Novel cell culture medium for use in oxidation experiments provides insights into mechanisms of endothelial cell-mediated oxidation of LDL.

Though one prominent theory of atherogenesis involves free-radical oxidation of low-density lipoprotein (LDL) within the vessel wall by one of the vascular cell types, the mechanism for cell-mediated LDL oxidation remains unclear[sn1]. In these studies we examined the effects of media phenols, thiols, and metals on endothelial cell-mediated oxidation. We found that cell culture media such as Dulbecco modified Eagle medium and minimal essential medium are unable to support cell-mediated oxidation of LDL because they contain high concentrations of phenol red (PR) and tyrosine, both of which strongly inhibit cell-mediated oxidation. Ham's F-10, a commonly used medium for cell-mediated oxidation experiments, is also not entirely appropriate, as it contains both PR and cysteine. Cysteine is not critical for endothelial cell-mediated oxidation, but does increase oxidation of LDL in the absence of cells. Finally, of utmost importance to cell-mediated oxidation was the presence of either micromolar concentrations of Fe(II) or physiological concentrations of holo-ceruloplasmin, the protein which carries copper in plasma. An appropriate culture medium for use in cell-mediated oxidation experiments should thus contain either micromolar concentrations of Fe(II) or physiological concentrations of holo-ceruloplasmin, and should be prepared without PR, cysteine, or large concentrations of tyrosine, all of which are shown here to inhibit endothelial cell-mediated LDL oxidation. These results are consistent with a mechanism of cell-mediated oxidation involving Fenton-type chemistry and redox cycling of the metal.

Dietary supplementation with beta-carotene, but not with lycopene, inhibits endothelial cell-mediated oxidation of low-density lipoprotein.

Carotenoids may protect low-density lipoprotein from oxidation, a process implicated in the development of atherosclerosis. Our previous studies showed that in vitro enrichment of low-density lipoprotein (LDL) with beta-carotene protected it from cell-mediated oxidation. However, in vitro enrichment with either lutein or lycopene actually enhanced oxidation of the LDL. In the present studies we have examined the impact of LDL carotenoid content on its oxidation by human aortic endothelial cells (EaHy-1) in culture, comparing the effects of in vivo supplementation with in vitro enrichments. The beta-carotene content in human LDL was increased three- to sixfold by daily supplementation with 15 mg beta-carotene for 4 weeks, and the lycopene content of LDL in other individuals was increased two- to threefold by ingestion of one glass (12 ounce) of tomato juice daily for 3 weeks. LDL isolated from these healthy, normolipidemic donors not taking supplemental carotenoid was incubated at 0.25 mg protein/ml with EaHy-1 cells in Ham's F-10 medium for up to 48 h. Following dietary beta-carotene supplementation, LDL oxidation (as assessed by formation of lipid hydroperoxides) was markedly inhibited, to an even greater extent than was observed for LDL enriched in vitro with beta-carotene (that resulted in an 11- to 12-fold increase in LDL beta-carotene). No effect on cell-mediated oxidation was observed, however, for LDL enriched in vivo with lycopene. Thus, beta-carotene appears to function as an antioxidant in protecting LDL from cell-mediated oxidation although lycopene does not. The fact that the three- to sixfold enrichments of LDL with beta-carotene achieved by dietary supplementation were more effective in inhibiting oxidation than the 11- to 12-fold enrichments achieved by an in vitro method suggests that dietary supplementation is a more appropriate procedure for studies involving the enrichment of lipoprotein with carotenoids.

Impact of LDL carotenoid and alpha-tocopherol content on LDL oxidation by endothelial cells in culture.

