Decreased Endoglin expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia rat model.
OBJECTIVE
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 β (TGFβ) signalling pathway, is expressed mainly by endothelial cells and has been found to be involved in angiogenesis and vascular remodelling. Genetic studies have linked TGFβ and Eng mutations to human arterial PH and other cardiovascular syndromes. Eng interacts with the TGFβ receptors 1 and 2 (Tgfβr1, Tgfβr2). 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 Tgfβr1 and Tgfβr2.


METHODS
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, Tgfβr1 and Tgfβr2 were assessed via qRT-PCR and western blotting. Immunofluorescence staining for Eng was combined with CD34 to evaluate Eng expression in the pulmonary vasculature.


RESULTS
Relative mRNA levels of Eng, Tgfβr1 and Tgfβr2 were significantly downregulated in CDH lungs compared to controls (Eng CDH 0.341 ± 0.022, Eng Ctrl 0.471 ± 0.031, p = 0.0015; Tgfβr1 CDH 0.161 ± 0.008, Tgfβr1 Ctrl 0.194 ± 0.01, p = 0.0114; Tgfβr2 CDH 0.896 ± 0.099, Tgfβr2 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.


CONCLUSIONS
This study demonstrates for the first time a reduced expression of Endoglin in the pulmonary vasculature of nitrofen-induced CDH. Abnormal Eng/Tgfβr1/Tgfβr2 signalling may contribute to impaired vascular remodelling and development of PH in this CDH animal model.
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland.
Julia
J
Zimmer
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland.
Toshiaki
T
Takahashi
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland.|Department of Paediatric Surgery, Hannover Medical School, Hannover, Germany.
Alejandro D
AD
Hofmann
National Children's Research Centre, Our Lady's Children's Hospital, Gate 5, Crumlin, Dublin, Ireland. prem.puri@ncrc.ie.|School of Medicine and Medical Science, Conway Institute of Biomedical Research, University College Dublin, Dublin, Ireland. prem.puri@ncrc.ie.
Prem
P
Puri
D000818:Animals; D015153:Blotting, Western; D004195:Disease Models, Animal; D000071063:Endoglin / Q000235:genetics* / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D065630:Hernias, Diaphragmatic, Congenital / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000098:blood supply* / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D010647:Phenyl Ethers; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000235:genetics
D000071063:Endoglin; D010647:Phenyl Ethers; C007350:nitrofen
Congenital diaphragmatic hernia; Endoglin; Nitrofen; Pulmonary hypertension; Tgf beta signalling