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Oxidized LDL (oxLDL) is a known risk factor for atherogenesis. This study aimed to reveal structural features of oxLDL present in human circulation related to atherosclerosis. When LDL was fractionated on an anion-exchange column, in vivo-oxLDL, detected by the anti-oxidized PC (oxPC) mAb, was recovered in flow-through and electronegative LDL [LDL(-)] fractions. The amount of the electronegative in vivo-oxLDL, namely oxLDL in the LDL(-) fraction, present in patients with acute MI was 3-fold higher than that observed in healthy subjects. Surprisingly, the LDL(-) fraction contained apoA1 in addition to apoB, and HDL-sized particles were observed with transmission electron microscopy. In LDL(-) fractions, acrolein adducts were identified at all lysine residues in apoA1, with only a small number of acrolein-modified residues identified in apoB. The amount of oxPC adducts of apoB was higher in the LDL(-) than in the L1 fraction, as determined using Western blotting. The electronegative in vivo-oxLDL was immunologically purified from the LDL(-) fraction with an anti-oxPC mAb. The majority of PC species were not oxidized, whereas oxPC and lysoPC did not accumulate. Here, we propose that there are two types of in vivo-oxLDL in human circulating plasma and the electronegative in vivo-oxLDL accompanies oxidized HDL.

Oxidative modification of lipoproteins is implicated in the occurrence and development of atherosclerotic lesions. Earlier studies have elucidated on the mechanisms of foam cell formation and lipid accumulation in these lesions, which is mediated by scavenger receptor-mediated endocytosis of oxidized low-density lipoprotein (oxLDL). Mounting clinical evidence has supported the involvement of oxLDL in cardiovascular diseases. High-density lipoprotein (HDL) is known as anti-atherogenic; however, recent studies have shown circulating oxidized HDL (oxHDL) is related to cardiovascular diseases. A modified structure of oxLDL, which was increased in the plasma of patients with acute myocardial infarction, was characterized. It had two unique features: (1) a fraction of oxLDL accompanied oxHDL, and (2) apoA1 was heavily modified, while modification of apoB, and the accumulation of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) was less pronounced. When LDL and HDL were present at the same time, oxidized lipoproteins actively interacted with each other, and oxPC and lysoPC were transferred to another lipoprotein particle and enzymatically metabolized rapidly. This brief review provides a novel view on the dynamics of oxLDL and oxHDL in circulation.

Oxidized low-density lipoprotein (oxLDL) and oxidized high-density lipoprotein (oxHDL), known as risk factors for cardiovascular disease, have been observed in plasma and atheromatous plaques. In a previous study, the content of oxidized phosphatidylcholine (oxPC) and lysophosphatidylcholine (lysoPC) species stayed constant in isolated in vivo oxLDL but increased in copper-induced oxLDL in vitro. In this study, we prepared synthetic deuterium-labeled 1-palmitoyl lysoPC and palmitoyl-glutaroyl PC (PGPC), a short chain-oxPC to elucidate the metabolic fate of oxPC and lysoPC in oxLDL in the presence of HDL. When LDL preloaded with d13-lysoPC was mixed with HDL, d13-lysoPC was recovered in both the LDL and HDL fractions equally. d13-LysoPC decreased by 50% after 4 h of incubation, while d13-PC increased in both fractions. Diacyl-PC production was abolished by an inhibitor of lecithin-cholesterol acyltransferase (LCAT). When d13-PGPC-preloaded LDL was incubated with HDL, d13-PGPC was transferred to HDL in a dose-dependent manner when both LCAT and lipoprotein-associated phospholipase A2 (Lp-PLA2) were inhibited. Lp-PLA2 in both HDL and LDL was responsible for the hydrolysis of d13-PGPC. These results suggest that short chain-oxPC and lysoPC can transfer between lipoproteins quickly and can be enzymatically converted from oxPC to lysoPC and from lysoPC to diacyl-PC in the presence of HDL.

