Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function

Abstract

The choroid in the eye provides vascular support for the retinal pigment epithelium (RPE) and the photoreceptors. Vascular endothelial growth factor (VEGF) derived from the RPE has been implicated in the physiological regulation of the choroidal vasculature, and overexpression of VEGF in this epithelium has been considered an important factor in the pathogenesis of choroidal neovascularization in age-related macular degeneration. Here, we demonstrate that RPE-derived VEGF is essential for choriocapillaris development. Conditional inactivation of VEGF expression in the RPE (in VEGFrpe-/- mice) results in the absence of choriocapillaris, occurrence of microphthalmia, and the loss of visual function. Severe abnormalities of RPE cells are already observed when VEGF expression in the RPE is only reduced (in VEGFrpe+/- mice), despite the formation of choroidal vessels at these VEGF levels. Finally, using Hif1arpe-/- mice we demonstrate that these roles of VEGF are not dependent on hypoxia-inducible factor-1alpha-mediated transcriptional regulation of VEGF expression in the RPE. Thus, hypoxia-inducible factor-1alpha-independent expression of VEGF is essential for choroid development.

Figure 1

VEGF expression during ocular development. Sections of VEGF-lacZ mice were stained for β-galactosidase activity: A, E12.5; B, E12.5; D, P1; E, P1. CD31 immunostaining of endothelial cells in choroidal vessels: C, E12.5; F, P1. Arrowheads indicate RPE cells. Arrows indicate choroidal vessels. L, Lens. Original magnifications: ×10 (A, D); ×40 (B, E); ×60 (C, F).

Figure 2

VEGF^rpe−/− mice display microphthalmia. A: Eye regions of 3-week-old VEGF^rpe−/− mouse and control littermate. B: The enucleated eye of an 8-week-old VEGF^rpe−/− mouse is much smaller in size than the eye of the control littermate. The posterior eyeball is lighter in appearance in the VEGF^rpe−/− eye. C: Cre-mediated excision of floxed DNA occurs in VEGF^rpe−/− eyes. PCR with DNA from posterior eyecup homogenates from VEGF^flox/flox mice (WT) reveals a single band (∼2000 bp), whereas Cre activity in the RPE of VEGF^rpe−/− mice (KO) leads to an additional smaller PCR product (∼550 bp). Immunostaining for Cre reveals positive staining (gray color of DAB/Ni) within the RPE cells (arrowhead; brown pigment is visible within the RPE cells). D: The optic nerve of an eye of an adult VEGF^rpe−/− mouse is indicated with an arrowhead (left). No vitreous or anterior chamber are visible; the lens is in direct contact with the retina and iris. Arrowhead shows clustered melanocytes surrounding the optic nerve (right). Original magnifications: ×4 (D, left); ×20 (D, right).

Figure 3

Loss of choroid in VEGF^rpe−/− mice. A and B: Methylene blue-stained section of an eye of a 4-week-old VEGF^rpe−/− mouse (B) and the control (A). B: Loss of choroid can be observed in VEGF^rpe−/− mice with absence of vessels and melanocytes underlying the RPE (arrowhead). A: Control littermate eye shows normal choroid (arrow) with choroidal vessels and melanocytes, and a continuous RPE cell layer (arrowhead). Photoreceptor outer segments (*) and inner segments are significantly shortened in VEGF^rpe−/− mice (B), when compared to those in control mice (A). C: CD31 immunostaining in a 4-week-old control eye shows staining for retinal vessels (arrowhead) and choroidal vessels underlying the RPE (arrow). D: Lack of choroidal vessels (arrow) in a VEGF^rpe−/− eye, with presence of retinal vessels (arrowhead). E: Vessels were already observed at E13.5 in control littermates, with an endothelial lining (arrowhead) and intraluminal erythrocytes (black arrow), which were lacking in VEGF^rpe−/− mice at E13.5 (F). RPE cell morphology shows no significant difference at E13.5 (white arrow). R, Retina. Original magnifications: ×20 (A, B); ×40 (E, F).

