Abstract
Purpose:
Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly, and its exudative subtype critically depends on local production of vascular endothelial growth factor (VEGF). In tumour models, mononuclear phagocytes (MPh) produce VEGF. MPh including dendritic cells, macrophages and microglia cells, express CX3CR1. MPh precursors, such as monocytes, can be recruited to sites of inflammation by CCR2. Here we studied VEGF production by MPh in a laser-induced murine model of choroidal neovascularisation (CNV) that mimics CNV in exudative AMD, and the impact of CCR2 on such production.
Methods:
CCR2-competent and -deficient CX3CR1-reporter mice expressing GFP in MPh were used to visualize these cells by 3 complementary fluorescence-based techniques. CNV was induced by laser injury rupturing Bruch's membrane beneath the retina. 3 and 6 days later, eyes were enucleated to prepare single cell suspensions from the retina and the choroid. Cellular subsets were identified and their VEGF-production quantified by flow cytometry. Scanning-Laser-Ophthalmoscopy (SLO) in the 488 nm autofluorescence mode was used to localize MPh in living animals. CNV areas were measured in choroidal flatmounts by isolectin staining.
Results:
VEGF-positive phagocytes increased 3 days post laser, with microglia being dominant in the retina (2fold) and macrophages in the choroid (>25fold). On day 6, VEGF-expressing microglia numbers remained increased, whereas macrophages had already declined. This temporary increase in macrophage numbers was abrogated in the absence of CCR2, whereas microglia was CCR2-independent. These macrophages were the only source of VEGF that upregulated their VEGF-production at day 3, but not later. Consistent with flow-cytometry, microscopy and in vivo SLO revealed that Mph accumulated in CNV areas. This area was reduced in CCR2-deficient mice on day 14, but not on day 21.
Conclusions:
We established an intracellular staining method for VEGF-production and describe a non-invasive in vivo imaging method for ocular MPh. These MPh were identified as relevant producers of VEGF as they accumulate responding to laser injury. The immune cell related source of VEGF decreases by an impaired recruitment to the choroid. Infiltrating CCR2-dependent macrophages specifically increase CNV, indicating that CCR2 inhibition might be a temporal strategy to prevent CNV in AMD.
Keywords: 453 choroid: neovascularization •
529 flow cytometry •
557 inflammation