Figure 3. Choroidal neovascularization and NLRP3 expression in aged VEGF-A^hypermice.

a.-b. Perfusion experiments in aged white VEGF-A^hyper mice with fluorescein-conjugated tomato lectin and subsequent wholemount staining for CD31 and phalloidin shows that proliferating neovessels (CD31, red) originate from the underlying perfused choroidal vasculature (green) and extend into the sub-RPE space. Autofluorescent round deposits can be seen at sites of CNV lesions. Scale bars 50μm. Inset in b. represents modeled z-stack of lesion.

c. Representative section of CNV lesion in aged VEGF-A^hyper mice shows massive subretinalneovascularization (arrow). Scale bar 20μm.

d. Representative image of a fully formed CNV lesion in a 7 months old VEGF-A^hyper mouse thatresembles a neovascular membrane in neovascular AMD (arrow). Neovascularization and fibrosishave completely replaced the photoreceptor layer at the site of CNV formation. Scale bar 50μm.

e. In age-matched control littermate mice such lesions were not seen. Scale bar 50μm.

f. Co-localization of RPE barrier breakdown with CNV lesions. Choroidal flatmount of a 24 monthsold white VEGF-A^hyper mouse in which choroidal perfusion is assessed by intracardiac administrationof a fluorescein-conjugated tomato lectin (green). Subsequent whole-mount staining for CD31 (red)highlights proliferating neovessels from underlying perfused choroidal vessels (green), and labelingfor β-catenin (white) shows RPE barrier breakdown at sites of neovascularization with cytoplasmicaccumulation of β-catenin. Scale bar 50μm.

g. Strongly increased NLRP3 expression in RPE cells in CNV lesions (arrows) with attenuation or lossof the overlying photoreceptor layer. 15 months old VEGF-A^hyper mice. Scale bars 50μm.

h. Perivascular accumulation of C1qA within CNV lesions (arrow). 15 months old VEGF-A^hyper mice. Scale bar 50μm.

i. Western blotting demonstrates accumulation of C1qA in choroid/RPE tissues of aged VEGF-A^hyper mice, while control littermate mice showed no accumulation of C1qA. Choroid/RPE or retinal tissues represent pools from three 25 months old VEGF-A^hyper mice or control mice. Normalized relative densitometric values are indicated.

j. Acrolein, a marker for ROS-induced lipid peroxidation, is increased focally in abnormal RPE cells in aged VEGF-A^hyper mice (green, arrow) (15 months old). DAPI nuclear staining; photoreceptor outer segments show autofluorescence (green). Scale bar 50μm.

k. VEGF-A¹⁶⁵ treatment of ARPE-19 cells loaded with 10 μM dihydroethidium (DHE) induces increased oxidative stress (superoxide indicator), measured by FACS (PerCP-Cy5-5 channel). Y-axis shows mean fluorescence intensity (MFI) of triplicate experiments (n=3/group). A representative histogram demonstrates the shift of fluorescence induced by VEGF-A¹⁶⁵, representing increased superoxide species. *P-value <0.05.

l. VEGF-A serum levels are increased in young (7 weeks old) SOD1^-/- mice, compared to age- and gender-matched control littermate mice (n=5). *P-value <0.05.

m. SOD1 is efficiently depleted using SOD1-targeted siRNA, demonstrated by Western blotting of ARPE-19 cell lysate. Cell culture supernatant of cells, primed with 4ng/ml IL-1α, shows that SOD1 knockdown increases levels of the inflammasome effector cytokine IL-1β in both ARPE-19 cells and the B-3 lens epithelial cells. N=4/group. *P-value <0.05. See also Figure S3.