VEGF-A165b is cytoprotective and antiangiogenic in the retina

Abstract

Purpose: A number of key ocular diseases, including diabetic retinopathy and age-related macular degeneration, are characterized by localized areas of epithelial or endothelial damage, which can ultimately result in the growth of fragile new blood vessels, vitreous hemorrhage, and retinal detachment. VEGF-A(165), the principal neovascular agent in ocular angiogenic conditions, is formed by proximal splice site selection in its terminal exon 8. Alternative splicing of this exon results in an antiangiogenic isoform, VEGF-A(165)b, which is downregulated in diabetic retinopathy. Here the authors investigate the antiangiogenic activity of VEGF(165)b and its effect on retinal epithelial and endothelial cell survival.

Methods: VEGF-A(165)b was injected intraocularly in a mouse model of retinal neovascularization (oxygen-induced retinopathy [OIR]). Cytotoxicity and cell migration assays were used to determine the effect of VEGF-A(165)b.

Results: VEGF-A(165)b dose dependently inhibited angiogenesis (IC(50), 12.6 pg/eye) and retinal endothelial migration induced by 1 nM VEGF-A(165) across monolayers in culture (IC(50), 1 nM). However, it also acts as a survival factor for endothelial cells and retinal epithelial cells through VEGFR2 and can stimulate downstream signaling. Furthermore, VEGF-A(165)b injection, while inhibiting neovascular proliferation in the eye, reduced the ischemic insult in OIR (IC(50), 2.6 pg/eye). Unlike bevacizumab, pegaptanib did not interact directly with VEGF-A(165)b.

Conclusions: The survival effects of VEGF-A(165)b signaling can protect the retina from ischemic damage. These results suggest that VEGF-A(165)b may be a useful therapeutic agent in ischemia-induced angiogenesis and a cytoprotective agent for retinal pigment epithelial cells.

Figure 1.

VEGF-A₁₆₅b inhibits neovascularization in the OIR model but does not block revascularization. (A) Intraocular injection of VEGF-A₁₆₅b has a half-life of 62.6 hours in the eye. ¹²⁵I-VEGF-A₁₆₅b was injected into the vitreous, the rats were killed, and the eyes, urine, and blood were assayed using a gamma counter. Biexponential clearance was expressed as gamma counts per gram of tissue; terminal half-life was 2.6 days (62.6 hours). Uptake into the urine and blood was seen within 30 minutes. Fluorescein-dextran did not leak after intraocular injection into mice (insets). (B) Mice were subjected to hyperoxia during postnatal development and were injected with increasing concentrations of VEGF-A₁₆₅b or HBSS as a control. Retinal vessels were visualized by isolectin B4 staining at low (Bi, 4×) and high (Bii, 40×) power. Left: HBSS-treated (control); right: VEGF-A₁₆₅b–treated retinas. The central ischemic avascular region (yellow line), preretinal proliferation region (neovascularization, red line), and total vascularized retina (blue line) were measured. (C–E) The area in square micrometers of each defined region was measured in Image J. Neovascularization was significantly reduced by VEGF-A₁₆₅b injection (C), and the amount of normal vascularization was increased (D). This was partly a result of blood vessels growing into the avascular area, reducing the avascular area (E). Thus, VEGF-A₁₆₅b is able to maintain normal revascularization while inhibiting neovascularization, making it an ideal agent for ischemia-induced angiogenesis.

Figure 2.

VEGF-A₁₆₅b inhibits human REC migration. (A) Human RECs were seeded onto polycarbonate filters, and migration toward increasing concentrations of VEGF-A₁₆₅b was measured. (B) Inhibition of REC migration in response to 1 nM VEGF-A₁₆₅b compared with 1 nM ranibizumab.

Figure 3.

