title: Decreased apelin and apelin-receptor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.
abstract: BACKGROUND
  The high morbidity and mortality in congenital diaphragmatic hernia (CDH) are attributed to severe pulmonary hypoplasia and persistent pulmonary hypertension (PH). PH is characterized by structural changes in pulmonary arteries, resulting in adventitial and medial thickness. These effects are triggered by abnormal apoptosis and proliferation of pulmonary vascular endothelial and smooth muscle cells (SMCs). Apelin (APLN), a target gene of bone morphogenic protein receptor 2 (BMPR2), is known to play an important and manifold role in regulating pulmonary homeostasis promoting endothelial cell (EC) survival, proliferation and migration. In addition to these autocrine effects of apelin, it displays a paracrine function attenuating the response of pulmonary SMCs to growth factors and promoting apoptosis. Apelin exerts its effect via its G-protein-coupled receptor (APLNR) and is solely expressed by pulmonary vascular EC, whereas APLNR is co-localized in pulmonary ECs and SMCs. Dysfunction of BMPR2 and downstream signalling have been shown to disturb the crucial balance of proliferation of SMCs contributing to the pathogenesis of human and experimentally induced PH. We designed this study to investigate the hypothesis that apelin and APLNR signalling are disrupted in the pulmonary vasculature of rats in nitrofen-induced CDH.
  
  
  METHODS
  Pregnant rats were exposed to nitrofen or vehicle on D9 of gestation. Foetuses were sacrificed on D21 and divided into nitrofen and control group (n = 32). Pulmonary RNA was extracted and mRNA levels of APLN and APLNR were determined by quantitative real-time PCR. Protein expression of apelin and APLNR was investigated by western blotting. Confocal immunofluorescence double staining for apelin, APLNR and SMCs were performed.
  
  
  RESULTS
  Relative mRNA level of APLN and APLNR were significantly decreased in the CDH group compared to control lungs. Western blotting and confocal microscopy confirmed the qRT-PCR results showing decreased pulmonary protein expression of apelin and APLNR in lungs of nitrofen-exposed foetuses compared to controls.
  
  
  CONCLUSIONS
  This study provides striking evidence of markedly decreased gene and protein expression of apelin and its receptor APLNR in the pulmonary vasculature of nitrofen-induced CDH. The disruption of the apelin-APLNR signalling axis in the pulmonary vasculature may lead to extensive vascular remodelling and contribute to PPH in the nitrofen-induced CDH model.
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Alejandro D
authors.initials: AD
authors.lastname: Hofmann
authors.forename: Florian
authors.initials: F
authors.lastname: Friedmacher
authors.forename: Hiromizu
authors.initials: H
authors.lastname: Takahashi
authors.forename: Manuela
authors.initials: M
authors.lastname: Hunziker
authors.forename: Jan-Hendrik
authors.initials: JH
authors.lastname: Gosemann
authors.forename: Prem
authors.initials: P
authors.lastname: Puri
mesh_terms: D000818:Animals; D000073861:Apelin; D000075244:Apelin Receptors; D015153:Blotting, Western / Q000379:methods; D002470:Cell Survival / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression / Q000235:genetics*; D006548:Hernia, Diaphragmatic / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D065630:Hernias, Diaphragmatic, Congenital; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics* / Q000378:metabolism; D008168:Lung / Q000098:blood supply* / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D010647:Phenyl Ethers; D011247:Pregnancy; D011651:Pulmonary Artery / Q000378:metabolism; D011667:Pulmonary Veins / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism
chemical_list: D000073861:Apelin; D000075244:Apelin Receptors; C498579:Apln protein, rat; C491284:Aplnr protein, rat; D036341:Intercellular Signaling Peptides and Proteins; D010647:Phenyl Ethers; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; C007350:nitrofen