The B-isoform of VEGFA-165 Does Not Cause Retinal Neovascularization In Vivo, but Does Trigger Inflammatory Response and Breakdown of the Blood Retinal Barrier.

Abstract

Purpose: VEGFA’s b-isoforms are reported to counter the angiogenic effects of VEGFA on cultured endothelial cells. There is speculation that the b-isoforms might be used to treat retinal neovascularization in diabetic retinopathy, AMD, ROP, and FEVR. However, while VEGFA b-isoforms cannot bind the NRP-1 co-receptor, they bind and activate VEGFR2. We hypothesize that NRP-1 binding is not required to disrupt the blood retinal barrier (BRB) in vivo, where many cell types contribute to cellular inflammatory response, including: endothelial cells, neurons, glia, and monocytes.

Methods: Intra-vitreal injection, of the Long Evans rat eye, compared the effects of human recombinant VEGFA-165b and VEGFA-165, in vivo. Functional assessment was accomplished by Fluorescein Angiography (FA) and Optical Coherence Tomography (OCT), comparing before and after injections. Real-time PCR gene-expression assays evaluated leukocyte endothelial cell adhesion, tight junction disruption, and macrophage activation.

Results: A single dose of VEGFA-165 (regular isoform) caused sustained dilation of the primary retinal veins at 24 hrs post-injection. Control injections of vehicle into the contralateral eyes had no effect. An equivalent dose of VEGFA-165b dilated retinal veins to the same extent. Co-injection of Aflibercept VEGF-trap blocked vein dilation caused by VEGFA-165b. Vessel dilation resolved slowly by 96 hrs. VEGFA-165b suppressed endogenous Vegfa gene expression (20%), but induced inflammatory response as indicated by the increased expression of the Tnfa, Cxcl1, and Cxcl2 genes (10-100 fold). Activation of leukocyte adhesion was revealed by elevated expression of the Icam1, and Selectin genes (Sele, Selp, Sell). Disruption of endothelial cell tight-junctions was confirmed by decreased Claudin-5 gene expression. Injections of VEGFA-165b twice per week for two weeks sustained chronic vascular dilation that did not resolve between injections. No neovascular growth was detected.

Conclusions: Either VEGFA-165b or VEGFA-165 can disrupt the BRB, therefore this process does not require binding of the NRP-1 co-receptor. Intra-vitreal VEGFA-165b reduced endogenous Vegfa gene expression and multiple doses did not cause neovascularization. However, BRB disruption and robust inflammatory response complicate the potential use of VEGFA b-isoforms to treat neovascularization.