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

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.

Highly potent VEGF-A-antagonistic DARPins as anti-angiogenic agents for topical and intravitreal applications.

The next-generation ophthalmic anti-VEGF therapeutics must aim at being superior to the currently available agents with regard to potency and improved drug delivery, while still being stable and safe to use at elevated concentrations. We show here the generation of a set of highly potent VEGF-A antagonistic DARPins (designed ankyrin repeat proteins) delivering these properties. DARPins with single-digit picomolar affinity to human VEGF-A were generated using ribosome display selections. Specific and potent human VEGF-A binding was confirmed by ELISA and endothelial cell sprouting assays. Cross-reactivity with VEGF-A of several species was confirmed by ELISA. Intravitreally injected DARPin penetrated into the retina and reduced fluorescein extravasation in a rabbit model of vascular leakage. In addition, topical DARPin application was found to diminish corneal neovascularization in a rabbit suture model, and to suppress laser-induced neovascularization in a rat model. Even at elevated doses, DARPins were safe to use. The fact that several DARPins are highly active in various assays illustrates the favorable class behavior of the selected binders. Anti-VEGF-A DARPins thus represent a novel class of highly potent and specific drug candidates for the treatment of neovascular eye diseases in both the posterior and the anterior eye chamber.

VEGF-A165b is cytoprotective and antiangiogenic in the retina.

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.