VEGF-A165b is an endogenous neuroprotective splice isoform of vascular endothelial growth factor A in vivo and in vitro

Abstract

Vascular endothelial growth factor (VEGF) A is generated as two isoform families by alternative RNA splicing, represented by VEGF-A165a and VEGF-A165b. These isoforms have opposing actions on vascular permeability, angiogenesis, and vasodilatation. The proangiogenic VEGF-A165a isoform is neuroprotective in hippocampal, dorsal root ganglia, and retinal neurons, but its propermeability, vasodilatatory, and angiogenic properties limit its therapeutic usefulness. In contrast, a neuroprotective effect of endogenous VEGF-A165b on neurons would be advantageous for neurodegenerative pathologies. Endogenous expression of human and rat VEGF-A165b was detected in hippocampal and cortical neurons. VEGF-A165b formed a significant proportion of total VEGF-A in rat brain. Recombinant human VEGF-A165b exerted neuroprotective effects in response to multiple insults, including glutamatergic excitotoxicity in hippocampal neurons, chemotherapy-induced cytotoxicity of dorsal root ganglion neurons, and retinal ganglion cells (RGCs) in rat retinal ischemia-reperfusion injury in vivo. Neuroprotection was dependent on VEGFR2 and MEK1/2 activation but not on p38 or phosphatidylinositol 3-kinase activation. Recombinant human VEGF-A165b is a neuroprotective agent that effectively protects both peripheral and central neurons in vivo and in vitro through VEGFR2, MEK1/2, and inhibition of caspase-3 induction. VEGF-A165b may be therapeutically useful for pathologies that involve neuronal damage, including hippocampal neurodegeneration, glaucoma diabetic retinopathy, and peripheral neuropathy. The endogenous nature of VEGF-A165b expression suggests that non-isoform-specific inhibition of VEGF-A (for antiangiogenic reasons) may be damaging to retinal and sensory neurons.

Figure 1

VEGF-A₁₆₅b is expressed in human and rat hippocampus. Human cortical sections from the Human Tissue Authority–licensed South West Dementia Brain Bank were stained with an anti–VEGF-A₁₆₅b antibody. Hippocampal staining in CA1 (A), CA2 (B), CA3, and dentate gyrus (DG; C). D: Negative control mouse IgG staining of the hippocampus. High-power view of DG stained with mouse IgG (right panel). E: Anti-VEGF₁₆₅b antibody preincubated with rhVEGF₁₆₅b reveals no staining of the hippocampus. High-power view (right panel). F: Anti-VEGF₁₆₅b antibody preincubated with rhVEGF₁₆₅a reveals similar staining of the hippocampus. G: Protein was extracted from cortex and hippocampus dissected from rat brains (n = 3) and subjected to ELISA for VEGF-A and VEGF-A_xxxb. VEGF-A_xxxa levels were estimated from the difference between total VEGF-A and VEGF-A_xxxb. H: Percentage of total VEGF-A that is VEGF-A_xxxb.

Figure 2

VEGF-A₁₆₅b is protective against glutamate-induced hippocampal neuronal excitotoxicity. A: Pseudocolored image of cultured hippocampal neurons exposed to glutamic acid, with and without VEGF-A₁₆₅b or galanin. Cells co-stained with Hoechst 33258 nuclear stain (blue) and Dead stain (red) are purple and represent approximately 75% of glutamic acid–treated neurons under control conditions. Also note effect of treatment with 10 nmol/L VEGF-A₁₆₅b on excitotoxicity in neurons co-incubated with 3 mmol/L glutamate. B: VEGF-A₁₆₅b has a concentration-dependent inhibitory effect on glutamatergic excitotoxicity in hippocampal neurons (P < 0.0001, n = 4, analysis of variance plus Newman-Keuls post hoc test, ^∗P < 0.01, ^∗∗P < 0.001). C: Effect of VEGF-A receptor inhibitors on the hippocampal neuroprotective effect of VEGF-A₁₆₅b [100 nmol/L PTK787 (nonspecific VEGFR2 antagonist), 10 nmol/L ZM323881 (VEGFR2-specific antagonist), and 100 nmol/L SU5416 (VEGFR1-specific antagonist); ^∗P < 0.05 compared with control, ^∗∗P < 0.01, n = 4 per group]. D: VEGFR2 (red) expression in β₃-tubulin–positive (green) neurons. Nuclei are stained blue. Higher-power individual color images (right panel) reveal membrane staining of the VEGFR2 outside tubulin.

