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Rosario Fernandez-Godino, Eric A Pierce; C3a triggers formation of sub-RPE deposits via the ubiquitin proteasome pathway.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):791.
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Progress in developing treatments for early age-related macular degeneration (AMD) has been hampered by the complexity and heterogeneity of the disease in patients, and by the difficulty in reproducing those features in animal models. We have developed a cell-based model that recapitulates the earliest clinical features of AMD, which includes formation of sub-retinal pigment epithelium (RPE) deposits mediated by the complement system. We hypothesize that C3a released upon complement activation plays a key role in the formation of sub-RPE deposits and can be a potential therapeutic target.
Freshly isolated human fetal (hf) RPE cells were cultured on transwells in serum-free media for 2 weeks. Next, cells were treated with different doses of recombinant human C3a in the presence or absence of a non-peptide C3a-Receptor (C3aR) antagonist for two to four additional weeks. Formation of basal deposits was characterized by transmission and scanning electron microscopy, and immunofluorescence, and quantified by measuring the integrated intensity using Image J. Ubiquitin proteasome pathway (UPP) activity was measured with fluorescent probes and matrix metalloproteinase-2 (MMP-2) activity was measured by zymography.
The addition of C3a caused normal hfRPE cells overexpress C3aR on the basal-lateral membrane in a dose-dependent fashion (p<0.01). Further, hfRPE cells treated with C3a for two weeks made basal deposits, which contained wide-spaced collagen and increased amounts of collagen IV (p=0.0040) and VI (p<0.0001) specifically. Additionally, MMP-2 activity was increased in conditioned media of cells treated with C3a. This response was not mediated by calcium influx, but by decreased activity of the ubiquitin proteasome (UP) upon binding of C3a to C3aR. The addition of C3aR antagonist prevented the formation of deposits and restored the UP function and ECM turnover by normalizing the MMP-2 activity.
The results demonstrate that C3a acts locally on RPE cells to stimulate production of basal deposits via interaction with C3aR, which in turn results in decreased UPP activity. Modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD. The cell-based model presented here can be used to study the mechanisms responsible for the RPE pathology in early AMD, and test potential therapeutic agents.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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