Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury

Abstract

Vascular endothelial growth factor-A (VEGF-A) has recently been recognized as an important neuroprotectant in the central nervous system. Given its position as an anti-angiogenic target in the treatment of human diseases, understanding the extent of VEGF's role in neural cell survival is paramount. Here, we used a model of ischemia-reperfusion injury and found that VEGF-A exposure resulted in a dose-dependent reduction in retinal neuron apoptosis. Although mechanistic studies suggested that VEGF-A-induced volumetric blood flow to the retina may be partially responsible for the neuroprotection, ex vivo retinal culture demonstrated a direct neuroprotective effect for VEGF-A. VEGF receptor-2 (VEGFR2) expression was detected in several neuronal cell layers of the retina, and functional analyses showed that VEGFR2 was involved in retinal neuroprotection. VEGF-A was also shown to be involved in the adaptive response to retinal ischemia. Ischemic preconditioning 24 hours before ischemia-reperfusion injury increased VEGF-A levels and substantially decreased the number of apoptotic retinal cells. The protective effect of ischemic preconditioning was reversed after VEGF-A inhibition. Finally, chronic inhibition of VEGF-A function in normal adult animals led to a significant loss of retinal ganglion cells yet had no observable effect on several vascular parameters. These findings have implications for both neural pathologies and ocular vascular diseases, such as diabetic retinopathy and age-related macular degeneration.

Figure 1

I/R in the rat induces temporally and spatially defined apoptosis of retinal cells. TUNEL staining of retinal sections at 3, 6, 12, 24, and 48 hours after I/R. In sham-operated eyes (depicted here 24 hours after the procedure), there was no observable TUNEL staining. In I/R injured eyes, small numbers of TUNEL-positive cells (green) could be detected in the GCL and the INL at 3 hours (I/R 3 hours) and 6 hours (I/R 6 hours) after injury. The number of TUNEL-positive cells was greatest at 12 hours (I/R 12 hours) and 24 hours (I/R 24 hours) after I/R in the GCL and INL, respectively. At 48 hours (I/R 48 hours), TUNEL-positive cells were detected mainly in the ONL. Cell nuclei are stained blue. Scale bar = 20 μm.

Figure 2

I/R injury in the rat induces retinal neuronal cell apoptosis, as demonstrated by co-localization of TUNEL staining with a neuronal cell marker. A: At 12 hours (I/R 12 hours) and 24 hours (I/R 24 hours) after injury, TUNEL staining (green) did not co-localize with isolectin B4 (red), an endothelial cell marker. B: At 12 hours (I/R 12 hours) and 24 hours (I/R 24 hours) after injury, TUNEL staining (green) did not co-localize with glutamine synthetase (red), a marker of retinal glial cells (Müller cells). C: At 24 hours (I/R 24 hours) after injury, TUNEL staining (green) co-localized with NeuN, a neuronal cell marker (red) in both the GCL and the INL. Cell nuclei are stained blue (DAPI). Scale bars = 50 μm.

Figure 3

Both VEGF120 and VEGF164 prevented retinal cell apoptosis resulting from I/R injury in the rat. A: Eyes injected immediately after I/R injury with either VEGF120 or VEGF164 had far fewer TUNEL-positive cells (green) in the INL 24 hours later compared with eyes injected with PBS. Quantification of TUNEL-positive cells per 200 μm of retina revealed that both VEGF120 (B) and VEGF164 (C) produce a significant and dose-dependent reduction in TUNEL-positive cells in the INL 24 hours after I/R. VEGF120 also significantly reduced the number of TUNEL-positive cells in the GCL at 12 hours (D, E). At least five animals were used per treatment condition. Cell nuclei are stained blue. *P < 0.05 and **P < 0.01. Scale bars = 20 μm.

Figure 4

VEGF120 provides partial protection against retinal thinning after I/R injury in the rat. A: Eyes in which I/R was induced showed a decrease in retinal thickness 14 days after injury compared with sham-operated eyes. The INL decreased dramatically in thickness, and edema was apparent in the IPL, which is located between the INL and the GCL. B: Quantification of the thickness of the retina (measured from the border of the ILM to the INL) revealed that administration of VEGF120 significantly reduced thinning of the retina when compared with PBS. At least five animals were used per treatment condition. **P < 0.01. Scale bar = 20 μm.

Figure 5

Retinal blood flow in the rat retina is elevated after I/R and further increased on treatment with VEGF120. A: Representative images of the major retinal vessels. Both arteries and veins were dilated 24 hours after I/R compared with the sham-operated eyes. Treatment with VEGF120 (40 pmol) seemed to increase the vasodilation when compared with PBPSS. B: Quantification of the diameter of major veins revealed a significant increase in vein diameter after I/R compared with sham; treatment with VEGF120 (40 pmol) further increased vein diameter. C: I/R and treatment with VEGF120 (40 pmol) significantly increase volumetric blood flow (calculated from vein diameter and velocity of flowing erythrocytes; see Materials and Methods). D: VEGF120 (40 pmol) reduced the number of apoptotic cells in the INL 24 hours after I/R injury; administration of the iNOS inhibitor 1400W reduced the protective effect of VEGF120 from ∼84 to ∼50% (P < 0.05). All treatment groups included at least five animals. One-way analysis of variance with a post hoc Bonferroni test was applied to all groups in each experiment. *P < 0.05 and **P < 0.01. Scale bar = 200 μm.

