Oxidatively-modified low-density lipoprotein (OxLDL) is thought to be involved in the early development of atherosclerotic lesions. The appearance of lipid-laden foam cells is known to be one of the typical features of atherosclerotic lesions, and accumulating evidence has demonstrated that foam cells are formed after taking up OxLDL by macrophages in vitro. However, the modified structures, distribution, and metabolism of OxLDL present in vivo are poorly understood. Recently, our studies, together with others, have demonstrated that OxLDL is actually present in circulating human plasma. Furthermore, we have provided evidence that foam cells accumulate modified apoB fragments derived from OxLDL in the cells. This article reviews recent progress in this field, including the intracellular metabolism of OxLDL in foam cells and the relevance of OxLDL as an in vivo ligand for macrophages.

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We investigated the influence of glycated low density lipoprotein (LDL) for vascular smooth muscle cell (SMC) proliferation or injury. We utilized glycated, slightly oxidized LDL (GLDL-LOX), glycated, auto-oxidized LDL (GLDL) and glycated, metal-induced extensively oxidized LDL (GLDL-OX) to examine the effect of glycation itself or combined glycation and oxidation on SMC. GLDL-LOX induced SMC proliferation and migration, and increased the number of platelet-derived growth factor receptor, β subunits, (PDGF-R) positive SMC. Also, GLDL-LOX promoted protease activity, compared with the other groups including native LDL (control). GLDL and GLDL-OX demonstrated SMC injury with apoptosis and Bax protein expression, compared with native LDL and GLDL-LOX. These results suggested that LDL glycation contributed to the progression of atherosclerosis by promoting SMC migration and proliferation, with little dependence on oxidative modification. Secondary auto-oxidation adding to glycation induced SMC apoptosis, and SMC injury occurred in the state of strong oxidation with glycation. We concluded that LDL glycation might play a key role in the progression of atherosclerosis in diabetes, and glycated LDL promoted atherosclerosis, even with little assistance from oxidation.

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The effect of various 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on the induction of HMG-CoA reductase and low density lipoprotein (LDL) receptor mRNA were quantitatively determined in the cultured human hepatoma cell line Hep G2 by means of a ribonuclease protection assay. Lipophilic inhibitors including mevastatin, simvastatin, atorvastatin and NK-104 were able to increase the levels of mRNAs for HMG-CoA reductase and the LDL receptor, but the hydrophilic inhibitor pravastatin was not effective in Hep G2 cells as had previously been reported. The LDL receptor mRNA was induced by NK-104 most effectively between 0.1 to 10, μM among the lipophilic inhibitors, whereas the degrees of induction of HMG-CoA reductase mRNA by these inhibitors did not differ significantly from each other. When cells were treated with a 200-fold excess of the IC50 concentration of each inhibitor, NK-104 was able to induce LDL receptor mRNA most effectively. These results indicate that the effect of HMG-CoA reductase inhibitors on the upregulation of mRNA for reductase and LDL receptor are different from each other and among these lipophilic inhibitors. NK-104 is most effective in inducing LDL receptor mRNA in Hep G2 cells.

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In order to identify changes in the gene expression profile during human monocyte/macrophage differentiation in the presence of GM-CSF, the expression level of various mRNA was studied using DNA microarray technology. We found LXR alpha (LXRa) to be the most highly induced transcriptional regulator during macrophage differentiation. The LXRa mRNA level was induced 40 fold which ranked it as the 10th highest among the approximately 5, 600 genes studied. Although only restricted hepatic expression of LXRa mRNA had been reported, the macrophage expressed the highest level of LXRa among the nine human tissues and cultured cells studied. To further investigate transcriptional control, we have characterized the genomic structure of the human LXRa gene and determined the structure of its promoter region. The human LXRa gene consists of eleven exons, and analysis of the promoter region indicated the presence of conserved binding sites for myeloid zinc finger protein 1, which may be related to the extrahepatic expression of LXRa. LXRa is known to be activated by oxysterols, and the induced expression of the gene may be related to the foam cell formation in atherosclerotic lesions.

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Cardiac death from atherosclerosis is common in hemodialysis patients. Human serum paraoxonase (PON1), an esterase, is associated with high-density lipoprotein and inhibits the susceptibility to oxidization of low-density lipoprotein (LDL). The PON1 genetic polymorphisms of 192 Gln/Arg and 55 Leu/Met in the amino acid sequence are partly involved in the PON1 enzyme activity. We investigated the PON1 enzyme activities for paraoxon (paraoxonase) and phenylacetate (arylesterase), and the two polymorphisms in 96 patients undergoing hemodialysis and in 136 normal controls. Both activities were significantly lower in the hemodialysis patients than in the controls (97±43 vs 155±57 μmol/min/l for paraoxonase, and 71±20 vs 92±22 mmol/min/l for arylesterase, respectively). There was no difference in the distribution of the two polymorphisms between patients and controls, and in every subgroup classified by the polymorphisms, both paraoxonase and arylesterase activities were lower in patients than in controls. This suggested that the enzyme activities of PON1 decreased in hemodialysis patients, independent of the genetic polymorphism. The decrease in PON1 enzyme activity in hemodialysis patients may modify a susceptibility to oxidization of LDL, which contributes to an acceleration of atherosclerosis.

