April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Role of Bestrophin in Basal Membranogenesis of Retinal Pigment Epithelium
Author Affiliations & Notes
  • A. Takase
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • T. Yasukawa
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • A. Nishiwaki
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • P. Wiedemann
    Ophthalmology, University of Leipzig, Leipzig, Germany
  • R. Sato
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • W. Eicher
    Ophthalmology, University of Leipzig, Leipzig, Germany
  • Y. Ogura
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • Footnotes
    Commercial Relationships  A. Takase, None; T. Yasukawa, None; A. Nishiwaki, None; P. Wiedemann, None; R. Sato, None; W. Eicher, None; Y. Ogura, None.
  • Footnotes
    Support  2006-2008 Grant-in-Aid for Scientific Research from Japan Society for the promotion of science
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 6008. doi:https://doi.org/
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      A. Takase, T. Yasukawa, A. Nishiwaki, P. Wiedemann, R. Sato, W. Eicher, Y. Ogura; Role of Bestrophin in Basal Membranogenesis of Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6008. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Best vitelliform macular dystrophy (Best disease) is one of early-onset forms of macular degeneration, linked to mutations in bestrophin-1 gene. The purpose of this study is to clarify the relationship between basal membranousgenesis of retinal pigment epithelium (RPE) and bestrophin, which is supposed to be a calcium-activated chloride channel, by use of a novel 3D culture system to observe basal functions of 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 96-well U-bottom culture plates to form spheroids, on the surface of which a monolayer of RPE was constructed. The spheroids 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 actin and elastin in human RPE spheroids were determined using each 1st antibody. The spheroids without bestrophin-1 interference were treated for an hour with Ca2+channel blocker, Cl-channel blocker and Ca2+ionophore at week 1, evaluated in the same manner.

Results: : While the surface of spheroids without siRNAs’ transfection became smooth in a day, reflecting lamellipodial crawling and fusion 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. In size, spheroids with siRNAs’ transfection became smaller than those without siRNAs’ transfection. Spheroids exposed to Cl-channel blocker had the similar rough surface. Immunohistochemistry showed that the expression of actin and elastin was reduced in RPE cells transfected with bestrophin-1 siRNAs or treated with Cl-channel blocker.

Conclusions: : The present study suggested that bestrophin might function as Cl-channel in spheroid culture. Cl-channel might play a role in biogenesis of Bruch’s membrane.

Keywords: Bruch's membrane • calcium • ion channels 
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