Nicotinamide and its role in reducing complement activation and oxidative stress in age-related macular degeneration

Abstract

Purpose : Recent work suggests that the vitamin B3 derivative nicotinamide (NA) ameliorates an age-related macular degeneration (AMD) phenotype in retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) (Saini et al., 2018). While NA is a frequent supplement in RPE culture media for its property to enhance iPSC-RPE differentiation, it may inadvertently disguise molecular phenotypes in cellular models of RPE pathologies. We therefore aimed to investigate the effects of NA depletion on RPE cell integrity and functionality as well as oxidative stress susceptibility in iPSC-RPE cell lines categorized into extremes of high or low genetic risk for AMD.

Methods : iPSC-RPE cells were cultivated on transwell inserts and depleted of NA one week after seeding. Oxidative stress was induced by sodium iodate (SI) treatment. RPE cell layer integrity and functionality was analyzed by transepithelial resistance (TEER) measurements, immunocytochemistry and a photoreceptor outer segment (POS) phagocytosis assay. mRNA expression of markers for RPE cell faith, complement cascade and oxidative stress response was measured by qRT-PCR and verified by Western Blot and ELISA.

Results : NA depletion had no influence on RPE monolayer integrity as shown by immunocytochemistry and TEER measurement. Upon NA depletion RPE markers BEST1 and RPE65 were down regulated, whereas expression of complement markers CFH and C3 and oxidative stress markers HMOX1 and NQO1 were increased. These changes were reversible by reintroducing NA. Intriguingly, cells depleted of NA showed an increased uptake of POS. Oxidative stress was induced by SI treatment. Cells depleted of NA did not show a more pronounced stress response than cells cultivated in presence of NA. Cell lines with different genetic AMD risk scores did not show differences for any of the parameters addressed.

Conclusions : While NA decreases baseline expression of oxidative stress response and complement cascade marker genes in iPSC-RPE the cellular responses to SI mediated oxidative stress remain unmodified. Additionally, RPE cells depleted of NA showed a much higher POS phagocytosis than cells cultivated with NA. It thus remains elusive if the beneficial effect of NA on oxidative stress and complement regulation makes it a suitable therapeutic agent for AMD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.