Proteomic analysis of cerebral microvascular endothelial cells under physiological normoxia and hyperoxia: implications for drug discovery

Traditional cell culture is performed under atmospheric oxygen levels (18 kPa), which do not accurately reflect the lower oxygen levels experienced by most cells in vivo (Keeley & Mann, Physiol. Reviews 2019;99:161-234). This is particularly important for brain endothelial cells, which are exposed to oxygen levels no higher than 5 kPa. Hyperoxia leads to sustained oxidative stress with significant implications for drug discovery and screening. In this study, we investigated the proteomic profiles of cerebral microvascular endothelial cells (hCMEC/D3) adapted for 5 days to either physiological normoxia (5 kPa O₂₎ or standard hyperoxic conditions (18 kPa O₂). Using an untargeted label-free proteomics approach, we identified significant alterations in protein expression profiles between 5 and 18 kPa O₂. The observed differential expression profile was associated with an enrichment of pathways related to energy metabolism, oxidative stress and protein degradation. Furthermore, we investigated the effects of the NRF2 activator sulforaphane (SFN) in cells adapted to 5 or 18 kPa O₂. Proteomic analysis indicated that SFN (2.5μM, 24h) had negligible effects in cells adapted to 5 kPa O₂, whereas under 18 kPa O₂ SFN treatment led to an enrichment of pathways related to cell cycle regulation and cell proliferation. These findings highlight the critical importance of culturing cells under physiologically relevant O₂ levels to improve the clinical translation of in vitro models. Understanding how pericellular oxygen levels affect the phenotype of cells is essential for the design of drug discovery and high throughput screening protocols.