Chemokines are secreted proteins that regulate cell migration and are involved in inflammatory and immune responses. Here, we sought to define the functional crosstalk between the lipid signaling and chemokine signaling. We obtained evidence that the induction of some chemokines is regulated by group VIA calcium-independent phospholipase A2 β (iPLA2 β) in IL-1β-stimulated rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with IL-1β elicited an increased release of chemotactic factor(s) for monocytic THP-1 cells into culture medium in a time-dependent manner. Inhibitor studies revealed that an intracellular PLA2 inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3 ), but not the cyclooxygenase inhibitor indomethacin, attenuated the release of chemotactic factor(s). The chemotactic activity was inactivated by treatment with either heat or proteinase K, suggesting this chemotactic factor(s) is a proteinaceous factor(s). We purified the chemotactic factor(s) from the conditioned medium of IL-1β-stimulated 3Y1 cells using a heparin column and identified several chemokines, including CCL2 and CXCL10. The inducible expressions of CCL2 and CXCL10 were significantly attenuated by pretreatment with AACOCF3 . Gene silencing using siRNA revealed that the inductions of CCL2 and CXCL10 were attenuated by iPLA2 β knockdown. Additionally, the transcriptional activation of nuclear factor of activated T-cell proteins (NFATs), but not nuclear factor-κB, by IL-1β stimulation was markedly attenuated by the iPLA2 inhibitor bromoenol lactone, and NFATc4 knockdown markedly attenuated the IL-1β-induced expression of both CCL2 and CXCL10. Collectively, these results indicated that iPLA2 β plays roles in IL-1β-induced chemokine expression, in part via NFATc4 signaling.

Background and objective Periodontal disease is a chronic inflammatory disease caused by bacterial infection that can lead to tooth loss. Gingival crevicular fluid can be collected easily and noninvasively. We previously discovered the presence of apolipoprotein B (apoB), the main constituent of low-density lipoprotein, and oxidized low-density lipoprotein (oxLDL) in the gingival crevicular fluid of healthy subjects. In this study, we investigated whether periodontal conditions affect the levels of apoB and oxLDL in gingival crevicular fluid. Material and methods The study population comprised 11 patients with chronic periodontitis. A pair of gingival crevicular fluid samples was collected from each patient at a healthy site and at a site with periodontitis (baseline samples). Thereafter, gingival crevicular fluid samples were collected from the same patients again at 4 and 8 wk after scaling and root planing (SRP). The levels of apoB, oxLDL, protein and cytokines in gingival crevicular fluid, in addition to gingival crevicular fluid volume, were measured. Results At baseline, the levels of apoB and oxLDL in gingival crevicular fluid were higher at the sites with periodontitis than at the healthy sites. The levels of apoB and oxLDL at periodontal sites decreased after SRP. The level of oxLDL in gingival crevicular fluid correlated well with the probing pocket depth. The oxLDL : apoB ratio in gingival crevicular fluid was significantly higher than that in plasma. Conclusions The levels of apoB and oxLDL in gingival crevicular fluid change according to the periodontal tissue conditions.

The function of oxidatively modified low-density lipoprotein (oxLDL) in the progression of cardiovascular diseases has been extensively investigated and well-characterized with regards to the activation of multiple cellular responses in macrophages and endothelial cells. Although accumulated evidence has revealed the presence of neutrophils in vascular lesions, the effect of oxLDL on neutrophil function has not been properly investigated. In the present decade, neutrophil extracellular traps (NETs) gained immense attention not only as a primary response against pathogenic bacteria but also due to their pathological roles in tissue damage in various diseases, such as atherosclerosis and thrombosis. In this study, we investigated if oxLDL affects NET formation and if it is a risk factor for inflammatory reactions in endothelial cells. HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA) for 30 min to induce NET formation, followed by incubation with 20 μg/mL native or oxidized LDL for additional 2 h. Culture media of the stimulated cells containing released NETs components were collected to evaluate NET formation by fluorometric quantitation of released DNA and detection of myeloperoxidase (MPO) by western blot analysis. NET formation of HL-60-derived neutrophils induced by PMA was significantly enhanced by additional incubation with oxLDL but not with native LDL. Treatment of HL-60-derived neutrophils with oxLDL alone in the absence of PMA did not induce NET formation. Furthermore, the culture media of HL-60-derived neutrophils after NET formation were then transferred to human aortic endothelial cell (HAECs) culture. Treatment of HAECs with the culture media containing NETs formed by HL-60-derived neutrophils increased the expression of metalloproteinase-1 protein in HAECs when HL-60-derived neutrophils were incubated with native LDL, and the expression was accelerated in the case of oxLDL. In addition, the culture media from NETs formed by HL-60-derived neutrophils caused the elongation of HAECs, which was immensely enhanced by coincubation with native LDL or oxLDL. These data suggest that oxLDL may act synergistically with neutrophils to form NETs and promote vascular endothelial inflammation.