Carotenoids and alpha-tocopherol are dietary, lipophilic antioxidants that may protect plasma lipoproteins from oxidation, a process believed to contribute to atherogenesis. Previous work demonstrated that after the Cu(II)-initiated oxidation of human low density lipoprotein (LDL) in vitro, carotenoids and alpha-tocopherol were destroyed before significant lipid peroxidation took place, and that alpha-tocopherol was destroyed at a much faster rate than were the carotenoids. Additionally, in vitro enrichment of LDL with beta-carotene, but not with lutein or lycopene, inhibited LDL oxidation. In the present studies the impact of LDL carotenoid and alpha-tocopherol content on LDL oxidation by human endothelial cells (EaHy-1) in culture was assessed. LDL isolated from 11 individual donors was incubated at 0.25 mg protein/mL with EaHy-1 cells in Ham's F-10 medium for up to 48 h. Formation of lipid hydroperoxides was assessed by chemical analysis and the contents of lutein, beta-cryptoxanthin, lycopene, beta-carotene and alpha-tocopherol were determined by high performance liquid chromatography. The extent of lipid peroxidation correlated with the endogenous alpha-tocopherol content of the LDL but not with its content of carotenoids. As in the Cu(II)-initiated system, carotenoids and alpha-tocopherol were destroyed before significant peroxidation took place, but, in the cell-mediated system, alpha-tocopherol and the carotenoids were destroyed at comparable rates. Also, like the Cu(II)-initiated oxidation, enrichment of the LDL with beta-carotene protected it from oxidation by the endothelial cells. However, enrichment with either lutein or lycopene actually enhanced the cell-mediated oxidation of the LDL. Thus, the specific content of carotenoids in low density lipoprotein (LDL) clearly modulates its susceptibility to oxidation, but individual carotenoids may either inhibit or promote LDL oxidation.

Comparison of the intracellular metabolism and trafficking of 25-hydroxycholesterol and cholesterol in macrophages.

Oxysterols arising from the diet or through lipid peroxidation may be important in the modulation of cellular cholesterol metabolism. In this study, the metabolism of one of the oxysterols, 25-hydroxycholesterol (25OHC), was examined in J774 and mouse peritoneal macrophages. Uptake of 25OHC from serum was rapid and substantial. Esterification of the cellular 25OHC was also rapid as was hydrolysis of pre-formed esters. Like cholesterol, 25OHC was removed from cells by an extracellular acceptor such as high density lipoprotein. Unlike cholesterol, 25OHC was also rapidly and extensively removed from cells by serum albumin, but not by ovalbumin. The differential removal of oxysterols and cholesterol from cells by albumin allows separation of cellular effects due to oxysterols and cholesterol. In order to understand more about this differential efflux of sterols, a computer model for sterol mass transport in cells was used to compare intracellular trafficking of cholesterol and 25OHC. The rate constants determined by this model for movement of sterols between cytoplasm and plasma membrane were similar for both cholesterol and 25OHC, whereas those for esterification and ester hydrolysis as well as those for bidirectional movement between plasma membrane and extracellular medium were greater for 25OHC than for cholesterol. For both sterols, the rate-limiting step for removal of cellular esters appeared to be the rate of cytoplasmic ester hydrolysis. As 25OHC and cholesterol differ significantly in aqueous solubility, the similarity in their rate constants for movement between cytoplasm and plasma membrane is consistent with facilitation of transport between these two loci.

Pancreatic carboxyl ester lipase: a circulating enzyme that modifies normal and oxidized lipoproteins in vitro.

Pancreatic carboxyl ester lipase (CEL) hydrolyzes cholesteryl esters (CE), triglycerides (TG), and lysophospholipids, with CE and TG hydrolysis stimulated by cholate. Originally thought to be confined to the gastrointestinal system, CEL has been reported in the plasma of humans and other mammals, implying its potential in vivo to modify lipids associated with LDL, HDL (CE, TG), and oxidized LDL (lysophosphatidylcholine, lysoPC). We measured the concentration of CEL in human plasma as 1.2+/-0.5 ng/ml (in the range reported for lipoprotein lipase). Human LDL and HDL3 reconstituted with radiolabeled lipids were incubated with purified porcine CEL without or with cholate (10 or 100 microM, concentrations achievable in systemic or portal plasma, respectively). Using a saturating concentration of lipoprotein-associated CE (4 microM), with increasing cholate concentration there was an increase in the hydrolysis of LDL- and HDL3-CE; at 100 microM cholate, the present hydrolysis per hour was 32+/-2 and 1.6+/-0.1, respectively, indicating that CEL interaction varied with lipoprotein class. HDL3-TG hydrolysis was also observed, but was only approximately 5-10% of that for HDL3-CE at either 10 or 100 microM cholate. Oxidized LDL (OxLDL) is enriched with lysoPC, a proatherogenic compound. After a 4-h incubation with CEL, the lysoPC content of OxLDL was depleted 57%. Colocalization of CEL in the vicinity of OxLDL formation was supported by demonstrating in human aortic homogenate a cholate-stimulated cholesteryl ester hydrolytic activity inhibited by anti-human CEL IgG. We conclude that CEL has the capability to modify normal human LDL and HDL composition and structure and to reduce the atherogenicity of OxLDL by decreasing its lysoPC content.

