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A. Takase, T. Yasukawa, P. Wiedemann, A. Nishiwaki, Y. Ogura; Role of Bestrophin in Basal Membranogenesis of Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2008;49(13):480.
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Best vitelliform macular dystrophy (Best disease) is one of early-onset forms of macular degeneration, linked to mutations in bestrophin-1 gene. Bestrophin is expressed predominantly in retinal pigment epithelium (RPE) and supposed to be a calcium-activated chloride channel. However, its roles in physiological functions of RPE and pathogenesis of Best disease are mostly unknown. The purpose of this study is to clarify the relationship between basal membranogenesis of RPE and bestrophin by use of a novel 3D-culture system.
Human RPE cells were seeded onto 6-well culture plates. On the day when they were confluent, transfection of bestrophin-1 specific siRNAs was performed by use of lipofectamine 2000. After 48 hours, RPE cells were collected and seeded onto 96-well U-bottom culture plates to form spheloids, on the surface of which a monolayer of RPE was constructed. The spheroids with and without siRNAs' transfection were compared morphologically. One week later, spheloids were sampled for western blotting or fixed in 4% paraformaldehyde for immunohistochemistry. The expression and distribution of bestrophin, actin, cytokeratin, and elastin were determined using corresponding 1st antibodies. Transfection of siRNAs was confirmed by use of fluoresceinated siRNAs and a fluorescein microscope.
Western blotting disclosed reduced expression of bestrophin in RPE cells transfected with siRNAs. The expression of actin was reduced in these RPE cells, while cytokeratin and elastin did not alter the expression. The surface of control spheroids without transfection became smooth in a day, representing lamellipodial crawling and fusion of microvilli to cover the surface of the spheroids and possibly initiate Bruch's membrane formation. On the other hand, spheroids interfered with bestrophin-1 revealed rough surface and membranous deposits. Immunohistochemistry showed reduced expression of actin on spheroids transfected with siRNAs. Spheroids with transfection decreased their sizes faster than those without transfection.
The present study suggested that bestrophin might play a role in the initiation of basal membranogenesis of RPE. This spheroid culture system might be useful to clarify physiological functions of bestrophin and elucidate pathogenesis of Best disease.
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