Neutrophil extracellular traps (NETs) significantly contribute to various pathophysiological conditions, including cardiovascular diseases. NET formation in the vasculature exhibits inflammatory and thrombogenic activities on the endothelium. NETs are induced by various stimulants such as exogenous damage-associated molecular patterns (DAMPs). Oxidatively modified low-density lipoprotein (oxLDL) has been physiologically defined as a subpopulation of LDL that comprises various oxidative modifications in the protein components and oxidized lipids, which could act as DAMPs. oxLDL has been recognized as a crucial initiator and accelerator of atherosclerosis through foam cell formation by macrophages; however, recent studies have demonstrated that oxLDL stimulates neutrophils to induce NET formation and enhance NET-mediated inflammatory responses in vascular endothelial cells, thereby suggesting that oxLDL may be involved in cardiovascular diseases through neutrophil activation. As NETs comprise myeloperoxidase and proteases, they have the potential to mediate oxidative modification of LDL. This review summarizes recent updates on the analysis of NETs, their implications for cardiovascular diseases, and prospects for a possible link between NET formation and oxidative modification of lipoproteins.

Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL–derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex–driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor–deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.

The events that trigger early onset of atherosclerotic lesion formation are poorly understood. Initially, microscopic atherosclerotic lesions appear in the aortic root in 10-week-old apoE-knockout mice that are fed normal chow. Using proteome and immunohistochemical analyses, we investigated proteins in aortic media whose expression changes in athero-prone regions at the beginning of lesion formation. Protein profiles of the root/arch and thoracic/abdominal regions of aortas in 10-week-old apoE-knockout mice were analyzed using 2D-gel electrophoresis. Proteins in 81 spots with different abundance were identified. Among them, we focused on proteins related to oxidative stress and smooth muscle cells (SMCs). The level of peroxiredoxin 2 (Prx2), a major cellular antioxidant enzyme that reduces hydrogen peroxide, was lower in aortic root/arch compared with thoracic/abdominal aorta. Immunohistochemical staining demonstrated that Prx2 expression in SMCs in the aortic root was high at 4 weeks and decreased at 10 weeks in apoE-knockout mice, while Prx2 expression in the aorta was unchanged in wild-type mice. The level of Prx2 expression correlated positively with the SMC differentiation markers, α-smooth muscle actin and transgelin, suggesting that a decline in Prx2 expression accompanies SMC dedifferentiation. Accumulated acrolein-modified proteins and the infiltration of macrophages in aortic media were observed in areas with low Prx2 expression. These results showed that Prx2 expression declines in athero-prone aortic root before lesion formation, and this reduction in Prx2 expression correlates with lipid peroxidation, SMC dedifferentiation, and macrophage recruitment.

