Article
The role of VEGF-expressing mononuclear phagocytes in a murine model of laser-induced choroidal neovascuarization
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Published: | May 30, 2012 |
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Background: The aim of this study was to evaluate, whether infiltrating or resident phagocytes are a source of VEGF that causes neovascularization in a murine CNV-model.
Methods: For visualization of mononuclear phagocytes CX3CR1-reporter mice were used.
These mice express GFP controlled by the CX3CR1-promoter, which is active in mononuclear phagocytes (dendritic cells, macrophages, microglia). In CX3CR1-reportermice with or without
CCR2-expression 10 laserspots were set (50 µm, 200 mW and 0.1 s duration) using a slitlamp applicator. The retinal pigment epithelium and Bruch“s membrane were ruptured. 3 and 6 days post laser eyes was enucleated, retina and choroid of both eyes collected, singlecellsuspensions prepared and stained with fluorescence-labeled antibodies for different celltypes (CD11b, CX3CR1, CD45, CD31) for analyzing VEGF-production by flow cytometry. CNV-areas were measured in choroidal flatmounts, while mononuclear phagocytes were localized in retinal flatmounts.
Results: The numbers of VEGF-producing mononuclear phagocytes (CD11b+) increased significantly 3 days post laser, especially those of retinal microglia (CX3CR1high CD45low) and choroidal macrophages (CX3CR1low CD45high). Microscopy revealed that these cells accumulated colocalized to laserspots. The number of VEGF-expressing choroidal macrophages increased on day 3 more than 28-fold and declined until day 6. The dramatic increase was absent in CCR2-deficient mice. In contrast the increase of VEGF-producing retinal microglia was CCR2-independent. CNV-area was diminished in flatmounts of CCR2-deficient mice.
Conclusions: VEGF-production of ocular phagocytes is quantifiable by flow cytometry.
CCR2-dependent macrophages are a relevant source of VEGF and accumulate post lasertreatment. CCR2-inhibition reduces CNV and may represent a therapeutical approach for an antineovascular therapy.