Esterification of oxysterols in human serum: effects on distribution and cellular uptake.

Oxysterols, oxidized derivatives of cholesterol, may enter the circulation as contaminants of cholesterol-containing food, arise through peroxidation of lipoproteins, or be generated by enzymatic reactions. They are found in serum associated either with lipoproteins or with albumin. In these studies, 25-hydroxycholesterol (25OHC) was used as a model oxysterol to investigate the effect of esterification on the association of oxysterols with serum components and their delivery to cultured cells. 25OHC added in vitro to fresh human serum was readily esterified during incubation at 37 degrees C, most likely by serum lecithin:cholesterol acyltransferase (LCAT) as it was blocked by known inhibitors of LCAT. The 25OHC-esters formed were identified as monoesters by comparing their elution on high performance liquid chromatography and thin-layer chromatography with that of chemically synthesized 25OHC mono- and diesters. Esterification doubled the percentage of 25OHC associated with lipoproteins, concomitantly decreasing the amount associated with albumin. Whereas unesterified 25OHC readily transferred between isolated lipoproteins, 25OHC-esters remained associated with donor lipoproteins unless human lipoprotein-deficient serum was added. That cholesteryl ester transfer protein (CETP) mediated transfer of 25OHC-esters was demonstrated by the ineffectiveness of rat lipoprotein-deficient serum as well as by the ability of IC-4, an anti-CETP monoclonal antibody, to suppress the transfer. Esterification of 25OHC in serum limited its entry into human erythrocytes and fibroblasts (GM 3468A cells) in vitro. Up-regulation of fibroblast low density lipoprotein (LDL)-receptors enhanced the uptake of esterified 25OHC from medium, but did little to enhance the total uptake of 25OHC. Thus, esterification of oxysterols in serum shifts their distribution away from albumin into LDL and high density lipoprotein (HDL) and limits their availability to cells in culture.

Distributions of carotenoids and alpha-tocopherol among lipoproteins do not change when human plasma is incubated in vitro.

Carotenoids and alpha-tocopherol are dietary, lipophilic antioxidants which may protect plasma lipoproteins from oxidation, a process believed to contribute to atherogenesis. In this study, the quantities and distributions of carotenoids, alpha-tocopherol and major lipids in the plasma and lipoproteins of seven normolipidemic humans were determined. Experiments were also conducted to determine if these antioxidants redistribute among lipoproteins when plasma is incubated in vitro. Virtually all of the total carotenoid in plasma associated with lipoproteins, primarily LDL [73 +/- 10% (mean +/- SD)], as did the more non-polar individual carotenoids, beta-cryptoxanthin (68 +/- 9%); lycopene (79 +/- 9%), and beta-carotene (72 +/- 12%), in patterns which closely resembled the distribution of total cholesterol. Xanthophyll, the most polar carotenoid examined, distributed equally between LDL (44 +/- 11%) and HDL (38 +/- 14%), whereas alpha-tocopherol associated with LDL (43 +/- 12%), HDL (26 +/- 10%), and VLDL (27 +/- 13%). These patterns closely resembled that of phospholipid. Approximately four carotenoid molecules associated with each VLDL and one with each LDL particle, whereas only 25 of every 1000 HDL particles contained carotenoid. Approximately 145 molecules of alpha-tocopherol associated with VLDL, 12 with LDL, and one with each HDL particle. Unlike triglyceride and cholesteryl ester, known to transfer among lipoproteins through the action of cholesteryl ester transfer protein, net transfer of carotenoids and alpha-tocopherol among lipoproteins did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol.

