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

• Published in: PloS one Vol. 8; no. 8; p. e71808
• Main Authors: Huang, Hu, Parlier, Rachel, Shen, Ji-Kui, Lutty, Gerard A, Vinores, Stanley A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.08.2013; Public Library of Science (PLoS)
• Subjects: Age; Alzheimer's disease; Angiogenesis; Animals; Antibodies; Antibodies, Monoclonal, Murine-Derived - administration & dosage; Arthritis; Atherosclerosis; Biology; Bone marrow; CD11b antigen; CD45 antigen; Cell Movement; Chemokines; Choroidal Neovascularization - drug therapy; Choroidal Neovascularization - metabolism; Choroidal Neovascularization - pathology; Dextran; Dextrans; Disease Models, Animal; Gene expression; Humans; Hypoxia; Immunofluorescence; Infiltration; Injections, Intraperitoneal; Laser Capture Microdissection; Lasers; Lesions; Leukocytes; Ligands; Macrophages; Macrophages - drug effects; Macrophages - immunology; Macular degeneration; Medicine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Microglia - drug effects; Microglia - immunology; Microscopy; Minocycline; Minocycline - administration & dosage; mRNA; Oxidative stress; Pathogenesis; Pathology; Photoreceptors; Polymerase chain reaction; Proteins; Receptors; Retina; Retina - drug effects; Retina - immunology; RNA; Tissue Culture Techniques; Tumor necrosis factor-TNF; Vascular endothelial growth factor; 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; Vascular endothelial growth factor receptors; Vascularization; Wet Macular Degeneration - drug therapy; Wet Macular Degeneration - metabolism; Wet Macular Degeneration - pathology; United States > US; Maryland; Baltimore Maryland
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0071808

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.