Physiological Hypoxia and Pharmacological HIF Stabilization Induce Distinct but Overlapping Responses in Retinal Pigment Epithelial Cells

Background/Objectives: Hypoxia is a major contributor to the development of retinal diseases by promoting metabolic adaptation and pathological angiogenesis through hypoxia-inducible factor (HIF) signaling. HIF-prolyl hydroxylase inhibitors (HIF-PHIs), which stabilize HIF under normoxic conditions, are widely used to treat anemia associated with chronic kidney disease. However, it remains unclear whether pharmacological HIF stabilization fully reproduces the cellular responses induced by physiological hypoxia. Methods: In this study, ARPE-19 cells were exposed to either physiological hypoxia or HIF-PHI treatment and analyzed using RNA sequencing, pathway enrichment, and pharmacological inhibition of PI3K/Akt, mTOR, and HIF-related signaling pathways. Results: Both treatments elicited a common transcriptional response marked by the induction of glycolytic and hypoxia-responsive genes together with increased vascular endothelial growth factor a VEGFA expression. Despite these similarities, only a small number of genes differed between the two conditions, suggesting that physiological hypoxia activates additional regulatory mechanisms beyond HIF stabilization alone. Rapamycin inhibited VEGFA production only under physiological hypoxia, further supporting mechanistic differences between the two models. Functionally, conditioned media from hypoxic ARPE-19 cells promoted endothelial tube formation, which was significantly reduced by pathway inhibition. Conclusions: Collectively, these findings show that HIF-PHIs reproduce many, but not all, aspects of the physiological hypoxic response in RPE cells.