The effect of oxysterols on efflux of cholesterol from mouse L-cell fibroblasts, rat Fu5AH hepatoma cells, J774 macrophages, and human EA.hy 926 endothelial cells was studied. Cells were preincubated with 25-hydroxycholesterol (25-OHC) either during labeling of the cells with [3H]cholesterol or during equilibration after labeling. Subsequently, the release of [3H]cholesterol into medium containing 0.2 mg HDL3/ml was measured and the fractional release of cellular [3H]cholesterol was calculated. Pretreatment with 25-OHC (1 microgram/ml) caused a 30% reduction in [3H]cholesterol efflux from L-cells during 8 h of incubation with HDL3. 25-OHC also inhibited cholesterol efflux from Fu5AH and J774 cells, but the effect was less marked. There was only a small, nonsignificant reduction of efflux from EA.hy 926 cells. The mechanisms of 25-OHC-induced inhibition of cellular cholesterol efflux was further studied in L-cells, because of their sensitivity to 25-OHC treatment. The effect of 25-OHC on cholesterol efflux was dose-dependent, with significant effects seen at 25-OHC concentrations as low as 50 ng/ml. The half-time for cholesterol efflux from 25-OHC-treated cells (5 micrograms/ml) was 13.0 +/- 3.3 h compared to 5.7 +/- 1.0 in control cells, corresponding to a 55% reduction in the rate of cholesterol release. Other oxysterols, including 7-ketocholesterol, 7 alpha- and 7 beta-hydroxycholesterol, and 22(S)-hydroxycholesterol also inhibited [3H]cholesterol efflux from L-cells significantly, but to a lesser degree. 25-Hydroxycholesterol (5 micrograms/ml) reduced efflux from both normal and cholesterol-enriched cells by 31 and 14%, respectively. Inhibition of efflux was similar when reconstituted HDL3-apolipoprotein/phosphatidylcholine particles or small unilamellar phosphatidylcholine vesicles were used as cholesterol acceptors instead of HDL3. The content of phospholipids, cholesterol and the FC/PL ratio of intact cells and from isolated plasma membrane vesicles were the same for control and 25-OHC-treated cells. Efflux of [3H]cholesterol from plasma membranes isolated from 25-OHC-treated cells was 20% less than efflux from membranes from control cells. The difference in efflux observed in intact cells is partially explained by the reduction in efflux from the plasma membrane. In conclusion, our studies suggest that oxysterols, especially 25-hydroxycholesterol, can reduce cellular cholesterol efflux in vitro. Therefore oxysterols, either endogenous or derived from the diet, may influence cellular cholesterol efflux in vivo, the first step in reverse cholesterol transport.

7 beta-hydroperoxycholest-5-en-3 beta-ol, a component of human atherosclerotic lesions, is the primary cytotoxin of oxidized human low density lipoprotein.

Modification of low density lipoprotein (LDL) by free radical oxidation renders this molecular complex cytotoxic. Oxidized lipoproteins exist in vivo in atherosclerotic lesions and in the plasma of diabetic animals, suggesting that lipoprotein-induced tissue damage may occur in certain diseases. We undertook purification and identification of the major cytotoxin in oxidized LDL. The lipid extract from oxidized LDL was subjected to multiple HPLC separations, and the fractions were assayed for cytotoxicity. Mass spectrometry and nuclear magnetic resonance identified the purified toxin as 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 beta-OOH-Chol). This molecule accounted for approximately 90% of the cytotoxicity of the lipids of oxidized LDL. We also found 7 beta-OOH-Chol in human atherosclerotic lesions from endarterectomy specimens obtained immediately after excision. These results are consistent with the hypothesis that the oxidized LDL present in lesions has the capacity to induce cell and tissue injury, leading to progression of the disease and the generation of the necrotic core of the lesion.

Treatment of cholesterol-fed rabbits with dietary vitamins E and C inhibits lipoprotein oxidation but not development of atherosclerosis.

