Pech-Amsellem, M.A., Myara, I., Storogenko, M., Demuth, K., Proust, A., and Moatti, N.
Objective: The aim of the study was to investigate LDL modifications by cultured human umbilical vein endothelial cells (HUVEC) from women smokers and non-smokers. Methods: Modifications of LDL by HUVEC were studied by determining the values of thiobarbituric acid-reactive substances (TBARS) and the percentage of the most electronegative oxidized LDL fraction (fraction C) by using an ion-exchange chromatographic method based on fast protein liquid chromatography (FPLC). We also studied the cellular production of superoxide anion, the effect of various inhibitors and cysteine, and determined total intracellular glutathione content and cell growth. Results: LDL exposed to HUVEC from smokers for 48 h showed significantly greater modifications than LDL exposed to HUVEC from non-smokers, as assessed by TBARS determination (19.4 ± 1.2, mean ± s.e.m., n = 20 versus 15.4 ± 0.7 nmol/mg LDL, n = 19; P < 0.01) and by FPLC (percentage of fraction C: 39 ± 7, n = 29 versus 14 ± 3, n = 34; P < 0.001). Moreover, HUVEC from smokers produced significantly more superoxide anion than those from non-smokers (0.46 ± 0.13 nmol/105 cell/min, n = 9 versus 0.22 ± 0.05, n = 10; P < 0.05). Superoxide production, like cell-induced modification of LDL, was strongly dependent on the presence of cysteine in the medium. Furthermore, HUVEC from smokers had a significantly (P < 0.05) higher total intracellular glutathione content than those from non-smokers (39.9 ± 3.1 nmol/mg, n = 9 versus 31.8 ± 2.2, n = 7). Finally, HUVEC from smokers and non-smokers showed similar growth at 48 h. Conclusion: HUVEC from smokers converted significantly more LDL into an atherogenic form than HUVEC from non-smokers, a phenomenon that was not due to altered cell growth. HUVEC-mediated LDL modifications were strongly thiol-dependent, as both LDL modifications and superoxide anion production were inhibited in cysteine-free medium. Stimulation of cystine uptake by HUVEC, reflected by the enhanced total glutathione content, could account for the enhanced superoxide anion production. All these observations may be relevant to the pathophysiology of smoking-related cardiovascular disease.