In this study, we measured the influence of cholesterol rigidification on oxygen permeability in human endothelial cell monolayer membranes (ECs). Cholesterol‐induced membrane rigidification was assessed at different membrane depths by a fluorescence polarization method with diphenyl‐hexatriene (DPH) and 1‐(4‐trimethylamino)‐6‐phenylhexatriene (TMA‐DPH). Fluorescence quenching by oxygen was probed in preferentially labelled membrane with pyrene butyric acid (PyC
_{4}
) and pyrene dodecanoic acid (PyC
_{12}
), as shown with a 3D fluorescence microscope (CellScanTM System). With both probes the experiments revealed a decrease in oxygen diffusion as the cholesterol concentration increased in the medium culture (from 3.42
\mu
M to 17.11
\mu
M). We showed that very low concentrations of cholesterol (about 1000 times below normal value, 6.2 mM) particularly decrease oxygen levels or diffusion rate in the middle region of the membrane. In conclusion, these findings prove in a direct manner that cholesterol significantly affect the endothelial barrier function and molecular oxygen transfer to underlying tissues. Risk factors (cholesterol) directly would contribute to tissue ischemia.
