title: Heparin binding VEGF isoforms attenuate hyperoxic embryonic lung growth retardation via a FLK1-neuropilin-1-PKC dependent pathway.
abstract: BACKGROUND
  Previous work in our laboratory demonstrated that hyperoxia suppressed the expression of vascular endothelial growth factor (VEGF) by the embryonic lung, leading to increased epithelial cell apoptosis and failure of explant airway growth and branching that was rescued by the addition of Vegf165. The aims of this study were to determine protective pathways by which VEGF isoforms attenuate hyperoxic lung growth retardation and to identify the target cell for VEGF action.
  
  
  METHODS
  Timed pregnant CD-1 or fetal liver kinase (FLK1)-eGFP lung explants cultured in 3% or 50% oxygen were treated ± Vegf121, VEGF164/Vegf165 or VEGF188 in the presence or absence of anti-rat neuropilin-1 (NRP1) antibody or GO6983 (protein kinase C (PKC) pan-inhibitor) and lung growth and branching quantified. Immunofluorescence studies were performed to determine apoptosis index and location of FLK1 phosphorylation and western blot studies of lung explants were performed to define the signaling pathways that mediate the protective effects of VEGF.
  
  
  RESULTS
  Heparin-binding VEGF isoforms (VEGF164/Vegf165 and VEGF188) but not Vegf121 selectively reduced epithelial apoptosis and partially rescued lung bud branching and growth. These protective effects required NRP1-dependent FLK1 activation in endothelial cells. Analysis of downstream signaling pathways demonstrated that the VEGF-mediated anti-apoptotic effects were dependent on PKC activation.
  
  
  CONCLUSIONS
  Vegf165 activates FLK1-NRP1 signaling in endothelial cells, leading to a PKC-dependent paracrine signal that in turn inhibits epithelial cell apoptosis.
authors.affiliation: Department of Pediatrics Section of Respiratory Medicine, Yale University School of Medicine, 333 Cedar Street, P,O, Box 208064, New Haven, CT 06520, USA. americo.esquibies@yale.edu.
authors.forename: Americo E
authors.initials: AE
authors.lastname: Esquibies
authors.forename: Anil
authors.initials: A
authors.lastname: Karihaloo
authors.forename: Susan E
authors.initials: SE
authors.lastname: Quaggin
authors.forename: Alia
authors.initials: A
authors.lastname: Bazzy-Asaad
authors.forename: Lloyd G
authors.initials: LG
authors.lastname: Cantley
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D005260:Female; D006493:Heparin / Q000378:metabolism*; D008168:Lung / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D039942:Neuropilin-1 / Q000502:physiology*; D009924:Organ Culture Techniques; D011247:Pregnancy; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D011897:Random Allocation; D015398:Signal Transduction / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000502:physiology*
chemical_list: D020033:Protein Isoforms; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D039942:Neuropilin-1; D006493:Heparin; D040301:Vascular Endothelial Growth Factor Receptor-2; D011493:Protein Kinase C