title: eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage.
abstract: OBJECTIVE
  In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O(2)(-)), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.
  
  
  METHODS
  Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.
  
  
  RESULTS
  In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH₄) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.
  
  
  CONCLUSIONS
  In hyperoxia, reduced BH₄ bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia.
authors.affiliation: Centre for Vision and Vascular Science, Queen's University Belfast, United Kingdom.
authors.forename: Kevin
authors.initials: K
authors.lastname: Edgar
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Gardiner
authors.forename: Rien
authors.initials: R
authors.lastname: van Haperen
authors.forename: Rini
authors.initials: R
authors.lastname: de Crom
authors.forename: Denise M
authors.initials: DM
authors.lastname: McDonald
mesh_terms: D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D052250:Nitric Oxide Synthase Type III / Q000096:biosynthesis*; D015861:Retinal Neovascularization / Q000201:enzymology* / Q000473:pathology; D012171:Retinal Vessels / Q000201:enzymology* / Q000473:pathology
chemical_list: D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse