Fate of Biotinylated Basic Fibroblast Growth Factor in the Retina Following Intravitreal Injection

Abstract

Exogenous basic fibroblast growth factor (bFGF) stimulates proliferation of non-neuronal retinal cells in vivo. To help understand how this proliferative effect is mediated, we followed the fate of biotinylated bFGF after injection into the vitreous of normal rabbit eyes. The retinal distributions, binding, and processing of biotinylated bFGF (bFGF-biotin) was examined from 2hr to 7 days after intravitreal injection using laser scanning confocal microscopy, electron microscopy and Western blot analysis. At 2hr, bFGF-biotin was detected throughout the extracellular space and on retinal basement membranes. At 6hr, discrete punctate material first appeared within the cytoplasm of Müller cells, astrocytes, endothelial cells, retinal pigment epithelial (RPE) cells, and ganglion cells. Labeling was also present in the invaginations of the photoreceptor synaptic terminals at this time. This general pattern persisted up to 4 days after injection but was greatly attenuated by post-injection day 7. Labeling in the inner retina decreased progressively over the seven days; whereas labeling in the outer retina, primarily within the RPE, increased at 4 days post-injection and then gradually decreased to nearly undetectable levels by 7 days. Western analysis of retinal protein homogenates following injection showed that an 18kDa component representing intact bFGF, can be identified up to 1 week following injection. This component, as well as a 15 and 9kDa biotinylated fragment, showed a progressive reduction during the one week post-injection period. Cross-linking experiments demonstrated that bFGF-biotin binds to three putative receptors with approximate molecular weights of 54, 62, and 110kDa. These data are consistent with binding of exogenous bFGF to: (a) low affinity bFGF receptors associated with retinal basement membranes; (b) invaginations at the base of photoreceptor synapses; and (c) putative high affinity bFGF receptors on the plasma membranes of glial cells, endothelial cells, RPE cells and ganglion cells. bFGF-biotin apparently binds to, and is then internalized by, the same non-neuronal cell types that are stimulated to proliferate following retinal injuries such as detachment.