Figure 4

Analysis of vasculature in flat mounts of control eyes (A–C), VEGF^rpe+/− eyes (D–F), and VEGF^rpe−/− eyes (G–I). A and B: A dense vascular network consisting mostly of capillaries (arrows) characterizes the choroid in control mice, supplied by choroidal arteries (arrowhead) (FITC-dextran). C: Concanavalin A lectin staining of the choroidal capillaries (arrow) and arteries (arrowhead) seen in flat mounts of control eyes. VEGF^rpe+/− eyes show areas with presence of choriocapillaries (D), while at some areas choriocapillaris was lacking and only thin vessels are seen at these areas (E, F) (FITC-dextran). G: Flat mount of a FITC-dextran-perfused VEGF^rpe−/− eye shows iris vessels (arrowhead) at the left. The area of the ora serrata is indicated (*), with the posterior eyeball being located at the right. Absence of choroidal vasculature is seen in most areas of the posterior eyeball in VEGF^rpe−/− mice, with the exception of only some small vessels (arrow). H: These vessels form a fine vascular network (arrow) that lacks the typical fenestrated vessels of the choriocapillaris (FITC-dextran). I: Concanavalin A lectin staining of the remaining vessels seen in flat mounts of VEGF^rpe−/− eyes. Original magnifications: ×20 (A, C, D–F, I); ×10 (B, H); ×2 (G).

Figure 5

RPE and retinal defects in VEGF^rpe−/− mice. Electron microscopic images of an eye of a 4-week-old VEGF^rpe−/− mouse and the control mouse. A: Control eye with normal RPE, photoreceptor outer segments (arrow), and choroid. Arrowhead points to Bruch’s membrane. B: RPE in the VEGF^rpe−/− eye is flattened, with loss of interdigitations between apical villi and photoreceptor outer segments (arrow), irregular basal infoldings, and reduced pigmentation with abnormal pigment granules. Arrowhead points to Bruch’s membrane. C and D: Ectopic localization of photoreceptor nuclei (arrows) next to RPE cells (arrowheads) in VEGF^rpe−/− mice. E: Photoreceptor outer segments in a 4-week-old VEGF^rpe−/− mouse eye with cystic defects and abnormal disks. F: Absence of Bruch’s membrane in the VEGF^rpe−/− eye. Collagen fibrils (arrow) are directly adjacent to the basal lamina (arrowhead) of the RPE (inset). Scale bars: 2.5 μm (A, B); 3.5 μm (D); 1.5 μm (E); 0.5 μm (F). Original magnification, ×40 (C).

Figure 6

Analysis of visual function in VEGF^rpe−/−, VEGF^rpe+/−, and control mice. Reduced ERG amplitudes are observed in VEGF^rpe+/− mice when compared to control mice. ERGs demonstrate an almost absent retinal function in VEGF^rpe−/− mice. Consistent with the ERG data, retinal rhodopsin levels are reduced in eyes of VEGF^rpe+/− mice, whereas rhodopsin is almost lacking in eyes of VEGF^rpe−/− mice. Mean ± SD, n = 5.

Figure 7

Morphological analysis of Hif1a^rpe−/− eyes. A: Control eye. B: Choroidal vessels and melanocytes are present in Hif1a^rpe−/− eyes. Methylene blue staining of a thin section. C: Electron microscopic image of a Hif1a^rpe−/− eye shows normal choroidal endothelial fenestrations (arrowhead). D: Cre-mediated excision of floxed DNA occurs in Hif1a^rpe−/− eyes. PCR with DNA from posterior eyecup homogenates from Hif1a^flox/flox mice (WT) reveals a single band (∼1200 bp), while Cre activity in the RPE of Hif1a^rpe−/− mice (KO) leads to an additional smaller PCR product (∼700 bp). Scale bar, 0.25 μm (C). Original magnifications, ×40 (A, B).

Figure 8

VEGF^rpe+/−mice show RPE cell defects. A: No microphthalmia is seen in VEGF^rpe+/− mice, but focal loss of pigmentation (arrow) of the posterior eyeball can be observed. Arrow indicates the optic nerve. B: This loss of pigmentation reflects localized absence of choroid tissue (arrowhead), while most areas of eyes of VEGF^rpe+/− mice have choroidal melanocytes and vessels (arrow). C: Flattening of RPE cells with loss of pigment granules (arrows) are seen next to choroid tissue (arrowhead) in VEGF^rpe+/− mice. D: Arrowhead indicates a choroidal vessel, underlying flattened RPE cells with loss of pigment granules (arrow). E: Fluorescein angiography in VEGF^rpe+/− mice demonstrates focal loss of RPE cell pigmentation as distinct areas of hyperfluorescence (arrows), due to the presence of fluorescein in the underlying choroidal vasculature. R, Retina; S, sclera. Original magnifications: ×20 (B); ×40 (C); ×100 (D).