Purchase this article with an account.
T. Yasukawa, P. Wiedemann, A. Takase, A. Nishiwaki, M. Hirata, J. Kacza, Y. Yafai, W. Eichler, J. Seeger, Y. Ogura; Drusen Formation in a Three-Dimensional Culture System of Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2177. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Age-related macular degeneration (AMD) is a major cause of blindness in the old. Drusen, a local extracellular deposit between retinal pigment epithelium (RPE) and Bruch's membrane, is a hallmark of AMD. However, its origin and biogenesis have been still controversial. Although there is some evidence suggesting local inflammation in or beneath drusen with some similarity to Alzheimer's disease, one cannot determine whether local inflammation is cause or effect of drusen formation. Previously, we reported an in vitro RPE model using a three-dimensional culture system of isolated human RPE cells. This model enables direct observation of basal functions of RPE. The object of this study is to demonstrate that drusen can be formed just by RPE.
RPE cells were seeded onto 98-well U-bottom culture plates to allow spheroid formation. A monolayer of RPE was constructed on the surface of spheroids. One week later, spheroids were sampled for western blotting or fixed in 4% paraformaldehyde for immunohistochemistry. The expression and distribution of key proteins involving vitronection, apoE, amyloid beta, matrix metalloproteinase 2, and complement factor H in human RPE spheroids were determined using corresponding 1st antibodies.
Spheroids composed of RPE cells showed the formation of Bruch's membrane, transmembranous deposition of lipoproteins, membranous debris, and, in some cases, drusen formation. Vitronectin, apoE, amyloid beta were distributed diffusely in the deposits with lipoproteins, suggesting that physiological transmembranous deposition might be mediated by these proteins. Amyloid beta, matrix metalloproteinase 2, and complement factor H were expressed in or around membranous debris and drusen, which suggested that these proteins might be related to drusen formation.
Our results demonstrated that RPE could solely exhibit drusen with expression of key proteins on drusen biogenesis, strongly suggesting local inflammation joined by other inflammatory cells might be not cause but effect of drusen formation. This model may have a variety of potentials to elucidate not only drusen biogenesis but also pathogenesis of AMD.
This PDF is available to Subscribers Only