Abstract
Purpose::
Best vitelliform macular dystrophy (Best disease) is one of early-onset forms of macular degeneration, linked to mutations in bestrophin-1 gene. Best disease and age-related macular degeneration (AMD) may share their pathogenesis in part.The purpose of this study is to clarify physiological and pathological roles of bestrophin, which is supposed do be a calcium-activated chloride channel, by use of a novel 3D culture system to observe basal functions of retinal pigment epithelium(RPE).
Methods::
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 98-well U-bottom culture plates to form spheroids, on the surface of which a monolayer of RPE was constructed. The sphroids with or without bestrophin-1 interference were compared morphologically. One week later, spheroids were sampled for western blotting or fixed in 4% paraformaldehyde for immunohistochemistry. The expression and distribution of Bestrophin-1 in human RPE spheroids was determined using the 1st antibody against human bestrophin-1. As a control, RPE cells were transfected with fluoresceinated siRNAs and observed by fluorescein microscopy.
Results::
Effective transfection of siRNAs was confirmed with enhanced fluorescence in RPE cells incubated with control siRNAs.Western blotting showed reduced expression of bestrophin-1 in RPE cells transfected with corresponding siRNAs. While the surface of spheroids without siRNAs' transfection became smooth in a day, reflecting lamellipodial crawling and fusion of microvilli to cover the surface of the spheroids and possibly initiate Bruch's membrane formation, spheroids interfered with bestrophin-1 revealed rough surface and membranous deposits. Immunohistochemistry showed that bestrophin was distributed on the basolateral side of RPE.
Conclusions::
The present study suggested that bestrophin might play a role in the morphogenesis of basal structure and the wound healing of RPE. This spheroid culture system might be useful to clarify roles of bestrophin and other key molecules in physiological functions of RPE and in some pathologic states such as in Best disease and AMD.
Keywords: age-related macular degeneration • drusen • retinal pigment epithelium