Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cells. II. Uptake and cytotoxicity of ultraviolet-treated LDL on lymphoid cell lines

A delayed and sustained rise of cytosolic calcium is elicited by oxidized LDL in cultured bovine aortic endothelial cells

“…LDL were isolated from human pooled fresh sera by ultraccntrifugation according to Havel [23], dialyzed, sterilized by filtration and their purity controlled as previously indicated [15,16]. Purified LDL were stored at 4"C under nitrogen (up to 2 weeks), For each experiment, 2-4 nag (as apoB) of LDL were exposed to UV-C radiations (254 nm, 0.5 roW/cruz, for 2 h, under the standard conditions) and immediately incorporated (at the concentrations indicated in the text) in the culture medium [16],…”

“…Purified LDL were stored at 4"C under nitrogen (up to 2 weeks), For each experiment, 2-4 nag (as apoB) of LDL were exposed to UV-C radiations (254 nm, 0.5 roW/cruz, for 2 h, under the standard conditions) and immediately incorporated (at the concentrations indicated in the text) in the culture medium [16],…”

“…The cytotoxicity was determined under the previously used conditions [16], with slight modifications, LDL (200/.tg apoB/ml), sterilized by filtration on 0.2#m Millipore membrane, were added to the culture medium. Two alternative procedures have been used: (i) in continuous pulse experiments, cells were kept in contact with LDL until the cell viability was determined (at the end of the 48 h-pulse period, under the standard conditions); (ii) in pulse-chase experiments, ¢zlls were in contact with LDL for 5 h, then the medium was discarded and replaced by a lipoprotein-free medium, until the cell viability was determined (up to 48 h when indicated).…”

“…genesis of necrotic lesions of the atheroma and defect of the endothelial cell lining [5,12,13]. In order to further investigate the mechanism of this 'cytotoxie' effect, we have recently developed a new experimental model system consisting of LDL irradiated by UV-C [14][15][16] which promote lipid peroxidation without detectable change in apoB-100 [14,15]. UV-treated LDL are taken up through the apoB/E receptor pathway and induce a membrane 'blebbing' and a marked loss of cell viability on lymph- old cells [14].…”

“…The bovine aortic endothelial cell line (GM 7372A) was obtained from the NIGM human genetic mutant cell repository (Camden, NJ, USA) and was grown in RPMI 1640 medium containing 10% fetal calf serum, penicillin, streptomycin and glutamine. 48 h before LDL ineorporatation, this medium was removed and replaced by RPMI 1640 containing 2% Ultroser G (a serum substitute devoid of lipoprotein), as previously used [16].…”