Figure 1. Increased VEGF-A levels result in a progressive RPE barrier breakdown.

a. The RPE is the main cell type of VEGF-A expression in the adult eye. Immunolabeling of an adultposterior eye of a VEGF-A^hyper mouse (15 months old) for β-galactosidase reflects cellular expressionof VEGF-A (green). Strong VEGF-A expression is observed in RPE cells (arrow), whereas low-levelVEGF-A expression is seen in retinal cells of the ganglion cell layer (GCL) and inner nuclear layer(INL) (arrowheads), but not the photoreceptor layer (outer nuclear layer; ONL). Scale bar 50μm.

b. VEGF-A ELISA of RPE/choroid and retinal tissue in adult VEGF-A^hyper mice (KI) and controllittermate (WT) mice (n=7/group) shows a significant increase of VEGF-A levels in the RPE/choroidtissues in VEGF-A^hyper mice. Serum levels of VEGF-A are elevated in mutant mice independently ofage as well (n=3/group). *P-value <0.05.

c. Mouse and human RPE cells express the VEGF-A receptors Flt1 and Flk1 and all major VEGF-Aisoforms, particularly VEGF-A¹⁶⁴ and VEGF-A¹²¹ (VEGF-A¹⁶⁵ and VEGF-A¹²¹ in human).

d. Focal RPE barrier breakdown is observed in VEGF-A^hyper mice with cytoplasmic and nuclearaccumulation of β-catenin. Co-labeling with Alexa488-conjugated phalloidin (green) shows that innormal RPE cells phalloidin and β-catenin (red) labeling is strongest along cell membranes, while inRPE cells with RPE barrier breakdown β-catenin labeling along the cell membranes is attenuatedand increased in the nuclei or cytoplasm. 7 months old VEGF-A^hyper mouse. Scale bar 20μm.

e. Co-labeling of cell junction proteins β-catenin (red) and ZO-1 (green) shows loss of membrane-bound ZO-1 and β-catenin with nuclear accumulation. 7 months old VEGF-A^hyper mouse. Scale bar50μm.

f. FITC-dextran flux assays with RPE cells demonstrate that VEGF-A¹⁶⁵ treatment induces barrierbreakdown and increased transepithelial flux of 10kDa FITC-dextran. (n=3), *P-value <0.05. y-axisindicates fluorescence units (AU).

g.-h. Choroidal flatmount staining of eyes from VEGF-A^hyper mice with β-catenin (red) shows that RPE-barrier breakdown is observed in small foci (arrow) in young VEGF-A^hyper eyes (2 months old, (g)), but that these areas expand and become confluent and affect most of the posterior eye (arrows) with progressive age of the mice (12 months old, (h)). Scale bars 200μm.

i. Magnification of area from Figure 1H that delineates normal appearing RPE with its honeycomb pattern from lesional abnormal appearing RPE cells. Scale bar 200μm.

j. Adult VEGF-A^hypo mice, hypomorphic for VEGF-A, express β-galactosidase (green) from the endogenous VEGF-A gene locus, but show normal RPE cells. Scale bar 100μm.

k. In contrast, adult VEGF-A^hyper mice, with increased VEGF-A levels, show the same expression of β-galactosidase (green) from the endogenous VEGF-A gene locus as VEGF-A^hypo mice, but show multifocal RPE barrier breakdown. Scale bar 100μm.

l. Mice that express VMD2-Cre specifically in RPE cells (white nuclear staining for Cre) that are heterozygous for VEGF-A^fl/fl and carry the VEGF-A^hyper allele; in these mice Cre⁺ RPE cells are expected to have lower levels of VEGF-A than Cre^- RPE cells. RPE barrier breakdown occurs predominantly within Cre^- RPE-patches (therefore expressing higher levels of VEGF-A). Scale bar 100μm. See also Figure S1.