New Zealand White rabbits were made hypercholesterolemic by feeding a high cholesterol diet (10 g/kg diet) with or without added antioxidants. The antioxidants used were either probucol (10 g/kg) or vitamin E (10 g/kg) plus vitamin C (0.6 g/kg). Serum cholesterol concentrations were monitored as a function of time. At the end of 10 wk, serum and lipoprotein vitamin E concentrations, the extent of oxidation of lipoprotein fractions (thiobarbituric acid reacting substances), the susceptibility of lipoprotein to oxidation in vitro (conjugated diene formation) and the extent of atherosclerosis (aortic area stained by Sudan IV and plaque thickness) were measured. Rabbits fed diets supplemented with vitamins E and C had markedly higher serum vitamin E concentrations, marked vitamin E enrichment in all lipoprotein fractions, less oxidation in VLDL and LDL and enhanced resistance of LDL to further in vitro oxidation, but did not have significantly less aortic atherosclerosis. Rabbits given supplemental probucol likewise exhibited reduced oxidation of lipoproteins. However, aortic atherosclerosis in these animals was significantly lower, as were serum cholesterol concentrations. Inhibition of lipoprotein oxidation itself was not sufficient to reduce atherosclerosis in cholesterol-fed New Zealand White rabbits.

Reduced cholesterol efflux to mildly oxidized high density lipoprotein.

Mild oxidation (< 6h) of human high density lipoprotein (HDL) induced by incubation with copper ions increased its thiobarbituric acid reactivity, decreased its phospholipid content, increased its free cholesterol to phospholipid ratio and reduced its ability to mediate cholesterol efflux from cells. While protein modification did occur with further oxidation (> 12h), diminished efflux of cell cholesterol was apparent for HDL samples without increased electrophoretic mobility or altered apolipoproteins. Thus, reduced cholesterol efflux to mildly oxidized HDL may reflect changes in its lipids rather than its apoproteins. By limiting cholesterol removal from cells, any mild oxidation of HDL which might occur in vivo could contribute to cellular cholesterol accumulation.

Activated human monocytes oxidize low-density lipoprotein by a lipoxygenase-dependent pathway.

Monocyte-mediated oxidation of low-density lipoprotein (LDL) converts the lipoprotein to a potent cytotoxin. The oxidation process requires monocyte activation and requires superoxide anion since it can be blocked by superoxide dismutase. In this study, the requirement for lipoxygenase activity is shown, in that 1) inhibitors of lipoxygenase prevent the alteration of LDL, 2) copper (II) (3,5-diisopropylsalicylic acid), an agent shown to enhance lipoxygenase activity in a cell-free system, similarly enhances monocyte-mediated LDL alteration, and 3) the (3,5-diisopropylsalicylic acid)-enhanced monocyte-mediated modification of LDL can be completely blocked by inhibitors of lipoxygenase or by superoxide dismutase. These data suggest an integral role for monocyte lipoxygenase in the generation by activated monocytes of the extracellular superoxide anion that participates in the oxidation of LDL and the conversion of LDL to a cytotoxin. Monocyte-modified LDL may be a mediator in tissue damage that accompanies atherosclerosis or occurs at sites of inflammation.

Antioxidant treatment of diabetic rats inhibits lipoprotein oxidation and cytotoxicity.

Increased lipid peroxidation products were detected in a lipoprotein fraction containing very low density lipoprotein (VLDL) and low density lipoprotein (LDL) obtained from rats made diabetic by streptozotocin injection. The enhanced oxidation in the diabetic VLDL plus LDL fraction correlated with the in vitro toxicity of this lipoprotein fraction to proliferating fibroblasts. In contrast, high density lipoprotein (HDL) was not cytotoxic. That the increased oxidation and development of cytotoxic activity in the diabetic VLDL + LDL was related to the diabetes was shown by the fact that insulin treatment of diabetic animals inhibited both oxidation and cytotoxicity of VLDL + LDL. In contrast, treatment of diabetic rats with the antioxidants vitamin E or probucol after diabetes was established also inhibited both the in vivo oxidation and in vitro cytotoxicity of diabetic VLDL + LDL, but without altering hyperglycemia. Vitamin E or probucol treatment thus allowed separation of the oxidation process from the hyperglycemia occurring in experimental diabetes. The mechanisms by which diabetes in humans or experimental animals leads to the various manifestations of tissue damage are unknown; however, these studies demonstrate for the first time that a relationship exists between the in vivo oxidation of lipoproteins in diabetes and the potential for tissue damage as monitored by in vitro cytotoxicity. Furthermore, these results suggest that the mechanism for certain aspects of tissue damage accompanying experimental diabetes may be mediated by lipid peroxidation products.

