VEGF receptor blockade markedly reduces retinal microglia/macrophage infiltration into laser-induced CNV

PLoS One. 2013 Aug 20;8(8):e71808. doi: 10.1371/journal.pone.0071808. eCollection 2013.

Abstract

Although blocking VEGF has a positive effect in wet age-related macular degeneration (AMD), the effect of blocking its receptors remains unclear. This was an investigation of the effect of VEGF receptor (VEGFR) 1 and/or 2 blockade on retinal microglia/macrophage infiltration in laser-induced choroidal neovascularization (CNV), a model of wet AMD. CNV lesions were isolated by laser capture microdissection at 3, 7, and 14 days after laser and analyzed by RT-PCR and immunofluorescence staining for mRNA and protein expression, respectively. Neutralizing antibodies for VEGFR1 or R2 and the microglia inhibitor minocycline were injected intraperitoneally (IP). Anti-CD11b, CD45 and Iba1 antibodies were used to confirm the cell identity of retinal microglia/macrophage, in the RPE/choroidal flat mounts or retinal cross sections. CD11b(+), CD45(+) or Iba1(+) cells were counted. mRNA of VEGFR1 and its three ligands, PlGF, VEGF-A (VEGF) and VEGF-B, were expressed at all stages, but VEGFR2 were detected only in the late stage. PlGF and VEGF proteins were expressed at 3 and 7 days after laser. Anti-VEGFR1 (MF1) delivered IP 3 days after laser inhibited infiltration of leukocyte populations, largely retinal microglia/macrophage to CNV, while anti-VEGFR2 (DC101) had no effect. At 14 days after laser, both MF1 and DC101 antibodies markedly inhibited retinal microglia/macrophage infiltration into CNV. Therefore, VEGFR1 and R2 play differential roles in the pathogenesis of CNV: VEGFR1 plays a dominant role at 3 days after laser; but both receptors play pivotal roles at 14 days after laser. In vivo imaging demonstrated accumulation of GFP-expressing microglia into CNV in both CX3CR1(gfp/gfp) and CX3CR1(gfp/+) mice. Minocycline treatment caused a significant increase in lectin(+) cells in the sub-retinal space anterior to CNV and a decrease in dextran-perfused neovessels compared to controls. Targeting the chemoattractant molecules that regulate trafficking of retinal microglia/macrophage appears to be a compelling therapeutic strategy to control CNV and treat wet AMD.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Murine-Derived / administration & dosage*
  • Cell Movement
  • Choroidal Neovascularization / drug therapy*
  • Choroidal Neovascularization / metabolism
  • Choroidal Neovascularization / pathology
  • Disease Models, Animal
  • Humans
  • Injections, Intraperitoneal
  • Laser Capture Microdissection
  • Lasers
  • Macrophages / drug effects
  • Macrophages / immunology*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Microglia / drug effects
  • Microglia / immunology*
  • Minocycline / administration & dosage
  • Retina / drug effects
  • Retina / immunology*
  • Tissue Culture Techniques
  • Vascular Endothelial Growth Factor Receptor-1 / antagonists & inhibitors*
  • Vascular Endothelial Growth Factor Receptor-1 / metabolism
  • Vascular Endothelial Growth Factor Receptor-2 / antagonists & inhibitors*
  • Vascular Endothelial Growth Factor Receptor-2 / metabolism
  • Wet Macular Degeneration / drug therapy
  • Wet Macular Degeneration / metabolism
  • Wet Macular Degeneration / pathology

Substances

  • Antibodies, Monoclonal, Murine-Derived
  • Vascular Endothelial Growth Factor Receptor-1
  • Vascular Endothelial Growth Factor Receptor-2
  • Minocycline