VEGF-A₁₆₅b is a survival factor for human endothelial cells. (A) HUVECs were serum starved (0.1% serum, SFM). LDH assay to measure cytotoxicity after 48-hour treatment with VEGF isoforms. VEGF-A₁₆₅ and VEGF-A₁₆₅b both inhibited cytotoxicity induced by serum starvation. (B) Cells were incubated either with VEGF-A₁₆₅b, VEGF-A₁₆₅, inhibitors, or VEGF-A₁₆₅b in the presence of VEGFR inhibitors and cytotoxicity determined by LDH activity in the media. Cytotoxicity is expressed relative to the appropriate control (i.e., inhibitor in SFM). The VEGFR inhibitors PTK787 (blocks both VEGFR) and ZM323881 (specific to VEGFR2) inhibited cytotoxicity. (C) Cells were treated with three different signal transduction inhibitors in the presence or absence of VEGF-A₁₆₅b, SB203580 (which blocks p38MAPK), PD98059 (which blocks p42/p44 MAPK phosphorylation by MEK), and LY294002 (which inhibits PI3K and cytotoxicity measured). MEK and PI3K inhibitors blocked the reduction in cytotoxicity but not the p38MAPK inhibitor. (D) Activation of VEGFR2, Tyr residue 1175 of VEGFR2, Akt, p42p44MAPK, and p38MAPK in endothelial cells by VEGF-A₁₆₅ and VEGF-A₁₆₅b. Cells were treated for 10 minutes with VEGFs. **P < 0.01 and ***P < 0.001 compared with control. One-way ANOVA, Student's Newman-Keuls post hoc test.

Figure 4.

VEGF-A₁₆₅b is a cytoprotective agent for RPE cells. (A–C) ARPE19 cells were treated with either Na butyrate (A) or hydrogen peroxide (B, C). Cells were incubated with VEGF-A₁₆₅b, VEGF-A₁₆₅, or EGF, and cytotoxicity was determined by measurement of LDH activity in the media. VEGF-A₁₆₅b inhibited cytotoxicity induced by Na butyrate (A) and H₂O₂ (B). Cells were treated with H₂O₂ and two different inhibitors in the presence or absence of VEGF-A₁₆₅b (C). PTK787, which blocks both VEGFR1 and VEGFR2, or ZM323881, which is specific for VEGFR2. Both inhibitors blocked the reduction in cytotoxicity induced by VEGF-A₁₆₅b. (D) RT-PCR of mRNA extracted from RPE cells indicated VEGFR2 expression. (E) VEGF₁₆₅b reduced loss of cell viability induced by 7-ketocholesterol, as assessed by WST1 assay. (F) VEGF₁₆₅b reduced LDH release from cells during treatment with 7-ketocholesterol. (G) VEGF-A₁₆₅b increased IGFBP3 expression in RPE cells, whereas VEGF-A₁₆₅ did not. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with no growth factor.

Figure 5.

VEGF-A₁₆₅b is an endogenous survival factor. (A) Immunofluorescence staining revealed expression of VEGF₁₆₅b (red) in RPE cells (Ai) that was confirmed by Western blot analysis (Aii) using a VEGF_xxxb-specific antibody, and mRNA was confirmed by RT-PCR (Aiii). Inhibition of endogenous VEGF_xxxb or all VEGF isoforms by bevacizumab increased cytotoxicity (Aiv). (B) Human endothelial cells show VEGF₁₆₅b expression (Bi) and inhibition of VEGF_xxxb increased cytotoxicity (Bii). ***P < 0.001 compared with control. Actin (green) and nucleus (blue).

Figure 6.

Pegaptanib binds VEGF-A₁₆₅ but not VEGF-A₁₆₅b and is not complementary to VEGF-A₁₆₅b. (A) VEGF protein (2 pmol) was incubated with 16 pmol pegaptanib or with a nonbinding scrambled aptamer for 30 minutes in HBS + 1 mM Ca²⁺/Mg²⁺, subjected to native SDS-PAGE, and probed using an antibody that detects both families of VEGF isoforms. The blot shows a band shift of VEGF-A₁₆₅ when aptamer was added but not when scrambled RNA or buffer, respectively, was added. VEGF-A₁₆₅b does not show a band shift when aptamer or when scrambled RNA was added under identical conditions. (B) Maximum inhibition of migration is seen at 10 nM pegaptanib. The same effect was seen with 1 nM VEGF-A₁₆₅b. The IC₅₀ for pegaptanib is 4 nM. (C) Adding in 0.5 nM VEGF-A₁₆₅b (close to IC₅₀) does not block the effect of pegaptanib but does cause a significant rightward shift in the dose-response curve, doubling the IC₅₀. (D) Adding 5 nM pegaptanib prevented the VEGF-A₁₆₅b mediated inhibition of migration induced by VEGF-A₁₆₅. **P < 0.01 compared with 1 nM VEGF-A₁₆₅ alone; ⁺⁺P < 0.01 compared with pegaptanib (ANOVA followed by Newman-Keuls test).