Generation of reactive oxygen metabolites by phagocytosing endothelial cells

Abstract

We have studied the ability of particulate stimuli to induce the release of reactive oxygen metabolites from sub-cultured monolayers of human endothelial cells. Basal release of superoxide (O 2 −) and hydrogen peroxide from undisturbed monolayers was very low (108 pmol (O 2 − and 75 pmol H202 in 3 h from dishes of 3 × 10 5 cells). Addition of 1-μm diameter polystyrene microspheres, which were phagocytosed by the cells progressively, caused a dramatic increase in release of both metabolites; by 3 h, a 13.5- and 6.6-fold increase over controls was observed respectively ( P < 0.001). Addition of formaldehyde-fixed human platelets or chylomicron-size lipid particles also increased production of reactive oxygen species. Similar rises in H 2O 2 and (O 2 − production were induced by treatment with 10 7 M phorbol myristate acetate. Pretreatment of endothelial cells with neuraminidase, heparinase or heparitinase to alter their glycocalyx composition substantially enhanced the effect of microspheres on H 2O 2 and (O 2 − generation. We conclude that the interactions of particles, including platelets and lipids, with endothelial cells leads to the generation of significant pericellular levels of reactive oxygen species. These metabolites can oxidise a wide variety of nearby molecules, leading to cell damage and altered uptake characteristics for lipoproteins containing peroxidised lipids. These effects are exacerbated when endothelial cell glycocalyx composition is disrupted.