title: Vascular endothelial growth factor-B gene transfer exacerbates retinal and choroidal neovascularization and vasopermeability without promoting inflammation.
abstract: OBJECTIVE
  The role of vascular endothelial growth factor (VEGF)-B in the eye is poorly understood. The present study was conducted to evaluate the effect of overexpression of VEGF-B via adeno-associated virus (AAV) gene transfer on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB).
  
  
  METHODS
  Three recombinant AAV vectors were prepared, expressing the 167 (AAV-VEGF-B167) or 186 amino acid isoform (AAV-VEGF-B186) of VEGF-B or the green fluorescent protein (GFP) reporter gene (AAV-GFP). Approximately 1 x 10⁹ viral genome copies of AAV-VEGF-B167, AAV-VEGF-B186, or AAV-GFP were intraocularly injected. The efficacy of the gene transfer was assessed by directly observing GFP, by immunohistochemistry, or by real-time PCR. A leukostasis assay using fluorescein isothiocyanate-conjugated Concanavalin A was used to evaluate inflammation. The BRB was assessed using a quantitative assay with ³H-mannitol as a tracer. Retinal neovascularization (NV) was assessed at postnatal day 17 in oxygen-induced ischemic retinopathy after intravitreal injection of AAV-VEGF-B in left eyes and AAV-GFP in right eyes at postnatal day 7. Two weeks after injection of AAV vectors, choroidal NV was generated by laser photocoagulation and assessed 2 weeks later.
  
  
  RESULTS
  GFP expression was clearly demonstrated, primarily in the retinal pigment epithelium (RPE) and outer retina, 1-6 weeks after delivery. mRNA expression levels of VEGF-B167 and VEGF-B186 were 5.8 and 12 fold higher in the AAV-VEGF-B167- and AAV-VEGF-B186-treated groups, respectively. There was no evidence of an inflammatory response or vessel abnormality following injection of the vectors in normal mice; however, VEGF-B increased retinal and choroidal neovascularization. AAV-VEGF-B186, but not AAV-VEGF-B167, enhanced retinal vascular permeability.
  
  
  CONCLUSIONS
  VEGF-B overexpression promoted pathological retinal and choroidal NV and BRB breakdown without causing inflammation, which is associated with the progression of diabetic retinopathy and age-related macular degeneration, showing that these complications are not dependent on inflammation. VEGF-B targeting could benefit antiangiogenic therapy.
authors.affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.
authors.forename: Xiufeng
authors.initials: X
authors.lastname: Zhong
authors.forename: Hu
authors.initials: H
authors.lastname: Huang
authors.forename: Jikui
authors.initials: J
authors.lastname: Shen
authors.forename: Serena
authors.initials: S
authors.lastname: Zacchigna
authors.forename: Lorena
authors.initials: L
authors.lastname: Zentilin
authors.forename: Mauro
authors.initials: M
authors.lastname: Giacca
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Vinores
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000502:physiology*; D020256:Choroidal Neovascularization / Q000150:complications / Q000235:genetics / Q000503:physiopathology*; D000229:Dependovirus / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007249:Inflammation / Q000150:complications* / Q000503:physiopathology; D007511:Ischemia / Q000150:complications / Q000503:physiopathology; D051379:Mice; D008810:Mice, Inbred C57BL; D010100:Oxygen; D011995:Recombination, Genetic / Q000235:genetics; D012160:Retina / Q000473:pathology / Q000503:physiopathology; D015861:Retinal Neovascularization / Q000150:complications / Q000235:genetics / Q000503:physiopathology*; D019076:Transgenes / Q000235:genetics; D042561:Vascular Endothelial Growth Factor B / Q000235:genetics*
chemical_list: D042561:Vascular Endothelial Growth Factor B; D049452:Green Fluorescent Proteins; D010100:Oxygen