Effects of crystallin-β-b2 on stressed RPE in vitro and in vivo.
BACKGROUND
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-β-b2 (cryβ-b2) and crystallin-β-b3 (cryβ-b3) on ARPE-19 cells in vitro and on the retinal pigment epithelium (RPE) in vivo.


METHODS
The influence of cryβ-b2 and cryβ-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 cryβ-b2, cryβ-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 cryβ-b2 and cryβ-b3 to stressed ARPE-19 cells, the cells were exposed to UV-light. In a rat model, cryβ-b2-expressing neural progenitor cells (cryβ-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.


RESULTS
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 cryβ-b2 and transplanted cryβ-b2-NPCs protected RPE from indirectly induced stress.


CONCLUSIONS
The data suggest that the RPE response to retinal ganglion cell denegeration is mediated via crystallins, which may thus be used therapeutically.
Institute of Experimental Ophthalmology, School of Medicine, Westfalian Wilhelms-University Münster, Albert-Schweitzer-Campus 1, D15, 48149 Münster, Germany. Michael.boehm@ukmuenster.de
Michael R R
MR
Böhm
Harutyun
H
Melkonyan
Patrick
P
Oellers
Solon
S
Thanos
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D053595:Embryonic Stem Cells; D005260:Female; D037502:Galectin 3 / Q000378:metabolism; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D008297:Male; D009409:Nerve Crush; D058953:Neural Stem Cells; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D012162:Retinal Degeneration / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays; D038206:beta-Crystallin B Chain / Q000378:metabolism*
C103908:CRYBB3 protein, human; D037502:Galectin 3; D051100:Glial Cell Line-Derived Neurotrophic Factor; D038206:beta-Crystallin B Chain; C113634:beta-crystallin B2