Figure 3

The effect of intracellular kinase–pharmacological inhibition on VEGF-A₁₆₅b neuroprotection. A: Representative images of hippocampal neurons subjected to the live/dead cell viability stain after 24-hour treatment with 3 mmol/L l-glutamic acid in the presence of test compounds or respective vehicles. Neurons treated in culture media alone (neurobasal media) or l-glutamic acid plus 2.5 nmol/L VEGF-A₁₆₅b with or without 10 μmol/L SB203580 (p38 MAPK inhibitor) or 15 μmol/L LY294002 (PI3K inhibitor) maintained neurite projections (arrows). In the presence of l-glutamic acid plus VEGF-A₁₆₅b plus 10 μmol/L PD098059 (MEK1/2 inhibitor) neurons retracted neurites (arrowheads). Scale bar = 25 μm. B: The percentage of red-stained (dead) nuclei per total nuclei stained was calculated. More neurons died when treated with the MEK1/2 inhibitor plus VEGF-A₁₆₅b (PD) than when treated with VEGF-A₁₆₅b and vehicle (control). Neither p38 MAPK (SB) nor PI3K (LY) inhibition had any effect on the neuroprotection exerted by VEGF-A₁₆₅b. Data are means ± SEM, n = 3 of 4 one-way analysis of variance plus Bonferroni post hoc comparison; increase of cell death in glutamic acid plus inhibitor/vehicle over media plus inhibitor/vehicle (white bars) compared with increase of cell death in glutamic acid plus VEGF-A₁₆₅b and inhibitor/vehicle (black bars), ^∗P < 0.05, ^∗∗P < 0.01. C: The level of phosphorylated p44/42 MAPK detected by immunoblotting was increased in cultured 50B11 neurons treated with 0.1 nmol/L rhVEGF₁₆₅b compared with control (n = 3). D: PD98059 blocked phosphorylation of p42/p44 MAPK induced by VEGF₁₆₅b in 50B11 neurons (^∗P < 0.05, ^∗∗P < 0.01 compared with untreated).

Figure 4

VEGF-A₁₆₅b protects retinal neurons from ischemia induced cell death in vivo. A: Pseudo-colored fluorescent images of retinal cells revealing the contralateral nonischemic retina, ischemic eye injected with HBSS, or ischemic eye injected with VEGF-A₁₆₅b. B: Staining of retinae of HBSS- or VEGF-A₁₆₅b–injected rats for activated caspase 3 (red) and nuclei (blue). C: Live RGC counts were significantly lower in ischemic eyes compared with nonischemic eyes. VEGF-A₁₆₅b treatment resulted in more viable RGCs (n = 8 HBSS, n = 12 VEGF-A₁₆₅b, P < 0.001, analysis of variance plus Bonferroni post hoc test). VEGF-A₁₆₅b treatment increased the numbers of live Fluorogold-labeled retinal cells compared with the HBSS and control untreated eyes, which can be clearly seen in D. The ratio of RGCs per field in the ischemic and nonischemic eyes were compared. E: Neuroprotection by VEGF-A₁₆₅b was mediated through an inhibition of apoptosis, as indicated by a reduction in active caspase-3 staining, in both the RGCs and inner nuclear layer (INL). Data are means ± SEM. ^∗∗P < 0.01, ^∗∗∗P < 0.001 compared with contralateral and ^†††P < 0.001 compared with HBSS.

