biological effects. There are increasing evidences that suggest transforming growth factor β1(TGF-β1) plays a crucial role in fetal lung growth and morphogenesis. The aim of this study was to evaluate the effect of prenatal administration of Tet and to investigate its possible mechanism on the expression of TGF-β1 in the lung of nitrofen-induced CDH rat model.

Methods

A CDH model was induced in pregnant Sprague-Dawley rats by administration of nitrofen on day 9.5 of gestation (Ed9.5 term, day 22). Tetrandrine (30 mg/kg) was given through gavage (once a day, for 3 days) on Ed11.5. Accordingly, there were 3 groups as follows: control (n = 9), CDH (n = 9), and CDH + Tet (n = 9). All the fetuses were delivered by cesarean delivery on Ed16.5, 18.5, and 21.5, respectively, to check if diaphragmatic hernia existed on each fetus, then the lung tissue weight (LW) and body weight (BW) of each fetus were recorded. Histologic evaluations and TGF-β1 immunohistochemistry staining in the lung sample were performed for image analysis.

Results

Diaphragmatic hernia was observed in 95 of the 112 rat fetuses in CDH and CDH + Tet groups on Ed18.5 and Ed21.5 (84.8%), the incidence between the 2 groups had no statistical significance (P = .642). Lung weight/body weight in the CDH group and the CDH + Tet group were lower than that in the control group (P < .01), and LW/BW in the CDH group was lower than that in the CDH + Tet group (P < .05). Observed under the light microscope and electron microscope, marked hypoplasia of the lungs in fetuses among the CDH groups was observed, in contrast to improvement of the lungs in CDH + Tet fetuses. Statistical differences in morphological parameters (percentage of alveoli area, counting bronchus) were found even on Ed16.5 when diaphragm had not closed (P < .01). The number of type II pneumocytes and lamellar bodies in each group had no significant difference (P > .05). The immunoreactivity of TGF-β1 in CDH group and CDH + Tet group were markedly stronger than that in the control group (P < .01). In addition, TGF-β1 expression in the CDH group was stronger than that in the CDH + Tet group (P < .01).

Conclusion

Nitrofen can interfere with lung development early in the fetal rat development before and separate from diaphragm development, and increased expression of TGF-β1 in the lung of CDH rat model may suppress lung growth and development. Prenatal treatment with Tet can improve the growth of the lung of the nitrofen-induced CDH fetuses and its mechanism seems to be involved in downregulating the expression of TGF-β1. It is a likely new approach to treat CDH and its coexistent lung hypoplasia by maternal Tet administration.     

Prenatal treatment with retinoic acid promotes pulmonary alveologenesis in the nitrofen model of congenital diaphragmatic hernia

Background/Purpose

Severe pulmonary hypoplasia remains the main cause of the high mortality in newborn infants with congenital diaphragmatic hernia (CDH). Retinoids are a family of molecules derived from vitamin A, which play an important role in lung development. We hypothesized that retinoids promote alveologenesis at the end of gestation and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs in CDH.

Methods

Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation. Retinoic acid 5 mg/kg was given intraperitoneally on days 18, 19, and 20 of gestation and fetuses were recovered on day 21. We had 4 study groups: control (n = 24), control + retinoic acid (n = 22), CDH (n = 24), and CDH + retinoic acid (n = 19). Lungs from the 4 study groups were fixed, and the following stereological measurements were performed on vertical random sections: total lung volume, volume density of airspaces, volume density of air walls, gas exchange surface area, alveolar volume, and total number of alveoli per lung. Total DNA content of each lung was measured using a spectrophotometer.

Results

Total lung volume increased in CDH lungs after the addition of retinoic acid but remained the same in the control group. Gas exchange surface area was larger in CDH lungs after the addition of retinoic acid but remained unchanged in the control group. The total number of alveoli per lung was higher after the addition of retinoic acid. Total DNA content as well as total DNA content-lung weight ratio of the left lung increased significantly in the CDH group after the addition of retinoic acid compared with CDH without retinoic acid.

Conclusions

Our results demonstrate that prenatal treatment with retinoic acid stimulates alveologenesis in hypoplastic lungs in CDH.     

Effect of VEGF on the branching morphogenesis of normal and nitrofen-induced hypoplastic fetal rat lung explants

Purpose

Changes in vascular structures as well as vascular endothelial growth factor (VEGF) downregulation have been reported in hypoplastic lungs associated with congenital diaphragmatic hernia. We hypothesized that VEGF may accelerate branching morphogenesis and thus may modulate lung growth in normal and nitrofen-induced pulmonary hypoplastic lungs.

Methods

A hypoplastic fetal lung model and a normal control lung model were induced by feeding pregnant rats with or without nitrofen, respectively. Fetal lungs harvested on day 13.5 were cultured at ambient oxygen tensions for 72 hours with 0, 25, 50, or 100 ng/mL of exogenous rat VEGF added daily in the serum-free medium. The rates of increase in bud count and airway contour were evaluated. Real-time polymerase chain reaction was carried out to evaluate the expression of surfactant protein C mRNA in the explants at the end of culture.

Results

Vascular endothelial growth factor accelerated the increase in bud count and airway contour in normal and hypoplastic lung explants compared to controls. Surfactant protein C mRNA expression was significantly increased at 50 ng/mL VEGF compared to controls in both normal and hypoplastic lung explants.

Conclusion

These data suggest that VEGF plays an important role in lung morphogenesis and may accelerate lung growth in nitrofen-induced hypoplastic lung.