Anaphylatoxin Complement Proteins C3a and C5a Induce AMD Cellular Endophenotypes in iPSC-RPE Through Apically Localized C3aR and C5aR Receptors

Abstract

Purpose : Age-related Macular Degeneration (AMD) is a leading cause of vision loss in the elderly population in the western world. The initiating events of AMD are linked to the degeneration of the retinal pigment epithelium coupled with over activation of the complement pathway in the outer retina/choroid. We derived iPSC-RPE from human fibroblasts to observe the role of complement activation in AMD pathogenesis in vitro.

Methods : iPSC-RPE were treated with complement competent human serum (CC-HS) or complement-incompetent human serum (CI-HS) to induce AMD cellular endophenotypes. RPE epithelial phenotype and functionality was measured using barrier resistance, immunostaining for RPE markers, ability to phagocytose photoreceptor outer segments (POS) and gene expression. C5 and C3 receptor blockers (PMX-53 and Compstatin) and C5 and C3 depleted sera were used to specifically assess the role of anaphylatoxin complement proteins in inducing AMD-like cellular endophenotypes in iPSC-RPE cells.

Results : CI-HS treatment does not induce a disease-like cellular phenotype, and thus serves as our control condition. Cellular stress due to CC-HS treatment resulted in iPSC-RPE losing their epithelial phenotype; which manifested with increased expression of EMT markers, and decreased junctional integrity/ decreased expression of tight-junctional proteins (Claudins and ZO1) between cells. The stressed iPSC-RPE monolayers exhibited a 6-fold decrease in their functional ability to digest outer-segments (p<0.0001). Cellular stress in iPSC-RPE also accelerated the accumulation of APOE positive sub-RPE deposits and increased cytoplasmic and extracellular lipid droplets. Sustained CC-HS stress ultimately resulted in cellular atrophy of the iPSC-RPE cells.
AMD-like cellular endophenotypes in iPSC-RPE cells were facilitated by the apically located C3aR and C5aR receptors. Neither the blockers nor the depleted sera treatments coupled with the CC-HS feeding conditions caused an AMD-like cellular endophenotype in iPSC-RPE; confirming the involvement of this signaling pathway in our stress model.

Conclusions : The notable cellular stress in CC-HS treated iPSC-RPE leads us to believe that apically located receptors, C3aR and C5aR likely initiate C3a and C5a mediated signaling in iPSC-RPE cells leading to AMD-like changes in the cells.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.