Previous studies have established that low density lipoprotein (LDL) incubated with endothelial cells (EC) undergoes extensive oxidative modification in structure and that the modified LDL is specifically recognized by the acetyl LDL receptor of the macrophage. Thus, in principle, EC-modified LDL could contribute to foam cell formation during atherogenesis. Oxidatively modified LDL is also potentially toxic to EC. The present studies show that addition of probucol during the incubation of LDL with EC prevents the increase in the electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation. It has also been shown that oxidation of LDL catalyzed by cupric ion induces many of the same changes occurring during EC modification. Addition of probucol (5 ,M) also prevented this copper-catalyzed modification of LDL. Most importantly, samples of LDL isolated from plasma of hypercholesterolemic patients under treatment with conventional dosages of probucol were shown to be highly resistant to oxidative modification either by incubation with endothelial cells or by cupric ion in the absence of cells. The findings suggest the hypothetical but intriguing possibility that probucol, in addition to its recognized effects on plasma LDL levels, may inhibit atherogenesis by limiting oxidative LDL modification and thus foam cell formation and/or EC injury. Other compounds with antioxidant properties might behave similarly.

The monocyte/macrophage appears to be the precursor of many of the lipid-laden cells in atherosclerotic lesions, but the mechanism by which these cells accumulate cholesterol to become foam cells remains unclear. We have previously reported that cultured endothelial cells can modify low density lipoprotein (LDL) in a manner that leads to rapid uptake by the acetyl LDL receptor of macrophages. This modification involves free radical-induced peroxidation of LDL and is accompanied by many changes in the physicochemical properties of LDL including increased electrophoretic mobility, increased density, decreased content of esterified cholesterol, hydrolysis of phosphatidylcholine, and fragmentation of apolipoprotein B. Under conditions highly favorable to oxidation, a similar modification can occur even in the absence of cells. In the present studies, oxidation of LDL simply by exposure to 5 microM Cu++ resulted in a modification that was indistinguishable from that produced by endothelial cells. Moreover, it was demonstrated that LDL oxidation by either method is accompanied by a marked decreased in amino group reactivity, comparable to that seen with the chemical modifications of LDL that lead to recognition by the acetyl LDL receptor. Inhibitors of proteolytic enzymes did not reduce fragmentation of apolipoprotein B during oxidation. The rate of catabolism of intravenously injected oxidized LDL in guinea pigs was very rapid, and over 80% of the degradation occurred in the liver. The studies demonstrate that all of the changes associated with endothelial cell modification of LDL can be attributed to oxidation. The cells can, however, promote oxidation under conditions where it would otherwise occur very slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative modification of low density lipoprotein (LDL) has been implicated as a factor in the generation of macrophage-derived foam cells in vitro and in vivo. However, the exact mechanism of LDL oxidation has not been established. The present studies show that cellular lipoxygenase activity is involved in endothelial cell-induced oxidation of LDL. Inhibitors of lipoxygenase (but not inhibitors of cyclooxygenase) reduced LDL oxidation by as much as 70-85% under the conditions used. In contrast, the addition of pure (recombinant) superoxide dismutase inhibited by only =25% under the same conditions. Oxidation ofLDL by smooth muscle cells, on the other hand, was effectively inhibited by superoxide dismutase, as was Cu2+-catalyzed oxidation of LDL. When LDL was added to endothelial cell cultures within a dialysis bag, it did not undergo oxidative modification, suggesting that cell-LDL contact is necessary. We propose that an important element in cell-induced oxidation of LDL depends on (i) lipoxygenase oxidation of cellular lipids, followed by their exchange into LDL in the medium; (ii) direct lipoxygenasedependent oxidation of LDL lipids during LDL-cell contact; (iii) or both.

Incubation of low density lipoprotein (LDL) with endothelial cells or smooth muscle cells overnight has resulted in an oxtdative modification of LDL that results in its recognition by macrophages by way of the acetyl LDL receptor. In the present study, we examined whether macrophages themselves can oxidize and modify LDL in a manner similar to that of endothelial cells. Incubation of 125 l-labeled LDL with resident or thioglycollate-elicited macrophages for 24 hours in Ham's F-10 medium resulted in the appearance of thiobarbituric acid (TBA) reactive materials and trichloroacetic acid (TCA) soluble radioactivity in the medium. The LDL harvested from these incubations showed increased electrophoretic mobility and was degraded rapidly when added to fresh macrophages as compared to LDL previously incubated in the absence of cells. T he accumulation of lipid-laden foam cells of monocvte origin in the aortic intima is an early event in the development of atherosclerosis.1 " 3 Monocyte-macrophages take up and degrade native low density lipoprotein (LDL) by way of the classical LDL (B/E) receptor, but only at rather low rates. 4 On the other hand, chemically acetylated LDL and other chemically modified forms of LDL 5 -6 are taken up much more rapidly by a distinct, alternative receptor, designated the acetyl LDL or scavenger receptor. 7Incubation of macrophages with these chemically modified forms readily generates foam cells whereas it is difficult to generate foam cells by incubation with native LDL 4 unless very long incubation times are used.8 Incubation of native LDL overnight with cultured endothelial cells has been shown to result in a modification that converts LDL to a form recognized by the same receptor that recognizes acetyl LDL.9 " 11 This modification has been shown to depend upon the presence of trace metals in the medium, to involve extensive lipid peroxidation, and to require the ac- tion of a phospholipase A 2 . 12 ' 13 The peroxidation of LDL lipids during such incubations has been shown to account for the cytotoxicity of LDL for cultured endothelial cells. 14 All of these changes can be blocked by the addition of alpha-tocopherol, butylated hydroxytoluene (BHT), or by probucol, a drug used in the management of hyperlipoproteinemia. 15 Since macrophages generate active oxygen species, which may play a role in their ability to scavenge and kill cells, it seemed likely that they might share the ability to oxidatively modify LDL. It was recently reported 16 ' 17 that LDL is oxidized by human monocytes and neutrophils and that the electrophoretic mobility of LDL is increased after incubation with cultured porcine monocytes. We report here that mouse peritoneal macrophages, like circulating human monocytes, can cause extensive oxidation of LDL lipids. We show further that the modified LDL is specifically recognized by the acetyl LDL receptor on the same cells that oxidized the LDL and, finally, that this macrophage-modified LDL competes with endothelial cellmodified LDL for uptake and degradation.

Hypercholesterolemia is a major risk for atherogenesis. Recent evidence suggests that oxidative modification of the major cholesterol-carrying lipoprotein, low-density lipoprotein (LDL), renders it more atherogenic. Not only does oxidized LDL (Ox-LDL) have enhanced uptake by macrophages, which contributes directly to foam cell formation, it may also adversely affect many other aspects of arterial wall metabolism and thus contribute further to the atherogenic process. Inhibition of the oxidation of LDL may be another approach to inhibiting atherogenesis, additive to or even synergistic with lowering of plasma LDL levels.