Figure 6

VEGF120 inhibits apoptosis in rat retinal explant cultures. A: Lectin staining of the developing retinal vasculature [postnatal day 1 to postnatal day 9 (P1 to P9)]. Arrows indicate the leading edge of the developing vasculature. The inset indicates the 1-mm² region of avascular retina used for the explant culture. B: TUNEL-positive cells (green) in the GCL of the retina immediately after explant from the retinal flatmount (top, control) and after culturing for 24 hours with no treatment (middle), or with the addition of 100 ng/ml VEGF120 (bottom). Cell nuclei are stained blue. C: Quantification of the percentage of apoptotic cells in the GCL as determined by dividing the number of TUNEL-positive cells by the number of DAPI-stained cells. All treatment groups included at least four animals. **P < 0.01. Scale bars: 1 mm (A); 10 μm (B).

Figure 7

VEGF receptors are involved in inhibiting rat retinal apoptosis after I/R injury. A: VEGF-E (40 pmol), a VEGFR2 ligand, but not PlGF-1 (40 pmol), which does not bind VEGFR2, reduced the number of TUNEL-positive cells (green) in the INL of eyes 24 hours after I/R. B: Quantification of the number of TUNEL-positive cells in the INL of eyes 24 hours after I/R. Only VEGF-E showed a statistically significant decrease in TUNEL-positive cells (P < 0.01). All treatment groups included at least five animals. C: Double labeling of VEGFR2 and isolectin B4, an endothelial cell marker, in the retina after ischemia showing isolectin B4-labeled vessels in the GCL, IPL, and INL (red) and VEGFR2 (green). Most retinal cells in the GCL and INL expressed VEGFR2 whereas neuronal cells in the ONL did not. VEGFR2-positive cells were also observed in the layer above the cells in the GCL, which may correspond to nerve fibers of the retina. Cell nuclei are stained blue. **P < 0.01. Scale bars = 20 μm.

Figure 8

Increased expression of VEGF120 and VEGF164 plays a direct role in the anti-apoptotic effects of ischemic preconditioning in the rat retina. A: Retinal VEGF mRNA expression after induction of I/R with (top row, labeled IP) or without (second row, labeled I/R) ischemic preconditioning; mRNA samples for semiquantitative RT-PCR analysis were obtained from retinas from sham-operated eyes (column 1), at 3 hours after 5-minute ischemic preconditioning (column 2), at 24 hours after ischemic preconditioning (column 3), at 3 hours, after 60-minute ischemia with ischemic preconditioning (column 4), at 6 hours after 60-minute I/R with ischemic preconditioning (column 5), at 12 hours after 60-minute I/R with ischemic preconditioning (column 6), and at 24 hours after 60-minute ischemia with ischemic preconditioning (column 7). Two bands show the expression of VEGF120 and VEGF164 mRNA. Up-regulation of VEGF120 and VEGF164 mRNA by ischemic preconditioning was observed at 3 hours and lasted until the 24-hour time point. B: Retinal VEGF protein levels after I/R injury were significantly up-regulated by ischemic preconditioning (P < 0.05). C: TUNEL staining (green) in retinal cross sections 24 hours after ischemic injury with or without ischemic preconditioning. Mice were injected intravitreally with PBS or with VEGFR1/Fc (5 μg, 12.5 pmol), a VEGF antagonist. Cell nuclei are stained blue. D: Ischemic preconditioning dramatically reduced induced apoptosis subsequent to I/R injury (P < 0.01). Blockade with VEGFR1/Fc protein significantly reduced the benefit of ischemic preconditioning (P < 0.05). Three to six animals were used per group. E: Intravitreous injection of an anti-VEGF-A neutralizing antibody (anti-VEGF, 5 pmol) after ischemic preconditioning (IP + anti-VEGF + IR) significantly reduced the protective effect of ischemic preconditioning, whereas antibody treatment after I/R (IP + I/R + anti-VEGF) nearly abolished the protective effect of ischemic preconditioning (P < 0.01). At least three rats were used per group; all data represent mean ± SEM. *P < 0.05 and **P < 0.01.

Figure 9

VEGF-A is required for the maintenance of the normal RGCs in the retina. A: Mice treated systemically three times per week for 8 weeks with shVEGFR1 had a reduction in the number of viable RGCs (to ∼50% of PBS or IgG controls), as determined by retrograde fluorogold labeling. The number of mice used per group is indicated within each bar; data represent mean ± SEM. **P < 0.01 B: Mice treated systemically with a polyclonal anti-VEGF antibody for three times per week for 8 weeks had a dose-dependent reduction in the number of viable RGCs (to ∼50% of IgG and PBS controls with 1.33 pmol anti-VEGF), as determined by retrograde fluorogold transport. The number of mice used per group is indicated within each bar; data represent mean ± SEM. **P < 0.01. C: Systemic administration of a polyclonal anti-VEGF antibody on alternate days for 14 days caused a significant decrease in the number of cells expressing p-Akt in the mouse retina compared with IgG controls, as determined by immunostaining. The mean (±SEM) number of p-Akt-positive cells in the retina was quantified from at least 10 images from three different eyes per group; administration of anti-VEGF antibody reduced the number of p-Akt-positive cells by ∼50% in the anti-VEGF antibody-treated mice as compared with IgG controls. **P < 0.01. D: Intravitreal injection of rats once weekly with a polyclonal anti-VEGF antibody at 1 pmol or 5 pmol for 6 weeks resulted in a dose-dependent loss of viable RGCs (of ∼60% in the high-dose group) compared with IgG and PBS controls, as determined by retrograde fluorogold transport. Pegaptanib injection at both 1 pmol and 5 pmol did not cause any detectable loss of viable RGCs in the rat retina. Number of rat eyes analyzed per group is shown within each bar; data represent mean ± SEM. Each comparison line represents P < 0.01.