Decreased Endoglin expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia rat model

Pulmonary hypertension (PH) remains a therapeutical challenge in neonates born with congenital diaphragmatic hernia (CDH). Endoglin (Eng), an auxiliary receptor component of the transforming growth factor beta (TGF beta) signalling pathway, is expressed mainly by endothelial cells and has been found to be involved in angiogenesis and vascular remodelling. Genetic studies have linked TGF beta and Eng mutations to human arterial PH and other cardiovascular syndromes. Eng interacts with the TGF beta receptors 1 and 2 (Tgf beta r1, Tgf beta r2). We designed this study to investigate the hypothesis that Eng is altered in the pulmonary vasculature of rats with nitrofen-induced CDH subjected to its interdependency with Tgf beta r1 and Tgf beta r2. After ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received either nitrofen or olive oil on gestational day (D9). The foetuses (n = 22) were sacrificed and divided into CDH and control group on D21. Gene and protein expressions of Eng, Tgf beta r1 and Tgf beta r2 were assessed via qRT-PCR and western blotting. Immunofluorescence staining for Eng was combined with CD34 to evaluate Eng expression in the pulmonary vasculature. Relative mRNA levels of Eng, Tgf beta r1 and Tgf beta r2 were significantly downregulated in CDH lungs compared to controls (Eng CDH 0.341 +/- 0.022, Eng Ctrl 0.471 +/- 0.031, p = 0.0015; Tgf beta r1 CDH 0.161 +/- 0.008, Tgf beta r1 Ctrl 0.194 +/- 0.01, p = 0.0114; Tgf beta r2 CDH 0.896 +/- 0.099, Tgf beta r2 Ctrl 1.379 +/- 0.081, p = 0.0006) Western blotting confirmed the reduced pulmonary protein expression of these three proteins in the CDH lungs. A markedly diminished endothelial expression of Eng in the pulmonary vasculature of nitrofen-exposed foetuses compared to controls was seen in laser scanning confocal-microscopy. This study demonstrates for the first time a reduced expression of Endoglin in the pulmonary vasculature of nitrofen-induced CDH. Abnormal Eng/Tgf beta r1/Tgf beta r2 signalling may contribute to impaired vascular remodelling and development of PH in this CDH animal model.