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Rosario Fernandez-Godino, Eric A Pierce, Donita Garland; The Role of Complement and the Extracellular Matrix in Early Stage Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3532.
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© ARVO (1962-2015); The Authors (2016-present)
Macular degenerations are a common cause of vision loss in developed countries. The hallmark of these diseases is the formation of drusen between the retinal pigment epithelium (RPE) and the Bruch’s membrane. Gene targeted Efemp1R345W/R345W knockin mice are a documented model of EFEMP1-associated inherited macular degeneration. These mice develop features of macular degenerations, including extensive basal deposits considered precursors to drusen, only when complement is active. The objective of this project was to use cultured primary RPE cells to investigate the mechanisms involved in the pathogenesis of basal deposit formation including the role of the complement system in this process.
RPE cells from wild type, Efemp1R345W/R345W knockin, and Efemp1R345W/R345W:C3-/- double mutant mice were grown on laminin coated transwells. Cell phenotypes were characterized by scanning and transmission electron microscopy. The presence of complement components and factors, extracellular matrix proteins (ECM) and other proinflammatory molecules were quantified by qRT-PCR, inmunofluorescence and ELISA. The activities of specific proteins were modulated with antibodies and recombinant proteins.
Electron micrographs showed that RPE monolayers from Efemp1R345W/R345W knockin but not from wild type or double mutant Efemp1R345W/R345W:C3-/- mice secrete deposit-like material (Deposit-LM) in vitro. The DLM contains high amounts of ECM and complement proteins. The formation of deposits can be inhibited by neutralizing C3a, IL-1B, IL-6 or EFEMP1 in Efemp1R345W/R345W cultures. Wild type cells make deposits in the presence of C3a or recombinant mutant EFEMP1.
We have developed a primary RPE cell culture system, which mimics the formation of deposits in vitro in only 2 weeks. RPE cells from Efemp1R345W/R345W knockin mice generate the extracellular deposits through a C3a dependent mechanisms that is activated by EFEMP1R345W. These cells thus provide a model system to investigate the early mechanisms responsible for RPE dysfunction in macular degeneration. 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.
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