Vitamin A improves Pax3 expression that is decreased in the heart of rats with experimental diaphragmatic hernia

Background/Aims

Rats with nitrofen-induced congenital diaphragmatic hernia (CDH) have hypoplasia and malformations of the heart. The mechanism of action of nitrofen involves changes in neural crest signaling. Pax3 function is required for cardiac neural crest cells to complete their migration to the developing heart. Vitamin A improves heart hypoplasia. The aims of this study were to examine whether Pax3 expression is decreased in the heart of E13 E15 and E21 rats exposed to nitrofen and if vitamin A reverts this effect.

Material and Methods

Pregnant rats received either 100 mg nitrofen or olive oil on E9.5. Each group was divided into 2 subgroups according to the subsequent treatment with intragastric vitamin A (15000 IU) or vehicle on E10.5 to E11.5. The pups were recovered on E13, E15 and E21 and the hearts were dissected out. Pax3 mRNA expression was determined by quantitative real time PCR. Comparisons among groups were made with ANOVA and Bonferroni post hoc tests with a threshold of significance of P < .05.

Results

Pax3 mRNA expression was significantly decreased on E13 and E15 in the hearts of nitrofen-treated embryos and it remained decreased although not significantly on E21. Vitamin A recovered this expression on E13, partially on E15 and above normal levels on E21.

Conclusions

Pax3 is underexpressed in the hearts of nitrofen exposed embryonal rats on days 13th and 15th of gestation and tends to be lower than normal near term. Vitamin A up-regulates this expression on the 3 end points. The mechanism of action of Pax3 should be further investigated because it could be one of the targets for future prenatal transplacental intervention.     

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.     

Fetal heart development in the nitrofen-induced CDH rat model: the role of mechanical and nonmechanical factors

Background/purpose

In congenital diaphragmatic hernia (CDH), it was recently shown that early and late gestational lung underdevelopment is caused by nonmechanical and mechanical factors, respectively. Heart underdevelopment, which might predict lung hypoplasia, is commonly attributed to mechanical factors. The authors analyzed whether nonmechanical and mechanical factors affect cardiac growth and correlations between lung and heart weights during gestation.

Methods

Left-sided CDH was induced in pregnant Wistar rats by administration of nitrofen on E9.5. At selected gestational ages (E18, E20, and E22), the lungs and heart were harvested, weighed, and analyzed for DNA and protein contents. Left lung and heart weights were correlated at those gestational ages. Two experimental groups: nitrofen without CDH (nitrofen), and nitrofen with CDH (CDH), were compared with normal controls.

Results

At E18, both nitrofen-exposed groups presented similar and significant left lung (LL) hypoplasia. As gestation progressed (E20 and E22), in the nitrofen group left lung (LL) hypoplasia decreased, whereas in the CDH group LL hypoplasia was exacerbated relative to normal controls. In contrast, at E18 and E20, heart-to-body weight ratios as well as cardiac DNA and protein contents were reduced significantly in all animals exposed to nitrofen, with no significant differences observed between nitrofen and CDH groups. As gestation progressed, the difference between cardiac parameters in nitrofen-exposed and normal control rats diminished, and at E22 no significant differences were documented. In the CDH group, significant correlations were seen between lung and heart weights at E18 (r = 0.65; P < .05) and E20 (r = 0.4; P < .05), whereas at term gestation (E22) no significant correlation was observed (r = 0.21, not significant).

Conclusions

Nonmechanical factors, which might be directed by nitrofen, play a role in the pathogenesis of lung and heart hypoplasia manifested precociously in fetal life, whereas mechanical compression might influence only lung growth during late gestation. Heart weight predicts lung weight only in early gestational ages.     

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.     

Murine nitrofen-induced pulmonary hypoplasia does not involve induction of TGF-beta signaling

Background/Purpose: In the murine nitrofen-induced model of congenital diaphragmatic hernia (CDH), the lungs are primarily hypoplastic and immature even before diaphragmatic closure. Because excess transforming growth factor-[beta ] (TGF-[beta ]) signaling induces pulmonary hypoplasia, the authors hypothesized that primary hypoplasia after nitrofen exposure may be caused by abberant signaling by the TGF-[beta ] pathway. Therefore, abrogation of TGF-[beta ] signaling might rescue the hypoplasia. Methods: The authors performed intratracheal microinjections of a recombinant adenoviral vector encoding a dominant-negative TGF-[beta ] type II receptor (AdIIR-DN) in nitrofen-exposed and control E12 mouse lungs, which then were cultured for 4 days in serumless chemically defined media. The mRNA expression of Smad2, 3, 4, and 7 in nitrofen-exposed and control E12 lungs after 4 days in culture were compared. Results: ADIIR-DN increased terminal branching in control lungs by 28% compared with lungs injected with control virus (61.8 [plusmn] 4.6 v. 48.4 [plusmn] 4.7, P = .004). However, there was no difference between nitrofen-exposed lungs injected with ADIIR-DN and those injected with control virus. Compared with control lungs, Smad mRNA expression was decreased markedly in nitrofen-exposed lungs: Smad2 (40%, P = .16), Smad3 (29%, P = .02), Smad4 (25%, P = .07), and Smad7 (36%, P = .04). Conclusions: Because abrogation of TGF-[beta ] signaling does not rescue the hypoplasia seen in the nitrofen model, and Smad expression is decreased in nitrofen-exposed lungs, the TGF-[beta ] pathway does not appear to play a role in nitrofen-induced pulmonary hypoplasia. J Pediatr Surg 37:1123-1127.