title: Peroxynitrite upregulates angiogenic factors VEGF-A, BFGF, and HIF-1α in human corneal limbal epithelial cells.
abstract: OBJECTIVE
  Corneal neovascularization (NV) is a sight-threatening condition often associated with infection, inflammation, prolonged contact lens use, corneal burns, and acute corneal graft rejection. Macrophages recruited to the cornea release nitric oxide (NO) and superoxide anion (O2(-)), which react together to form the highly toxic molecule peroxynitrite (ONOO(-)). The role of ONOO(-) in upregulating multiple angiogenic factors in cultured human corneal limbal epithelial (HCLE) cells was investigated.
  
  
  METHODS
  Human corneal limbal epithelial cells were incubated with 500 μM of ONOO(-) donor for various times. VEGF-A, BFGF, and hypoxic-inducible factor-alpha (HIF-1α) were investigated via Western blot and RT-PCR was performed for VEGF. Functional assays using human umbilical vein endothelial cells (HUVEC) used conditioned media from ONOO(-)-exposed HCLE cells. Secreted VEGF from conditioned media was detected and analyzed using ELISA.
  
  
  RESULTS
  Increased angiogenic factors were observed as early as 4 hours after HCLE exposure to ONOO(-). HIF-1 expression was seen at 4, 6, and 8 hours post-ONOO(-) exposure (P < 0.05). BFGF expression was elevated at 4 hours and peaked at 8 hours after treatment with ONOO(-) (P < 0.005). Increased VEGF-A gene expression was observed at 6 and 8 hours post-ONOO(-) treatment. Functional assays using conditioned media showed increased HUVEC migration and tube formation.
  
  
  CONCLUSIONS
  Exposure to elevated extracellular concentrations of ONOO(-) results in upregulation of angiogenic factors in HCLE cells. It is possible that, in the setting of inflammation or infection, that exposure to ONOO(-) could be one contributor to the complex initiators of corneal NV. Validation in vivo would identify an additional potential control point for corneal NV.
authors.affiliation: Department of Ophthalmology, Jules Stein Eye Institute, University of California Los Angeles, Los Angeles, California.
authors.forename: Negin
authors.initials: N
authors.lastname: Ashki
authors.forename: Ann M
authors.initials: AM
authors.lastname: Chan
authors.forename: Yu
authors.initials: Y
authors.lastname: Qin
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Meagan
authors.initials: M
authors.lastname: Kiyohara
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Jonathan
authors.initials: J
authors.lastname: Braun
authors.forename: Madhuri
authors.initials: M
authors.lastname: Wadehra
authors.forename: Lynn K
authors.initials: LK
authors.lastname: Gordon
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D016510:Corneal Neovascularization / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000235:genetics*; D005434:Flow Cytometry; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis / Q000235:genetics*; D016850:Limbus Corneae / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D030421:Peroxynitrous Acid; D012313:RNA
keywords: Oxi; corneal epithelium; epithelial cells; neovascularization