Pigment epithelium‐derived factor supports normal Müller cell development and glutamine synthetase expression after removal of the retinal pigment epithelium

Abstract

In conditions in which the retinal pigment epithelium (RPE) is dystrophic, carries a genetic mutation, or is removed physically, Müller cells undergo degenerative changes that contribute to the retinal pathology. We previously demonstrated that pigment epithelium-derived factor (PEDF), a glycoprotein secreted by the RPE cells with neuroprotective and differentiation properties, protects against photoreceptor degeneration induced by RPE removal. The purpose of the present study was to analyze the putative gliosupportive activity of PEDF on Müller cells of RPE-deprived retinas and assess whether protection of Müller cells was correlated with improved photoreceptor outer segment assembly. Eyes were dissected from Xenopus laevis tadpoles, and the RPE was removed before culturing in medium containing purified PEDF, PEDF plus anti-PEDF, or medium alone. Control eyes matured with an adherent RPE or in medium containing PEDF plus nonimmune serum. Müller cell ultrastructure was examined. Glial fibrillary acidic protein (GFAP) and glutamine synthetase were localized immunocytochemically, and the corresponding protein levels were quantified. In control retinas, Müller cells were structurally intact and formed adherens junctions with neighboring photoreceptors. In addition, they did not express GFAP, whereas glutamine synthetase expression was high. RPE removal dramatically altered the ultrastructure and biosynthetic activity of Müller cells; Müller cells failed to form adherens junctions with photoreceptors and glutamine synthetase expression was suppressed. PEDF prevented the degenerative glial response; Müller cells were ultrastructurally normal and formed junctional complexes with photoreceptors. PEDF also preserved the expression of glutamine synthetase at near-normal levels. The morphogenetic effects of PEDF were blocked by the anti-PEDF antibody. Our study documents the glioprotective effects of PEDF and suggests that maintenance of the proper Müller cell ultrastructure and expression of glutamine synthetase may be necessary to support the proper assembly of photoreceptor outer segments.

A novel function forHedgehogsignalling in retinal pigment epithelium differentiation

“…The RPE also seems to be involved in the maintenance of Müller cells. In conditions in which the RPE is dystrophic because of a genetic mutation, or when it is removed physically, Müller cells undergo degenerative changes (Jablonski et al, 2001). During development, signals from the RPE are involved in retinal histogenesis and layer formation (Layer et al, 1998;Jensen et al, 2001).…”

Contribution of Müller cells toward the regulation of photoreceptor outer segment assembly

“…Previously we demonstrated that in addition to its neuro-Rportive properties, PEDF also had a powerful morpho-genetic effect upon Müller cells and that it was able to prevent many of the dysmorphic and biosynthetic alterations in the glial cell population that were induced by removal of the RPE (Jablonski et al, 2001b). These data lead us to hypothesize that the glioprotective effects of PEDF may further Rport the proper folding of photoreceptor outer segments (Jablonski et al, 2001b), given the positive influence that Müller cells have upon outer segment assembly .…”

“…These data lead us to hypothesize that the glioprotective effects of PEDF may further Rport the proper folding of photoreceptor outer segments (Jablonski et al, 2001b), given the positive influence that Müller cells have upon outer segment assembly . To our surprise, PEDF was able to exert its neuro-Rportive effects in the absence of a Müller cell contribution.…”

“…Concentrations of supplements were 50 ng/ml purified bovine PEDF or 5 × 10 -5 M IPTG. In previous studies, these concentrations were shown to be those yielding the most effective Rport to photoreceptor outer segment assembly Jablonski et al, 2001b;Wang et al, 2003). In the parallel Müller cell inhibition experiments, 1 × 10 -5 M α-AAA was added to culture, i.e.…”

“…The RPE was left intact in RPE-supported control eye rudiments, while it was gently peeled away from the neuro-retina in RPE-deprived eye rudiments. Purified bovine PEDF (generously provided by Joyce Tombran-Tink, University of Missouri, Kansas City), IPTG (Sigma Chemical Co., St Louis, MO), and alpha-aminoadipic acid (α-AAA) (Sigma Chemical Co., St Louis, MO) were prepared as previously described Jablonski et al, 2001b;Wang et al, 2003).…”