Abstract
Purpose :
VEGF165 is the pathological VEGF isoform that causes retinal leukostasis in different rodent disease models including retinopathy of prematurity and diabetic retinopathy. Experimental evidence supports that the heparin-binding VEGF165 isoform, but not the VEGF121 isoform that lacks the heparin-binding domain (HBD), is responsible for inducing retinal leukostasis. Preliminary data from cell-based experiment suggest that the purified HBD protein can interfere with the pro-inflammatory activity VEGF165, likely by binding to the VEGF165 co-receptors heparan sulfate proteoglycans and neuropilins. We hypothesize that the HBD protein could be use to inhibit VEGF165-induced leukostasis and pathogenesis angiogenesis.
Methods :
Intravitreal injection of VEGF165 with and without recombinant HBD protein in wild type C57BL/6 mice was used for the leukostasis assay. Treated-eyes were perfused with FITC-conjugated Concanavalin-A, fixed in paraformaldehyde and retinas were flat-mounted for visualization of retinal leukostasis by fluorescent microscopy. For the angiogenesis experiment, P7 mice were housed in 75 % oxygen for 5 days then returned to room air for 5 days to establish an oxygen-induced retinopathy (OIR) model. Intravitreal injection of the HBD protein was given at P12 and pathological neovascular tufts were analyzed at P17 by FITC-conjugated isolectin B4 staining.
Results :
Injection of VEGF165 (2 pmol) significantly induced leukostasis, while co-injection with HBD protein (10 pmol) significantly suppressed VEGF165-induced retinal leukostasis (33.86±1.97 vs. 8.75±2.20, P<0.001), and to the levels comparable to the PBS injected negative control (5.67±1.11) (Figure 1A). In the OIR model intravitreally injected HBD protein (50 pmol) significantly suppressed formation of pathological vascular tufts compared to the vehicle control (4.19±0.52% vs. 8.85±0.41%, P<0.001) and promotes normal revascularization as measured by the reduction of avascular area in the retina compared to control (8.30±1.19% vs. 15.05±1.18%, P<0.01) (Figure 1B).
Conclusions :
Recombinant HBD protein is a potential therapeutic for pathological leukostasis and angiogenesis induced by VEGF165.
This is a 2020 ARVO Annual Meeting abstract.