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Insulin resistance is a possible major metabolic cause of atherosclerosis. Endothelial dysfunction is commonly found in patients with insulin resistance, and primary treatment of insulin resistance with troglitazone should improve such endothelial dysfunction. Thus, the effects of troglitazone on endothelial function were investigated. Thirteen non-diabetic male subjects with hyperinsulinemic response to oral glucose load (n=7) and normal (n=6) subjects were investigated. Flow-mediated dilatation (FMD) of the brachial artery was examined by high resolution ultrasonography before and after the administration of troglitazone of 400 mg for 4 weeks. In insulin resistant subjects, fasting glucose (4.9±0.3 to 4.7±0.3 mmol/L, p<0.05), insulin (45±30 to 25±15 pmol/L, p<0.05) and response to oral glucose load (AUC glucose : 15.0±3.5 to 13.0±2.2 mmol·h/L, p<0.05 ; AUC insulin : 965±560 to 475±275 pmol·h/L, p<0.05) were significantly reduced. FMD was significantly improved in insulin resistant subjects. A significant negative correlation was observed between FMD and AUC insulin (r=-0.64, p<0.05). The present study demonstrates that FMD is impaired in insulin resistant subjects, and troglitazone improves the blunted vascular response and impaired insulin response. This finding suggests that primary treatment of insulin resistance could prevent the development of atherosclerosis by improving endothelial dysfunction.

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The effects of cacao liquor polyphenols (CLP) on the susceptibility of low-density lipoprotein (LDL) to oxidation in hypercholesterolemic rabbits were examined. Six Japanese white rabbits which had been fed a high cholesterol diet (HCD) for 3 weeks were fed HCD containing 1% CLP for the following 10 days. The susceptibility of LDL to oxidation induced by 2-2'-azobis (4-methoxy-2, 4-dimethylvaleronitrile) (V-70) was evaluated by measuring the production of conjugated dienes and thiobarbituric acid reactive substances (TBARS). The lag time was significantly prolonged from 37.7 min before intake of CLP to 42.9, 44.2 and 45.8 min after 4, 7 and 10 days of CLP intake. TBARS production after intake of CLP was also markedly reduced compared with the level before intake. There was no difference in plasma lipid concentrations comparing the levels before and after CLP intake. In conclusion, in hypercholesterolemic rabbits, orally administered CLP was absorbed and distributed to the blood, and the resistance of LDL to oxidation was thereby increased.

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The aim of this study was to evaluate the effects of advanced glycation end-products (AGEs) on the proliferative activity and fibronectin production of smooth muscle cells (SMCs). AGE-bovine serum albumin (AGE-BSA) was prepared by incubation with D-glucose at 37°C for 60 days. Cultured SMCs were obtained from explants isolated from porcine abdominal aorta and used between passages 3 and 10. The proliferative activity of SMCs was examined by MTT (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) assay and by incorporation of ³H-thymidine into DNA. Fibronectin production was assessed by competitive ELISA assay for both fibronectin secreted into the culture medium (M-FN) and cell-associated fibronectin (C-FN), i.e., both intra- and peri-cellular fibronectin. The MTT assay revealed that AGE-BSA did not produce any change in optical density (A₅₇₀) of SMCs at concentrations of up to 20 μg/ml, but decreased that of SMCs at a concentration of 40μg/ml. The addition of PDGF (5 ng/ml) induced an increase in ³H-thymidine incorporation into DNA of quiescent SMCs, while the addition of AGE-BSA (20 μg/ml) had no effect. In contrast, AGE-BSA significantly increased C-FN of SMCs (30.8±8.58 ng/μg TP), compared to unmodified BSA (16.5+4.19 ng/μg TP). However, no difference in M-FN levels was observed between cells treated with AGE-BSA and unmodified BSA. The addition of anti-transforming growth factor (TGF) -β antibody restored the levels of C-FN in SMCs cultured in 20 μg/ml of AGE-BSA, suggesting that TGF-β might act as an intermediate factor in AGE-induced fibronectin production by SMCs. Our results suggest that interaction of AGE-modified proteins with SMCs may play a role in the development of atherosclerosis in diabetic and non-diabetic patients.

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