Effects of crystallin-?-b2 on stressed RPE in vitro and in vivo.

Böhm MR, Melkonyan H, Oellers P, Thanos S

Abstract

Crystallins are thought to play a cytoprotective role in conditions of cellular stress. The aim of this study was to determine the effects of crystallin-?-b2 (cry?-b2) and crystallin-?-b3 (cry?-b3) on ARPE-19 cells in vitro and on the retinal pigment epithelium (RPE) in vivo.The influence of cry?-b2 and cry?-b3 on the viability, proliferation and dying of ARPE-19 was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, bromo-2-deoxyuridine assay and life/death assay. The expressions of cry?-b2, cry?-b3, glial-derived neurotrophic factor (GDNF), and galectin-3 (Gal-3) in ARPE-19 cells were evaluated using immunohistochemistry (IHC), Western blotting (WB) and real-time-quantitative-PCR (qRT-PCR). To evaluate the response of cry?-b2 and cry?-b3 to stressed ARPE-19 cells, the cells were exposed to UV-light. In a rat model, cry?-b2-expressing neural progenitor cells (cry?-b2-NPCs) were injected intravitreally after retinal stress induced by optic nerve axotomy to examine whether they influence the RPE. Protein expression was examined 2 and 4 weeks postsurgery using IHC and WB.Detectable alterations of GDNF, and Gal-3 were found in ARPE-19 cells upon exposure to UV light. Adding the crystallins to the medium promoted proliferation and increased viability of ARPE-19 cells in vitro. The obtained data support the view that these crystallins possess epithelioprotective properties. Likewise, in vivo, intravitreally injected cry?-b2 and transplanted cry?-b2-NPCs protected RPE from indirectly induced stress.The data suggest that the RPE response to retinal ganglion cell denegeration is mediated via crystallins, which may thus be used therapeutically.