title: Endothelin-1 impairs angiogenesis in vitro through Rho-kinase activation after chronic intrauterine pulmonary hypertension in fetal sheep.
abstract: BACKGROUND
  Endothelin-1 (ET-1) and Rho-kinase (ROCK) increase vascular tone in experimental persistent pulmonary hypertension of the newborn (PPHN). Whether ET-1 activates ROCK to decrease angiogenesis in the developing lung remains unknown.
  
  
  METHODS
  Proximal pulmonary artery endothelial cells (PAECs) were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and controls. Growth and tube formation were assessed after ET-1 treatment. The effect of ET-1 antagonism on tube formation was studied using ET-1 small interfering RNA (siRNA), ET-1 monoclonal antibodies (ET-1mAbs), BQ-123 (an ET(A) blocker), and bosentan (an ET(A)/ET(B) blocker). ET-1 gene and protein and ET(A)/ET(B) receptor protein expression were measured in normal and PPHN PAECs. ET-1-ROCK interactions were assessed by measuring ROCK activity after ET-1, ET-1 siRNA, and bosentan treatments, and tube formation with ET-1 and Y-27632 (ROCK inhibitor).
  
  
  RESULTS
  ET-1 did not affect growth but decreased tube formation in normal and PPHN PAECs. ET-1 protein and gene expression were increased and ET(B) receptor protein decreased in PPHN PAECs. ET-1 siRNA, ET-1mAbs, and bosentan, but not BQ-123, increased tube formation. ROCK activity was increased in PPHN PAECs and decreased with ET-1 siRNA and bosentan treatments. Y-27632 prevented the decrease in tube formation with ET-1.
  
  
  CONCLUSIONS
  ET-1 activation of ROCK impairs angiogenesis of fetal PAECs. Disruption of ET-1-ROCK interactions may increase vascular growth in PPHN.
authors.affiliation: Department of Neonatology, University of Colorado School of Medicine, Aurora, Colorado, USA. jason.gien@ucdenver.edu
authors.forename: Jason
authors.initials: J
authors.lastname: Gien
authors.forename: Nancy
authors.initials: N
authors.lastname: Tseng
authors.forename: Gregory
authors.initials: G
authors.lastname: Seedorf
authors.forename: Gates
authors.initials: G
authors.lastname: Roe
authors.forename: Steven H
authors.initials: SH
authors.lastname: Abman
mesh_terms: D000577:Amides; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004373:Ductus Arteriosus / Q000601:surgery; D042783:Endothelial Cells / Q000187:drug effects; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects*; D004797:Enzyme-Linked Immunosorbent Assay; D005333:Fetus / Q000503:physiopathology; D006801:Humans; D066298:In Vitro Techniques; D007231:Infant, Newborn; D008026:Ligation; D008168:Lung / Q000098:blood supply*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D010547:Persistent Fetal Circulation Syndrome / Q000378:metabolism*; D011725:Pyridines; D034622:RNA Interference; D060888:Real-Time Polymerase Chain Reaction; D012756:Sheep; D054460:rho-Associated Kinases / Q000378:metabolism*
chemical_list: D000577:Amides; D017931:DNA Primers; D019332:Endothelin-1; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases