Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors

Abstract

Background: Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical.

Methods and findings: Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective.

Conclusions: These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

Figure 1. VEGF signaling in the adult retina.

(A) Sections of eyes from adult VEGF-lacZ mice stained for LacZ using x-gal (blue) revealed VEGF expression in the GCL (arrowheads) and INL layer (asterisks). (B) Flat-mounted retinas from adult VEGF-lacZ mice stained for lacZ (blue) and NG2 (brown) demonstrated VEGF expression by NG2-positive cells associated with microvessels (arrowhead) and by some astrocytes (arrow) in the GCL. (C) Sections of eyes from adult VEGFR2-lacZ mice stained for lacZ revealed expression of VEGFR2 in the GCL, in the INL (asterisks), and in the photoreceptors where a strong lacZ staining was observed in the inner segments (arrows). (D) Immunohistochemistry for VEGFR2 in sections of adult retina revealed expression in vascular cells (arrowhead), in Müller cells processes, and in the IS of the photoreceptors (arrows). (E) Immunohistochemistry for VEGFR1 revealed a spotty expression in the IPL and OPL. VEGFR1 was also detected in the photoreceptor IS. (F) Staining of Müller cells using CRALBP revealed an expression pattern similar to VEGFR2 (compare with D). (G) Expression of VEGF and its receptors, VEGFR1 and VEGFR2, by Müller cells was confirmed by RT-PCR of RNA from adult rat retina and isolated rat Müller cells. (H) Expression of VEGFR1 and VEGFR2 in photoreceptors (PR) isolated from adult mouse retinas. (I) Immunoprecipitation (IP) of VEGFR2 from pooled adult mouse retinas, followed by immunoblotting for phosphorylated tyrosine revealed VEGFR2 expression (bottom panel) and activation (top panel) in the adult retina. As a control, lysates of porcine aortic endothelial (PAE) cells overexpressing VEGFR2 either untreated or stimulated with VEGF were immunoprecipitated and immunoblotted as described above. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, IS: inner segment, OS: outer segment. Scale bar is 100 µm.

Figure 2. VEGF neutralization does not affect retinal vascular perfusion or permeability.

Mice infected with Ad-null or Ad-sFlt1 for 14 days were perfused with h.m.w fluorescein dextran (green), then eyes were dissected and (A) flat mounted or (B) sectioned and immunostained for collagen IV, and visualized with a Cy3-conjugated secondary antibody (red). (A) Flat mounts of retinas from mice expressing sFlt1 perfused with fluorescein dextran shows no gross abnormalities or perfusion defects, and no changes in capillary density in the inner retina compared to the control mice expressing Ad-null (n = 6 mice/condition). (B) Immunofluorescent localization of collagen IV in sections of fluorescein-perfused retinas revealed nearly complete overlay of FITC-dextran and collagen IV of the inner retinal vessels in both Ad-null and Ad-sFlt1 (arrows, third column). (C) Quantification of fluorescein- and collagen IV-positive vessels of the innermost retinal vasculature in Ad-sFlt1 (n = 5) compared to Ad-null (n = 4) showed no reduction in the number of perfused vessels, indicating the absence of vascular damage. (D) Fluorescein angiography of experimental mice seven days after infection showing no opacity in the vitreous 4-5 min after injection in either group, indicating no change in retinal vascular permeability (n = 6 mice/condition). Scale bar is 1 mm in A and 100 µm in B.

Figure 3. Systemic VEGF blockade does not alter retinal vessels ultrastructure or choriocapillaris fenestration.

(A) TEM of a microvessel located in the GCL of sFlt1 expressing mouse at day 14 showed a normal ultrastructure of the endothelial nuclei and cytoplasm. Characteristically electron -dense tight junctions were well defined (arrows). (B) TEM micrograph of the outer segment of a retina, 14 days post-infection of Ad-sFlt1, revealed a normal RPE-choroid complex. The RPE basal infoldings were dense and well associated with the Bruch's membrane (BM). The choriocapillaris showed no thickening of the membrane. No sign of thrombosis were detected. (C) Higher magnification of the choriocapillaris revealed numerous fenestrations on the endothelial membrane facing the RPE (arrows). (D) Endothelial fenestrations of the choriocapillaris (arrows) were quantified at 28 days post-infection on TEM micrographs (n = 3 mice/condition). No change in the number of choriocapillaris fenestration was observed between Ad-null and Ad-sFlt1 mice. Scale bar is 5 µm in A–B, 0.5 µm in C and 1 µm in D.

Figure 4. VEGF neutralization leads to increased retina cell death.

(A) TUNEL staining showed markedly increased apoptosis in both the INL and ONL of Ad-sFlt1 mice 14 days post-infection (n = 4 mice/condition). (B) Higher magnification images of retinal sections of sFlt1-expressing mice stained for TUNEL and counterstained with hematoxylin revealed an significant increase in the number of apoptotic cells in the INL and ONL. GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OPL: outer plexiform layer, Ch: choroid. Scale bars are 100 µm in A and 50 µm in B.

Figure 5. Decreased retina thickness and loss of visual function in sFlt1 expressing mice.

(A) Semi-thin epon sections from retinas of experimental mice expressing sFlt1 for 28 days showed substantial thinning of the INL (black double arrowheads line) and the ONL (white double arrowheads line) (n = 3 for Ad-null and n = 4 for Ad-sFlt1). (B–D) TEM of retinas from sFlt1 expressing mice at 28 days. (B–C) Micrographs taken in the INL region revealed apoptotic Müller cells (MC) and amacrine cells (A). Both cells displayed condensation of the chromatin, membrane swelling and rupture mitochondria (arrows). (D) In the ONL, a high percentage of photoreceptors appeared apoptotic (white asterisk). Cell death left numerous empty spaces containing membranous debris (arrowhead). Processes from the Müller cells that normally fill the intercellular space appeared shrunken (MP). (E) Scotopic ERG recordings of experimental mice 28 days post-infection using a flash intensity of +10 dB revealed a marked reduction of both a- and b-wave amplitudes in sFlt1-expressing mice (n = 6 for Ad-null and n = 10 for Ad-sFlt1). GCL: ganglion cell layer, INL: inner nuclear layer, ONL: outer nuclear layer, OPL: outer plexiform layer, Ch: choroid. Scale bars are 100 µm in A and 5 µm in B to D.

Figure 6. Inhibition of the autocrine VEGF signaling in Müller cells leads to increased cell death.

VEGF expression by MIO-M1 Müller cells was inhibited by transfection of siRNA into sub-confluent cells. (A–B) Seventy-two hr following transfection of the control and VEGF siRNA, VEGF mRNA levels were determined by qPCR (A) and VEGF protein secretion was quantified by ELISA (B). siVEGF transfection led to a 75% inhibition of VEGF mRNA and more than 90% reduction of VEGF protein (n = 3). (C) Inhibition of VEGF was associated with an increase in TUNEL-positive cells after three days of culture in serum-free conditions (n = 4). (D) Quantification of cell death by FACS analysis demonstrated a doubling of the number of annexin-V positive apoptotic cells in siVEGF transfected cells compared to siControl (n = 3). (E) Increased apoptosis was accompanied by a significant up-regulation of the pro-apoptotic gene, bax, and by the increase of the Bax/Bcl-2 ratio (F). Scale bar is 100 µm.

Figure 7. VEGF is a direct survival factor for photoreceptors.

(A) Photoreceptors isolated from adult C57BL/6J mice were cultured on laminin-coated wells in absence or presence of 10 ng/ml VEGF165 for up to 72 hr. Addition of 10 ng/ml of VEGF165 decreased the number of dead cells identified by trypan blue staining. (B) Quantification of the percentage of dead photoreceptors based on the trypan blue exclusion assay revealed a significant protection from apoptosis by VEGF165 (n = 9). (C) ONL explants containing only the photoreceptor nuclei and IS were cultured in basal medium±10 ng/ml of VEGF165 for 24, 48 and 72 hr. Apoptotic cells were detected by TUNEL staining. (D) Quantification of TUNEL-positive cells per sheet revealed a significant decreased in the number of apoptotic photoreceptors in presence of VEGF (n = 6).