Figure 5

VEGF-A₁₆₅b is cytoprotective for primary sensory neurons. A: Effect of VEGF-A₁₆₅b in a model of chemotherapeutic-induced neurotoxicity. Primary adult rat DRG cultures were treated with increasing concentrations (0, 5, 10, and 20 μg/mL) of the chemotherapeutic oxaliplatin for 24 hours with or without 2.5 nmol/L VEGF-A₁₆₅b (after 16 hours of pretreatment). The percentage of activated caspase-3–positive NeuN-positive cells was determined after immunofluorescence analysis. The percentage of neurons positive for activated caspase-3 was determined after treatment with 0 to 20 μg/mL of oxaliplatin for 24 hours with or without 2.5 nmol/L VEGF-A₁₆₅b. VEGF-A₁₆₅b treatment inhibited oxaliplatin-induced caspase-3 expression. Data are means ± SEM (n = 3). B: Representative images of NeuN-positive cells after treatment. NeuN-negative cells (only blue or blue and red) were not counted. Arrows signify activated caspase-3 detected both around and in neuronal nuclei. C: Primary DRG neurons were pretreated with concentrations of a neutralizing VEGF₁₆₅b antibody for 20 hours before oxaliplatin treatment (0, 10 μg/mL; 24 hours). IgG content was equalized for the groups by addition of control mouse IgG. Pretreatment with anti-VEGF₁₆₅b did not affect the proportion of live neurons without oxaliplatin treatment. The percentage of live neurons was significantly decreased by anti-VEGF₁₆₅b after oxaliplatin treatment. D: Cell viability was measured by PrestoBlue absorbance from DRG neurons treated as above. Oxaliplatin treatment significantly decreased the viability without anti-VEGF₁₆₅b pretreatment compared with control. VEGF₁₆₅b pretreatment reduced the viability further still compared with control, which was significant from oxaliplatin treatment without anti-VEGF₁₆₅b. Data are means ± SEM, n = 6, one-way analysis of variance plus Bonferroni. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, compared with no oxaliplatin plus IgG control; ^†P < 0.05 compared with oxaliplatin IgG control.

Figure 6

Neuroprotection is mediated through VEGFR2 not VEGFR1. A: Primary cultured DRG were stained for βIII tubulin (green), VEGFR2 (red), and Hoechst (blue). βIII tubulin–positive cells were also positive for VEGFR2, with the receptor detected in the soma (white arrows) and along cellular projections (arrowheads). Some VEGFR2 detection did not colocalize to βIII tubulin (cyan arrows). The matched-species IgG-negative control confirms the detection of VEGFR2 expression. B: Treatment of DRG cultures with oxaliplatin plus 2.5 nmol/L VEGF-A₁₆₅b and 10 nmol/L VEGFR2 inhibitor ZM323881 blocked the neuroprotection exerted by VEGF-A₁₆₅b in the presence of ZM vehicle. Treatment of DRG cultures with 10 nmol/L PlGF, a competitor for VEGFR1, did not affect rhVEGF₁₆₅b neuroprotection against oxaliplatin. C: Treatment of DRG cultures with oxaliplatin plus 2.5 nmol/L VEGF-A₁₆₅b and 10 μg/mL of VEGFR2 blocking antibody DC101 blocked the neuroprotection exerted by VEGF-A₁₆₅b in the presence of rat IgG. Data are means ± SEM, (n = 3), one-way analysis of variance plus Newman Keuls. ^∗∗P < 0.01, ^∗∗∗P < 0.001, compared with no oxaliplatin; ^†P < 0.05, compared with respective concentration matched vehicle; and ns are compared with respective oxaliplatin without VEGF₁₆₅b; ^‡‡P < 0.01, ^‡‡‡P < 0.001, compared with respective oxaliplatin without VEGF₁₆₅b; ^§P < 0.05, compared with oxaliplatin and VEGF₁₆₅b. Scale bar = 50 μm.

Figure 7

VEGF-A₁₆₅b is neuroprotective for DRG neurons in vivo. PSNI was performed on anesthetized C57BL/6 mice, test compounds were administered biweekly by i.p. injection after surgery, and L4 DRG was harvested 14 days later. For each DRG a complete cross section profile was analyzed for ATF3 immunofluorescence intensity. A: Low-power representative images of ATF3 immunofluorescence intensities from whole ipsilateral L4 DRG sections and a species- and concentration-matched IgG negative control. B: Mean pixel intensity was calculated for each DRG profile and expressed relative to the mean pixel intensity for L4 DRG in the sham surgery group. PSNI increased ATF3 staining, which was blocked by VEGF-A₁₆₅b treatment. Results are means ± SEM, n = 3 per group. Statistical analysis was performed with Kruskal-Wallis and Dunn's multiple comparison test: test group ipsilateral DRG intensity versus ipsilateral DRG in the sham surgery group. ^∗P < 0.05. C: DRG ipsilateral to the injury was stained for ATF3 and the neuronal marker NeuN to identify nerve injury–induced neuronal ATF3 expression. Scale bar = 500 μm.