Figure 1. Quantifications of CNV lesions in Vegfa^hyper mice.

(A) Disrupted honeycomb pattern morphology of RPE cells and sub-RPE protrusions of CD31⁺ neovessels demarcate a choroidal neovascular lesion in choroidal flat mounts of Vegfa^hyper mice. A choroidal flat mount of a Vegfa^hyper mouse is shown. Phalloidin staining highlights RPE cell membranes and shows the regular honeycomb pattern of RPE cells at sites devoid of CNV lesions (white arrows). At the site of a CNV lesion, the typical RPE honeycomb pattern is disrupted (phalloidin, green; red arrows). CD31 (red) staining identifies neovessels of a CNV lesion that protrude into the sub-RPE space (yellow arrows). Disruption of regular RPE cell morphology and CD31 staining of neovessels determine the size of the lesion. A polygon is used to quantify the lesion area. Scale bars, 50 µm. 6-weeks-old mouse. (B) CNV lesion in a 6-weeks-old Vegfa^hyper mouse shows that RPE disruption occurs only at sites of choroidal neovascularization due to CD31⁺ neovessels (green) protruding into the sub-RPE space, whereas RPE cells show a normal honeycomb pattern cellular morphology at sites without CNV lesions (white arrow). Labeling for active β-catenin outlines RPE cell membranes (red), similar to phalloidin staining. F4/80⁺ cells (white) infiltrate sites of CNV lesions. Pigmented cells cover part of the CNV lesion (yellow arrow). Scale bars, 100 µm. (C) Section through a CNV lesion (yellow arrows) in an aged Vegfa^hyper mouse eye (22-months-old) shows CD31⁺ neovessels in CNV lesions. Scale bars, 50 µm. (D) Representative image of how CNV lesions were quantified in choroidal flat mounts in mice with the Vegfa^hyper allele. Measurements of multiple lesions in a choroidal flat mount. A choroidal flat mount of a Vegfa^hyper mouse is shown in which each CNV lesion is counted (numbered, arrows) and each lesion area shown by a polygon. Phalloidin staining (green) outlines CNV lesions and CD31 immunolabeling (red) detects CD31⁺ neovessels. Increased phalloidin signal at sites of disruption of the regular RPE morphology occurs only at sites of CD31⁺ neovessels. Scale bars, 500 µm. 6-weeks-old mouse. (E) Enlarged confluent CNV lesions can be observed in aged Vegfa^hyper mice. Even at an advanced age, disruption of the honeycomb pattern RPE morphology is strictly localized to the site of CD31⁺ neovessel protrusions into the sub-RPE space in CNV lesions (red arrow), whereas RPE areas with no CNV maintain their honeycomb pattern (green arrow). The left image shows Z-stack 3D-rendering of CNV lesions in a 12-months-old Vegfa^hyper mouse eye and the right image shows the projection of this z-stack. CD31⁺ neovessels of CNV lesions in red; phalloidin in white. Scale bars, 50 µm. (F) RPE/choroid lysates from 6-weeks-old Vegfa^hyper mice and their WT littermates show that the inflammasome activation product caspase-1 p10 can already be detected at this young age at which CNV lesions were quantified. A > 2 fold increase in p10 levels relative to WT (normalized to β-actin levels) are observed at 6 weeks of age in RPE/choroid lysates of Vegfa^hyper mice. RPE/choroids of both eyes from four Vegfa^hyper mice versus four of their WT littermates were pooled. An anti-caspase-1 p10 antibody from Thermo Fisher Scientific was used (PA5-105049) to detect p10.