GENERATION OF REACTIVE OXYGEN METABOLITES BY PHAGOCYTOSING ENDOTHELIAL CELLS: REGULATORY ROLE OF THE GLYCOCALYX

We have studied the ability of particular stimuli to induce the release of reactive oxygen metabolites from sub-cultured monolayers of human umbilical vein endothelial cells. Superoxide production was measured as the superoxide dismutase-inhibitable reduction of exogenous cytochrome C, and H₂O₂ production was measured by the horseradish peroxidase catalysed oxidation of scopoletin. Basal release of either metabolite from undisturbed monolayers was low (80pmol O₂ and 65pmol H₂O₂ in 3 h from dishes of 3×10⁸ cells). Addition of 1μM diameter polystyrene microspheres (4.5×10⁸ particles/dish) which were phagocytosed by the cells progressively over 3h, caused a dramatic increase in release of both metabolites (2nmol O₂ and 490pmol H₂O₂ at 3h). Release of O₂ was linear, whereas release of H₂O₂ was more rapid in the first hour. Addition of formaldehyde-fixed human platelets (2.5×l0⁶/dish) significantly enhanced O₂ production (490pmol at 2h) and raised, thought not significantly, H2O2 release (130pmol at 2h); addition of a stabilized lipid emulsion (lipofundin-S ; particle diameter 1μm, 3.6×10⁸ particles/dish) caused a significant rise in O² production (1.3nmol at 2h) but measurement of its effect on H₂O₂ release was prevented by interference in the assay. Similar rises in H₂O₂ and O₂ production were induced by treatment with 10^™7M phorbol myristate acetate. Pretreatment of endothelial cells with 12.5mU /ml neuraminidase or 0.5U/ml heparinase for 60 min to alter their glycocalyx composition substantially enhanced the effect of microspheres on H₂O₂ and O₂ generation, particularly at early times (lh). We conclude that the interactions of particulates, including platelets and lipids, with endothelial cells can lead to the generation of significant pericellular levels of reactive oxygen species. These metabolites may 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.