April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
The Mechanism of Basal Membranousgenesis of Retinal Pigment Epithelium and Bestrophin
Author Affiliations & Notes
  • A. Takase
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • T. Yasukawa
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • P. Wiedemann
    Eye Clinic, University of Leipzig, Leipzig, Germany
  • A. Nishiwaki
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • A. Kato
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • Y. Ogura
    Ophthalmology, Nagoya City Univ Med Sch, Nagoya, Japan
  • Footnotes
    Commercial Relationships  A. Takase, None; T. Yasukawa, None; P. Wiedemann, None; A. Nishiwaki, None; A. Kato, 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 2010, Vol.51, 6002. doi:
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      A. Takase, T. Yasukawa, P. Wiedemann, A. Nishiwaki, A. Kato, Y. Ogura; The Mechanism of Basal Membranousgenesis of Retinal Pigment Epithelium and Bestrophin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6002.

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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 membranogenesis 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 spheloids, 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, spheloids were sampled for western blotting or fixed in 4% paraformaldehyde for immunohistochemistry. The expression and distribution of actin and elastin in human RPE spheloids were determined by immunohistochemistry. The spheroids without bestrophin-1 interference were treated for an hour with Ca2+ channel blocker, Cl- channel blocker and Ca2+ ionophore at week 1 and 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 • retinal pigment epithelium • retinal degenerations: cell biology 
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