BACKGROUND: Endothelial cell injury mediated by activated polymorphonuclearleucocytes (PMN) occurs during inflammation or reperfusion after brain ischemia. Protein oxidation causedby activated PMN may lead to functional disturbances, degeneration and death of the endothelial cells.The aim of this study was to detect protein oxidation in endothelial cells induced by activated neutrophilsby using a novel fluorescent probe.
MATERIAL AND METHODS: Protein oxidation of Human Umbilical Vein EndothelialCells (HUVEC) in culture was investigated by a 15-min incubation with human neutrophils activated byphorbol myristate acetate (PMA) in the presence of tyramine coupled to the succinimidyl ester of (fluorescein-5 (and-6)-carboxamido) hexanoic acid. Dityrosine bond formation as reflected by the linkage of the fluorescenttyramine to proteins was determined by Western-blotting.
RESULTS: The oxidative burst generated by activatedneutrophils induced dityrosine formation in the extracellular proteins (ECP) of HUVEC. Similar resultswere obtained, when horseradish peroxidase (HRP) was used for the induction of oxidative stress. However,when hydrogen peroxide (0.1 mM) was used, dityrosine formation was not detected.
CONCLUSIONS: Fluorescentlylabelled tyramine is a powerful tool for the detection of ECP oxidation in endothelial cells. As longas the oxidation by the activated neutrophils is limited to ECP, the endothelial cells may be protectedby antioxidants.

Keywords: HUVEC, Neutrophils, protein oxidation, Dityrosine, Fluorescent probe