title: Prenatal administration of all-trans retinoic acid upregulates leptin signaling in hypoplastic rat lungs with experimental congenital diaphragmatic hernia.
abstract: OBJECTIVE
  Pulmonary hypoplasia (PH), characterized by alveolar immaturity, is one of the leading causes of respiratory insufficiency in newborns with congenital diaphragmatic hernia (CDH). Leptin (Lep) and its receptor (Lep-R) play an important role in fetal lung growth by stimulating alveolar differentiation and maturation. Lep and Lep-R are strongly expressed by alveolar cells during the saccular stage of fetal lung development. Lep-deficient mice exhibit decreased alveolarization with reduced pulmonary surfactant phospholipid synthesis, similar to human and nitrofen-induced PH. Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced PH. Recent studies have demonstrated that Lep and Lep-R expression in developing lungs is regulated by ATRA. We hypothesized that prenatal treatment with ATRA increases pulmonary Lep and Lep-R expression in the nitrofen model of CDH-associated PH.
  
  
  METHODS
  Time-mated rats received either 100 mg nitrofen or vehicle via oral-gastric lavage on embryonic day 9.5 (E9.5). Control and nitrofen-exposed dams were randomly assigned to either intraperitoneal ATRA (5 mg/kg/d) or placebo administration on E18.5, E19.5 and E20.5. Fetal lungs were harvested on E21.5, and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo and Nitrofen+ATRA. Alveolarization was assessed using stereo- and morphometric analysis techniques. Surfactant phospholipid synthesis was analyzed by labeling for surfactant protein B (SP-B). Pulmonary gene expression levels of Lep and Lep-R were determined using quantitative real-time polymerase chain reaction. Immunohistochemical staining for Lep and Lep-R was performed to evaluate alveolar protein expression and localization.
  
  
  RESULTS
  In vivo administration of ATRA resulted in significantly increased lung-to-body weight ratio with enhanced radial alveolar count and decreased mean linear intercept compared to placebo treatment. Immunofluorescence analysis demonstrated markedly increased pulmonary SP-B expression in Nitrofen+ATRA compared to Nitrofen+Placebo. Relative mRNA expression of Lep and Lep-R was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo. Lep and Lep-R immunoreactivity was markedly increased in interstitial and alveolar epithelial cells of Nitrofen+ATRA compared to Nitrofen+Placebo.
  
  
  CONCLUSIONS
  Increased Lep and Lep-R expression after prenatal administration of ATRA in nitrofen-induced PH suggests that ATRA may have therapeutic potential in attenuating CDH-associated PH by stimulating alveolarization and de novo surfactant production.
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Florian
authors.initials: F
authors.lastname: Friedmacher
authors.forename: Alejandro Daniel
authors.initials: AD
authors.lastname: Hofmann
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Hiromizu
authors.initials: H
authors.lastname: Takahashi
authors.forename: Balazs
authors.initials: B
authors.lastname: Kutasy
authors.forename: Prem
authors.initials: P
authors.lastname: Puri
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D065630:Hernias, Diaphragmatic, Congenital / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D007150:Immunohistochemistry; D020738:Leptin / Q000096:biosynthesis / Q000235:genetics*; D008168:Lung / Q000196:embryology* / Q000378:metabolism; D038081:Organogenesis / Q000187:drug effects; D011247:Pregnancy; D011270:Pregnancy, Animal; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction; D014212:Tretinoin / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D020738:Leptin; D012333:RNA, Messenger; D014212:Tretinoin