Therefore, on embryonic day (E) 9.5, nitrofen (100mg) or vehicle was administered to pregnant Sprague-Dawley rats. In the 1

SUMMARY

Congenital diaphragmatic hernia (CDH) is characterized by a lack of closure of the diaphragm leading to the herniation of the abdominal viscera into the thorax. This compromises lung development in utero. CDH is a cause of severe respiratory distress in the newborn, mainly due to pulmonary hypoplasia and pulmonary vascular remodeling leading to persistent pulmonary hypertension of the newborn (PPHN). These two factors mainly limit the prognosis and contribute to the high mortality rate in the newborn (30-40%). The pathobiology of pulmonary hypoplasia and pulmonary vascular remodeling in CDH remain largely unknown. CDH can be reproduced experimentally by maternal nitrofen administration in pregnant rats. This experimental model particularly recapitulates pathological features of pulmonary vascular remodelling observed in human CDH.

In this context and because bone morphogenetic proteins (BMP) have been shown to play crucial roles in the pathogenesis of pulmonary vascular remodeling in pulmonary hypertension (PH), we explored the BMP signaling in nitrofen-induced congenital diaphragmatic hernia (CDH).Moreover, we also tested the efficacy of antenatal simvastatin (compared to antenatal sildenafil) to prevent the development of pathological features of PPHN in nitrofen-induced CDH.

Therefore, on embryonic day (E) 9.5, nitrofen (100mg) or vehicle was administered to pregnant Sprague-Dawley rats. In the 1

part of the study, foetuses were delivered, on E17 and E21, by caesarean section, killed, checked for left-sided CDH and the tissue was harvested for pathobiological evaluation. In the 2

part of the study, nitrofen-treated rats were randomly assigned to antenatal simvastatin (20 mg·kg-1·day-1 orally), antenatal sildenafil (100 mg·kg-1·day-1 orally), or placebo administration from E11 to E21.Foetuses were then delivered by caesarean section, killed, checked for left-sided CDH and the tissue was harvested for pathobiological evaluation.

In the first part of our work, we showed that fetuses suffering from nitrofen-induced CDH presented with:

- pulmonary hypoplasia characterized by pulmonary immaturity, with a decreased lung weight to the total body weight ratio, a reduced radial alveolar count and a thickening of alveolar septa;

- pulmonary vascular remodeling, characterized by an increased pulmonary arteriolar medial thickness, with a decrease in size and in number of pulmonary arteries;

- decreased pulmonary expression of different members of the bone morphogenetic protein (BMP) and type 2 receptor, the BMPR2 signaling pathway, characterized by decreased expressions of BMPR2 and BMP agonists (including BMP4 in embryos of 17 and 21 days and BMP7 in embryos of 21 days of gestation), associated with an increased BMP antagonist (gremlin) expression;

- decreased pulmonary activation (by phosphorylation) of Smad1/5/8 signaling pathway and decreased pulmonary expression of the inhibitor of the DNA binding protein 1 (ID1), a target gene of BMP/BMPR2 pathway in embryos of 21 days, confirming a reduced activation of the BMP /BMPR2 signaling pathway. Moreover, there was a correlation between pulmonary expression of Id1 and of BMP4 and BMPR2. In the lungs, the expression of Id1 was inversely correlated to gremlin expression, suggesting reduced BMP/BMPR2 activation in the lungs of embryos with nitrofen-CDH;

- decreased activation of the apoptotic processes with a decreased expression of the

pro-apoptotic Bax/Bcl-2 ratio in the lungs.

They also presented with heart hypoplasia with a decreased heart weight to the total body weight ratio; decreased myocardial expression of various members of the BMP/BMPR2 signaling, characterized by decreased myocardial expressions of BMPR2 and BMPR1A receptors, of Id1 and BMP7 agonist, associated with an increased myocardial expression of BMP antagonists, gremlin-1 and noggin in embryos of 17 and 21 days, suggesting a decreased activation of the BMP/BMPR2 signaling in the hearts of embryos suffering from CDH. Myocardial expression of Ca

ATPase of the endoplasmic reticulum (SERCA-2A), a protein involved in the calcium-dependent relaxation cycle contraction was decreased in the embryos of 17 days. Moreover, a correlation was observed

between myocardial expressions BMPR2 and BMPR1A and myocardial expression of SERCA-2A. There was an inverse correlation between the expression of myocardial BMPR2 and BMPR1A and the pro- apoptotic Bax-to-Bcl-2 ratio, suggesting a potential link between the alteration of the BMP/BMPR2 signaling and the impaired myocardial contraction Ca

-dependent, as well as the activation of the apoptotic process in the heart level (Makanga M et al. Down regulated bone morphogenetic protein signaling in nitrofen-induced congenital diaphragmatic hernia. Pediatr Int Surg 2013; 29: 823-834).

In the second part of our work, we showed that antenatal administration of simvastatin neither allows to reduce the incidence of nitrofen-induced CDH in fetal rat pups; neither to increase the total weight of the fetus. In fetuses with nitrofen-induced CDH, antenatal simvastatin administration:

- improved the pathological features of pulmonary hypoplasia and persistent pulmonary hypertension;

- improved lung weight to the total body weight ratio of pups and lung parenchyma structure (radial alveolar count increased with a decreased thickness of the alveolar septa);

- restored the pulmonary vascular density and the external diameter of the pulmonary arteries;

- reduced the pulmonary arterial remodeling (by decreasing the thickness of the media of small pulmonary arteries, ≤ 25 microM), and increased the external diameter of the pulmonary arteries;

- restored the expression of various members of the BMP/BMPR2 signaling, characterized by an increased gene expression of BMPR2, of the inhibitor of the binding protein to the DNA (ID1), a BMP signalling target gene and a decreased gremlin-1 expression;

- restored pulmonary expressions of endothelin precursor (the preproendothelin-1), the endothelin converting enzyme 1 (ECE-1) and the protein expression of endothelin-1, together with restored expressions of type A and B receptors of endothelin, endothelial and inducible nitric oxide synthase and pulmonary levels of Nox representing nitric oxide;

- restored the activation of apoptotic processes in lung epithelium (restoration of the

percentage of apoptotic nuclei in pulmonary epithelial level evaluated by the

TUNEL method) and of the expression of pro-apoptotic Bax/Bcl-2 ratio.

Antenatal administration of sildenafil showed similar effects on nitrofen-induced CDH than antenatal simvastatin (Makanga et al. Prevention of pulmonary hypoplasia and pulmonary vascular remodeling by antenatal Simvastatin treatment in nitrofen-induced congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 2015; 308: L672- 682).

In conclusion, our study on the pathobiology of nitrofen-induced CDH in rats, showed that the

BMP/BMPR2 signaling was impaired at the pulmonary and myocardial levels in this experimental

model. Simvastatin and sildenafil showed beneficial effects in the lungs: antenatal administration of

simvastatin could prevent the development of pulmonary hypoplasia and pulmonary vascular

remodeling in fetuses with CDH.