Invest Ophthalmol Vis Sci. 2026 Feb 2;67(2):9. doi: 10.1167/iovs.67.2.9.
ABSTRACT
PURPOSE: To investigate whether the alternative pathway (AP) of complement inhibition restores the epithelial phenotype and ameliorates dry AMD biomarkers in AMD patient induced pluripotent stem cell-derived RPE (iPSC-RPE) monolayers.
METHODS: iPSC-RPE monolayers were established from an AMD patient iPSC line; ARPE-19 monolayers were used for comparison. For AP inhibition, monolayers were treated with CR2-fH (fusion protein composed of the iC3b/C3d/C3dg-binding region of complement receptor type 2 [CR2] linked to the inhibitory domain of human factor H [fH]) either as a soluble protein or by adeno-associated virus transduction. Complement challenge used 5% complement competent human serum (CC-HS); 5% heat-inactivated serum served as a control. Membrane attack complex, apolipoprotein E, lipid deposits, and autophagy were analyzed by immunostaining. Morphology and integrity were evaluated in brightfield images, using zonula occludens-1 (ZO-1) immunostaining, followed by ImageJ analysis, and transepithelial electrical resistance measurements.
RESULTS: iPSC-RPE monolayers expressed the RPE-specific markers zonula occludens-1 and RPE65 and exhibited a honeycomb pattern, as confirmed by nearest-neighbor analysis. Exposure to CC-HS significantly reduced transepithelial electrical resistance; increased membrane attack complex, apolipoprotein E, and lipid deposits; and altered levels of autophagy-related proteins. Preincubation with soluble CR2-fH or CR2-fH expression reversed these features. Quantitatively, similar changes were observed in ARPE-19 cell monolayers.
CONCLUSIONS: The AP inhibitor CR2-fH delivered either as a soluble protein or via gene therapy is efficacious in preventing serum-induced complement activation that led to monolayer disruption and AMD-like pathology in iPSC-RPE cells. These results contribute to existing data highlighting the importance of the AP of complement in dry AMD pathology and its potential role in AMD treatment.
PMID:41631752 | DOI:10.1167/iovs.67.2.9