Abstract
Purpose :
Cell-based models are valuable tools to study the mechanisms of pathology at early stages of age-related macular degeneration (AMD), including the formation of sub-retinal pigment epithelial (RPE) deposits mediated by the activation of the complement system. We propose that alterations in the extracellular matrix (ECM) of Bruch’s membrane (BrM) caused by aging or mutations accelerate the rate of hydrolysis of complement C3 to C3(H2O) (tick-over), which forms a stable C3(H2O)-convertase on the ECM that activates C3 chronically and ultimately leads to deposit formation.
Methods :
To test our hypothesis, we have used CRISPR-based gene editing tools to generate induced Pluripotent Stem Cells (iPSCs) carrying the mutation p.923delDG in C3, which allows complement activation via tick-over only. iPSC C3-/- and C3WT/WT were used as controls. The edited iPSCs were differentiated into RPE cells using direct differentiation methods and cultured on transwells coated with RPE-derived ECM that mimics BrM with AMD. To investigate the potential of C3 as a therapeutic target, C3 was inhibited with short hairpin (sh) RNAs in the iPSC-RPE cells grown on AMD-like ECM. Complement activation by tick-over or alternative pathway was quantified by the presence of C3(H2O) and C3a respectively in conditioned media measured via ELISA. Formation of basal deposits was characterized by immunofluorescence and electron microscopy.
Results :
iPSC-RPE cells C3WT/WT and C3WT/923DG grown on AMD-like ECM secreted elevated levels of C3(H2O) and C3a compared to the same cells grown on normal ECM (ANOVA, **p<0.01). Abnormal deposition of collagen fibers underneath the RPE cultured on AMD-like ECM was also observed by immunostaining and electron microscopy after 3 months. Treatment with shRNA anti-C3 decreased the mRNA levels of C3 (ANOVA, ****p<0.0001) as well as the amount of C3(H2O) secreted to the media of wild type cells grown on AMD-like ECM (ANOVA, **p<0.01). The deposition of collagens underneath the RPE was also diminished after the treatment with shRNAs anti-C3.
Conclusions :
The results are consistent with our hypothesis that abnormalities in the ECM result in increased activation of C3 via tick-over, which can be inhibited with shRNAs. Thereby, the tick-over could be used as a therapeutic target to prevent increased complement activation in patients with early AMD.
This is a 2021 ARVO Annual Meeting abstract.