A primary RPE cell culture model to study early mechanisms of macular degeneration

Abstract

Purpose: Macular degenerations, inherited and age-related (AMD), share some common features of pathogenesis. A hallmark of these diseases is the formation of drusen. Gene targeted Efemp1 ^R345W/R345W knockin mice are a documented model of the inherited macular degeneration DHRD/ML. These mice develop extensive basal deposits, which are considered precursors to drusen. Efemp1 ^R345W/R345W:C3 ^-/- double mutant mice do not develop the basal deposits. This demonstrates a critical role for the complement system in the formation of basal deposits. The purpose of this study was to use a primary RPE cell culture system to investigate the mechanisms involved in the pathogenesis of basal deposit formation in the Efemp1 ^R345W/R345W mutant mice and the role of the complement system in this process.

Methods: Primary RPE cells from wild type, Efemp1 ^R345W/R345W knockin, and Efemp1^R345W/R345W:C3^-/- double mutant mice were grown on transwells. Cell phenotype was characterized by SEM and TEM. Expression levels of complement and extracellular matrix genes were quantified by qRT-PCR. Protein expression was determined by Western blot analysis and immunofluorescence using confocal microscopy.

Results: As demonstrated by qRT-PCR and immunofluorescence experiments, RPE cells from each strain express components of the complement system including C1q, C2, C3, C4, C5, C6, C7, Cfb, Cfd and Cfh, and extracellular matrix proteins such as Efemp1, elastin, fibronectin, collagens and laminins. Electron micrographs showed that RPE monolayers from Efemp1^R345W/R345W knockin but not from wild type or double mutant Efemp1^R345W/R345W:C3^-/- mice secrete deposit-like material in vitro. Immunostaining of the deposits revealed that EFEMP1, C3 and other complement components and extracellular matrix proteins co-localize in the deposits.

Conclusions: The results demonstrate that primary RPE cells from Efemp1 mutant mice produce deposit-like material, and recapitulate in vivo observations. These cells thus provide a model system to investigate the early mechanisms responsible for RPE dysfunction in macular degeneration. The results suggest that complement expressed locally by the RPE is important in basal deposit formation in Efemp1^R345W/R345W mutant mice. This model may be a useful system to test drugs, such as complement inhibitors, to prevent basal deposit formation in inherited macular degeneration as well as the more common age-related macular degeneration.

Keywords: 412 age-related macular degeneration • 557 inflammation • 701 retinal pigment epithelium