title: Down-regulation of N-deacetylase-N-sulfotransferase-1 signaling in the developing diaphragmatic vasculature of nitrofen-induced congenital diaphragmatic hernia.
abstract: BACKGROUND
  Congenital diaphragmatic hernia (CDH) has been attributed to various developmental abnormalities of the underlying tissue components. N-deacetylase-N-sulfotransferase-1 (Ndst1) is a strongly expressed biosynthetic enzyme in endothelial cells, which has recently been identified as an important factor during diaphragmatic vascularization. Loss of endothelial Ndst1 has been demonstrated to cause angiogenic defects in the developing diaphragm and disrupt normal diaphragmatic development. Furthermore, deficiency of Ndst1 diminishes the expression of slit homolog 3 (Slit3), a known CDH-related gene that has been associated with reduced vascular density and muscle defects in the diaphragm of Slit3-/-mice. We hypothesized that expression of Ndst1 and Slit3 is decreased in the diaphragmatic vasculature of fetal rats with nitrofen-induced CDH.
  
  
  METHODS
  Time-mated rats received either nitrofen or vehicle on gestational day 9 (D9). Fetal diaphragms were microdissected on D13, D15 and D18, and divided into control and nitrofen-exposed specimens. Gene expression levels of Ndst1 and Slit3 were assessed using qRT-PCR. Immunofluorescence-double-staining for Ndst1 and Slit3 was performed to evaluate protein expression and localization.
  
  
  RESULTS
  Relative mRNA expression of Ndst1 and Slit3 was significantly decreased in pleuroperitoneal folds (D13), developing diaphragms (D15) and fully muscularized diaphragms (D18) of nitrofen-exposed fetuses compared to controls. Confocal-laser-scanning-microscopy revealed markedly diminished Ndst1 and Slit3 expression in endothelial cells within the diaphragmatic vasculature on D13, D15 and D18 compared to controls.
  
  
  CONCLUSIONS
  Down-regulation of Ndst1 signaling in the developing diaphragm may impair endothelial cell migration and angiogenesis, thus leading to defective diaphragmatic vascular development and CDH.
  
  
  METHODS
  Ib.
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Department of Paediatric Surgery, King's College Hospital, Denmark Hill, London, UK.
authors.forename: Florian
authors.initials: F
authors.lastname: Friedmacher
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland.
authors.forename: Julia
authors.initials: J
authors.lastname: Zimmer
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland. Electronic address: prem.puri@ncrc.ie.
authors.forename: Prem
authors.initials: P
authors.lastname: Puri
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D003964:Diaphragm / Q000098:blood supply / Q000196:embryology / Q000201:enzymology*; D015536:Down-Regulation; D042783:Endothelial Cells / Q000201:enzymology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D065630:Hernias, Diaphragmatic, Congenital / Q000139:chemically induced / Q000196:embryology / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D010647:Phenyl Ethers; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism
chemical_list: D015415:Biomarkers; D005819:Genetic Markers; D008565:Membrane Proteins; D010647:Phenyl Ethers; C498991:Slit3 protein, rat; D015238:Sulfotransferases; C021350:heparitin sulfotransferase; C007350:nitrofen
keywords: Congenital diaphragmatic hernia; Diaphragm; N-deacetylase-N-sulfotransferase-1; Nitrofen; Slit homolog 3; Vasculature