Differential Effects of Retinal Fatty Acids Under Oxidative Stress Reveal DHA's Susceptibility to Ferroptosis and Polarity Disruption in iPSC Derived RPE Cells.

Age-related macular degeneration (AMD) is driven by chronic oxidative stress and lipid dysregulation in the retinal pigment epithelium (RPE), yet the specific contributions of individual retinal fatty acids remain unclear. Among these, docosahexaenoic acid (DHA)—the most abundant polyunsaturated fatty acid in the retina—may play a dual role, supporting visual function under physiological conditions but predisposing RPE cells to injury under oxidative stress. Here, we investigated how DHA and other retinal fatty acids modulate oxidative stress responses in induced pluripotent stem cell–derived RPE (iRPE) cells. Cells were exposed to DHA, arachidonic acid (ARA), or α-linolenic acid (ALA) with or without hydrogen peroxide (H₂O₂) to assess their effects on viability, lipid peroxidation, ferroptosis, necroptosis, barrier integrity, and VEGF secretion. Combined DHA and H₂O₂ exposure markedly reduced iRPE viability and trans-epithelial resistance, whereas ARA caused moderate and ALA minimal cytotoxicity. DHA uniquely induced hallmark features of ferroptosis, including lipid peroxidation, GPX4 downregulation, glutathione depletion, and iron accumulation. Treatment with ferroptosis inhibitors (ferrostatin-1, liproxstatin-1) restored viability, preserved polarity, and normalized VEGF secretion, while necroptosis inhibitors offered only partial protection. Notably, DHA-induced polarity loss occurred without activation of EMT transcription factors, revealing a ferroptosis-driven mechanism of barrier disruption. These findings identify DHA as a distinct sensitizer of ferroptosis in iRPE cells under oxidative stress, linking retinal lipid metabolism to ferroptotic vulnerability and suggesting that targeting ferroptosis may help preserve RPE integrity and slow AMD progression.Clinical Trial Number: Not applicable.