MECHANISMS INVOLVED IN THE IN-VITRO MODIFICATION OF LOW-DENSITY-LIPOPROTEIN BY HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS AND COPPER IONS

In this study we evaluated the time course and mechanism of low densit y lipoprotein (LDL) oxidation induced by human umbilical vein endothel ial cells (HUVECs), cell-free medium (CFM) and Cu2+. After incubating LDL (200 mu g/ml) with HUVECs, CFM and Cu2+ (concentration adjusted to obtain the same degree of LDL modification as with HUVECs), the exten t of LDL lipid peroxidation and apoprotein B modification was monitore d at different times from 0 to 24 h, This involved evaluating the time course of LDL conjugated diene, peroxide, malonyldialdehyde (MDA), fl uorescence, relative electrophoretic mobility (REM), vitamin E and mon ounsaturated and polyunsaturated fatty acids. After incubation with HU VECs, the LDL REM was significantly higher than that obtained in CFM ( p < 0.01). When balanced for the same degree of LDL modification as ob tained with HUVECs, Cu2+ gave a REM similar to that obtained with HUVE Cs. At the different times of incubation there was no statistical diff erence between conjugated diene and peroxide values after incubation w ith HUVECs and with CFM. The values obtained with Cu2+ were significan tly higher than those obtained with HUVECs and CFM (p < 0.01). MDA and LDL fluorescence were significantly higher after exposure to HUVECs t han to CFM (p < 0.01), values being similar to those obtained with Cu2 +. There was no statistical difference between the values of LDL oleic , linoleic, arachidonic and eicosapentaenoic acids after incubation wi th HUVECs and CFM. Eicosatetraynoic acid (ETYA), a lipoxygenase inhibi tor, determined dose-dependent reduction of MDA formation induced by t he incubation of LDL with HUVECs; it did not affect LDL conjugated die ne. ETYA did not have any effect on the MDA derived from LDL after inc ubation with Cu2+ or CFM. The results of this study demonstrate that, unlike Cu2+, the contribution of HUVECs to LDL modification does not i nvolve only lipid peroxidation of the lipoprotein; it also includes in tracellular radical and non-radical processes.