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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3469-3474.)
© 1997 American Heart Association, Inc.

Articles

Endothelial Cells Prevent Accumulation of Lipid Hydroperoxides in Low-Density Lipoprotein

David M. Smalley; Neil Hogg; B. Kalyanaraman; ; Kirkwood A. Pritchard, Jr

From the Department of Pathology (D.M.S., K.A.P.), Cardiovascular Research Center (D.M.S., K.A.P.), Department of Pharmacology and Toxicology (K.A.P.), and the Biophysics Research Institute (N.H., B.K.), Medical College of Wisconsin, Milwaukee.

Correspondence to Kirkwood A. Pritchard, Jr, PhD, Medical College of Wisconsin, Cardiovascular Research Center, 493D, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail kpritch{at}post.its.mcw.edu

Abstract A variety of cell types, including endothelial cells, oxidize low-density lipoprotein (LDL). To investigate the mechanisms by which endothelial cells modulate LDL oxidation states, endothelial cell cultures were incubated with LDL (240 mg cholesterol/dL) for 24 hours in M199 supplemented with fetal bovine serum (FBS, 16.7%). These conditions were not toxic to endothelial cells over the time frame of the study. Changes in LDL oxidation were monitored by measuring thiobarbituric acid–reactive substances (TBARS), lipid hydroperoxide (LOOH), and conjugated dienes (A_234nm). LDL medium incubated in the absence of endothelial cells contained higher TBARS than did LDL medium incubated with endothelial cells (0.35±0.08 versus 0.23±0.08 nmol MDA/mg, respectively). LOOHs were higher in LDL medium incubated without endothelial cells than in LDL medium incubated with endothelial cells (6.8±4.4 versus 0.49±0.89 nmol/mg, respectively). Conjugated diene formation, based on changes in absorbance at 234 nm, increased to a greater extent in LDL medium incubated in the absence of endothelial cells than when endothelial cells were present. To increase oxidative stress on the endothelial cell cultures, increasing concentrations of Cu²⁺ (0 to 4 µmol/L) were added to LDL medium. Endothelial cells prevented LOOH accumulation until the concentration of Cu²⁺ exceeded 0.75 µmol/L. At 1.5 and 4 µmol/L Cu²⁺, endothelial cells enhanced LOOH formation nearly 3 and 2.5 times the LOOH values in the corresponding medium incubated in the absence of endothelial cells. This loss of protective function however, was not permanent. Endothelial cells, preincubated for 24 hours with Cu²⁺-containing LDL medium, were still able to prevent LOOH accumulation in fresh LDL medium. Endothelial cells prevented LOOH accumulation even when exposed to LDL medium that contained low concentrations of LOOHs (<22 nmol/mg). However, endothelial cells accelerated the accumulation of LOOHs in LDL when exposed to LDL medium that contained slightly higher concentrations of preexisting LOOHs (33 nmol/mg). These data indicate that endothelial cells have a limited capacity for preventing LOOH formation and that small increases in LOOHs may play a critical role in enhancing the potential of endothelial cells for oxidative modification of LDL.

Key Words: LDL • endothelial cells • lipid peroxides

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