Superoxide anion participation in human monocyte-mediated oxidation of low-density lipoprotein and conversion of low-density lipoprotein to a cytotoxin.

Human monocytes, upon activation with opsonized zymosan, altered low-density lipoprotein (LDL) during a 24-h co-incubation, resulting in its oxidation and acquisition of cytotoxic activity against target fibroblast cell lines. Both the oxidation of LDL and its conversion to a cytotoxin were enhanced with time of incubation, with the most substantial changes occurring after 6 h of culture of LDL with activated monocytes. Unactivated monocytes did not mediate either alteration. Superoxide anion (O2-) participated in both the oxidation of LDL and its conversion to a cytotoxin since addition of superoxide dismutase (SOD) at the beginning of the co-incubation inhibited, in a concentration dependent fashion, both the monocyte-mediated oxidation and the monocyte-mediated conversion of LDL to a cytotoxin. As expected, the rate of superoxide anion release was greatest during the respiratory burst, very early in the 24-h incubation (0 to 2 h); however, exposure of LDL to monocytes during the respiratory burst was not required for LDL oxidation. The lower levels of O2- released by the cells hours after the respiratory burst had subsided were sufficient to lead to the initiation of LDL oxidation. Three results indicated that the oxidative modification of LDL into a cytotoxin required O2(-)-independent free radical propagation after O2(-)-dependent initiation. First, oxidation of LDL exposed to the activated, superoxide anion-releasing monocytes for 6 h could be almost completely blocked by the addition at 6 h of the general free radical scavenger butylated hydroxytoluene, but not by SOD. Second, LDL oxidation proceeded even after removal of LDL from the superoxide anion-producing, activated cells after various durations of exposure. Third, the development of substantial levels of lipid peroxidation products and the development of greater cytotoxicity occurred after 6 h of exposure of LDL to activated cells, long after peak O2- release had subsided. These results lead us to conclude that monocyte-mediated oxidation of LDL, leading to its transformation into a cytotoxin, requires release of O2- occurring as a result of activation but not necessarily during the respiratory burst, and also requires O2(-)-independent free radical propagation. The modification of LDL into a potent toxin by activated monocytes may explain the tissue damage in atherosclerotic lesions and other pathologic sites in which inflammatory cells congregate.

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60.

Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (greater than 30,000 Daltons) and in a lipoprotein-free, low molecular weight fraction (less than 30,000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury.

Lipoprotein oxidation and cytotoxicity: effect of probucol on streptozotocin-treated rats.

For a number of years, the cell-damaging effects of oxidized low-density lipoproteins (LDL) have been studied. Oxidized LDL-induced tissue damage may be important in vivo; there is mounting evidence for the occurrence of oxidized lipoproteins in various pathologic conditions such as in atherosclerotic lesions and in the plasma of diabetic humans and experimental animals. These developments led to the current study of lipoprotein oxidation in streptozotocin-induced diabetes in the rat. This presentation will first review investigations of the toxicity of LDL to cells grown in tissue culture that occurs when LDL becomes oxidized. Then the results are presented indicating that lipoprotein oxidation occurs in vivo in experimental diabetes and renders diabetic lipoproteins cytotoxic in vitro. Both the oxidation and the cytotoxicity of diabetic lipoproteins are inhibitable by treating the diabetic rats with lipophilic antioxidants such as probucol.

Toxicity of oxidized low-density lipoprotein to cultured fibroblasts is selective for S phase of the cell cycle.

Oxidized LDL (o-LDL) is toxic to a variety of cultured cells. Preliminary results suggested that susceptibility is enhanced by cell proliferation. As a step toward determining the mechanism of cytotoxicity, we chose to identify the cell cycle phase(s) during which exposure of cultured human fibroblasts to o-LDL leads to death. Cytochalasin B, which blocks cell migration and proliferation, and irradiation, which prevents mitosis but not migration, both blocked cytotoxicity. Colchicine, which arrests cells in mitosis but does not inhibit DNA synthesis, did not block cytotoxicity. Treatment of cells with hydroxyurea, which blocks cells prior to S phase, prevented cell death. Addition of o-LDL to cells immediately after S phase allowed mitosis without death. The above results coupled with results using cells synchronized by three different means indicate that cell death is selective for proliferating cells and occurs after exposure to o-LDL during S phase. Understanding the mechanism of o-LDL-induced death may have implications for tissue damage in vivo in the numerous instances of pathology in which oxidized lipoproteins or lipids are present.

