PGC-1beta promotes oxidative damage and dysfunction of the retinal pigment epithelium

Abstract

Purpose : Oxidative damage and mitochondrial dysfunctions of the retinal pigment epithelium (RPE) are key early events in age-related macular degeneration but the root mechanisms for RPE pro-angiogenic switch remain unclear. The transcriptional co-activators, PGC-1α and β, are two master regulators of oxidative metabolism in many tissues. We recently demonstrated that PGC-1α controls RPE cells oxidative metabolism and protects cells from cytotoxic oxidative damage. Conversely to PGC-1α, PGC-1β expression in RPE is repressed during RPE maturation in a PGC-1α-dependent manner. Here we further examined the role of PGC-1β in RPE biology and response to oxidative damage.

Methods : Human fetal RPE and ARPE-19 cells were maintained and matured using standard procedures. PGC-1 isoforms gain of function was performed by adenoviral transduction. Mitochondrial respiration was measured with the Seahorse extracellular flux analyzer. Gene expression changes were analyzed by qPCR. Cell death was quantified by LDH assay. Oxidative damage was induced by treatment with H₂O₂ (0.5 mM for 18 hrs) and oxidized LDL (500 µg/ml for 24 hrs).

Results : Oxygen consumption rate analysis showed that forced expression of PGC-1β stimulates RPE mitochondrial respiration similarly to PGC-1α. Contrary to PGC-1α, PGC-1β significantly repressed the expression of critical antioxidant transcription factors (FOXO3, p<0.05 and NFE2L2, p<0.01) and enzymes (HMOX1, CAT, p<0.01) leading to increased oxidative damage and cell death (p<0.01) in RPE exposed to cytotoxic levels of H₂O₂ (0.5mM for 18hours). We next evaluated whether AMD-related toxicants could induce PGC-1β expression in cultured RPE and demonstrated that PGC-1β, but not PGC-1α, is selectively induced by cytotoxic doses of oxidized LDL (p=0.0101) while non-oxidized LDL had no effect. In cells exposed to H₂O₂, PGC-1β was also specifically induced at 6 (p<0.001) and 18 hours (p<0.05) post-treatment. Finally we demonstrated that PGC-1β overexpression in RPE robustly induced Placenta Growth Factor expression (p<0.01) but not VEGFA (p>0.05) and strongly repressed the RPE-secreted anti-angiogenic factors, Thrombospondin-1 (p<0.001) and PEDF (p<0.01).

Conclusions : All together, these findings indicate that PGC-1s exert paradoxical functions in RPE and that pathologic induction of PGC-1β may promote RPE oxidative damage and choroidal neovascularization.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.