Modulation of endotoxin-induced endothelial cell toxicity by low density lipoprotein.

Bacterial endotoxins (lipopolysaccharides (LPS] have been reported to the toxic to endothelial cells in vivo. In vitro they have been shown to be toxic to bovine endothelial cells but not to human endothelial cells. In this report we demonstrate that the presence of plasma low density lipoprotein (LDL) protected bovine endothelial cells from LPS-induced toxicity whereas the presence of LDL actually promoted LPS-induced toxicity to human endothelial cells. These effects of LPS were independent of its source or method of preparation. High density lipoprotein also inhibited LPS-induced toxicity to bovine endothelial cells but unlike LDL, did not enhance LPS-induced toxicity to human cells. The toxicity of LPS to human endothelial cells in the presence of LDL required the oxidation of LDL by free radicals produced by the endothelial cells. LDL modified by acetylation enhanced LPS-induced toxicity to both human and bovine endothelial cells. The toxicity to human endothelial cells of LPS plus either LDL (after endothelial cell-mediated oxidation) or acetyl-LDL was inhibited by fucoidin and polyinosinic acid, blockers of the acetyl-LDL (scavenger) receptor. Polymyxin B, a specific LPS antagonist, inhibited the toxicity of LPS to bovine endothelial cells but not the toxicity of LPS plus LDL to human endothelial cells. These results are consistent with our hypothesis that LDL prevents the toxicity of LPS to bovine endothelial cells by binding the LPS and making it less accessible to the cells. Human endothelial cells are not directly susceptible to LPS-induced toxicity but, unlike bovine cells, produce oxygen free radicals in sufficient quantity to oxidize LDL and render the LDL-LPS complex recognizable for uptake by a scavenger receptor-like process similar to that for acetyl-LDL. LPS thus enters the human endothelial cells via this complex and kills the cells. These findings may have important implications for the study of LPS-induced toxicity to endothelial cells in vitro and for understanding the phenomenon in vivo.

Monocytes and neutrophils oxidize low density lipoprotein making it cytotoxic.

Free radicals are believed to be involved in leukocyte induced tissue injury. The present studies were performed to determine whether low density lipoprotein (LDL) might serve as a mediator of tissue injury after leukocyte induced free radical oxidation of LDL. Our results show that incubation of LDL with monocytes or polymorphonuclear leukocytes (PMN) leads to oxidation of the lipoprotein rendering it toxic to proliferating fibroblasts. Monocyte activation enhances these effects. Butylated hydroxytoluene (BHT), vitamin E (vit E) and glutathione (GSH) virtually prevent the oxidation of LDL and the formation of cytotoxic LDL, indicating that these alterations are mediated by leukocyte-derived free radicals. This is the first demonstration that short-lived free radicals emanating from phagocytic cells could mediate cell injury through the action of a stable cytotoxin formed by the oxidation of LDL. The fact that lipoproteins can transfer a cytotoxic effect from leukocytes to proliferating cells reveals a pathway for cell destruction which may have implications in atherosclerotic plaque progression, macrophage mediated toxicity to tumor cells and tissue injury by inflammatory processes.

Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation.

Our purpose was to determine whether the action of oxidative free radicals released by endothelial cells and vascular smooth muscle cells grown in culture could be responsible for certain modifications to low density lipoprotein (LDL). In these experiments we showed that after a 48-hour incubation with human umbilical vein endothelial cells or bovine aortic smooth muscle cells, human LDL: 1) became oxidized, as evidenced by reactivity to thiobarbituric acid; 2) lost variable amounts of sterol relative to protein (up to 20%); 3) had an increased relative electrophoretic mobility (by 30% to 70%); and 4) became toxic to proliferating fibroblasts. None of these changes occurred after a 48-hour incubation with confluent fibroblasts or bovine aortic endothelial cells, and all could be virtually prevented by the presence of butylated hydroxytoluene or other free radical scavengers. The results suggest that cells modifying LDL do so in part by an oxidation of LDL subsequent to cellular generation of free radicals.