Lipid droplets (LDs) are functional subcellular organelles involved in multiple intracellular processes. LDs are found in nearly all types of eukaryotic cells, but their properties are highly variable in different types of tissues. Steroidogenic cells synthesize steroid hormones de novo from the cholesterol deposited in cytosolic LDs. However, the roles of LD proteins in steroidogenesis under pituitary hormone stimulation have not been well elucidated. The protein profile of isolated LDs from the mouse Leydig tumor cell line MLTC-1 was distinct from that of hepatic cells or macrophages. By proteomic analysis of the components using mass spectrometry, two enzymes for steroidogenesis, 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1) and 17 β-hydroxysteroid dehydrogenase type 11 (17βHSD11), were identified in two strong bands in the LD fractions. The LD fraction of MLTC-1 cells also included CYP11A1 and CYP17, suggesting that the LDs contain all the enzymes needed for testosterone synthesis. The steroidogenesis in Leydig cells is activated by luteinizing hormone through a PKA-dependent pathway. Stimulation of MLTC-1 cells with luteinizing hormone or 8-bromo-cAMP caused drastic changes in the morphology of the LDs in the MLTC-1 cells. Upon stimulation, large perinuclear LDs are turned into much smaller LDs and dispersed throughout the cytosol. These results raise the possibility that LDs are involved in a regulatory pathway of steroidogenesis, not just by serving as a storage depot for cholesterol esters, but also by providing enzymes and generating sites for enzymatic activity.

Aims The adhesion of circulating monocytes to endothelial cells (ECs) is an early and critical event in the formation of atherosclerotic plaques. Hydrogen peroxide-inducible clone 5 (Hic-5) serves as an adaptor molecule in cell adhesion complexes. However, the role of endothelial Hic-5 in monocyte–EC interaction and atherogenesis remains unclear. We examined the roles of endothelial Hic-5 in monocyte–EC interaction and atherogenesis using mouse models of atherosclerosis and cultured human umbilical vein endothelial cells (HUVECs). Methods and results Hic-5 was expressed in ECs, but not in monocytes/macrophages. An ex vivo monocyte adhesion assay revealed that adhesion of THP-1 monocytes to aortas isolated from Apoe −/− and LDLR −/− mice stimulated by TNF-α or oxidized LDL was suppressed by Hic-5 deficiency. Scanning electron microscopic observations of aortas harvested from Apoe −/− mice revealed that TNF-α- or oxidized LDL-induced microvilli-like structures were markedly suppressed by Hic-5 deficiency. Relative Hic-5 deficiency suppressed 60% of the atherosclerotic lesions in aortas from Apoe −/− and LDLR −/− mice. In contrast, overexpression of Hic-5 in HUVECs promoted induction of microvilli-like structures and adherence of THP-1 cells in an adhesion receptor such as intercellular adhesion molecule-1- and vascular cell adhesion molecule-1-dependent manner. Conclusion Hic-5 in ECs plays an important role in the formation of microvilli-like structures and in the interaction between ECs and monocytes, leading to monocyte recruitment and subsequent development of atherosclerosis.

End-stage renal disease (ESRD) patients undergoing hemodialysis (HD) have a high prevalence of cardiovascular events. Low-density lipoprotein (LDL) in dialysis patients has been shown to be susceptible to in vitro peroxidation; therefore, oxidized-LDL (ox-LDL) could be generated in these patients. Moreover, myeloperoxidase (MPO) released from activated neutrophils may play a role in the induction of LDL oxidation. The purpose of this study was to investigate the relationship between plasma ox-LDL levels, plasma MPO levels, and serum high-sensitivity C-reactive protein (hs-CRP) levels during initial HD in patients with diabetic ESRD. Patients (n=28) had serial venous blood samples drawn before and after HD at the initial, second, and third sessions. Plasma ox-LDL levels were measured using a specific monoclonal antibody (DLH3), and plasma MPO levels were measured using an enzyme-linked immunosorbent assay kit. Plasma ox-LDL levels and MPO levels after a single HD session increased significantly (ox-LDL, P

Agaricus blazei (A. blazei) Murrill mycelia-dikaryon has attracted the attention of scientists and clinicians worldwide owing to its potential for the treatment of cancer. However, little is known about its effect on other pathologies. This study sought to extend the potential medical usefulness of A. blazei for preventing vascular damage and to unravel its mechanism of action. The A. blazei extract showed estrogen-like activity in both gene expression profiling and a luciferase assay. Indeed, the extract inhibited oxidized low-density lipoprotein-stimulated activation of Erk1/2, Akt and p38 in HUVECs and macrophage-derived TIB-67 cells. Moreover, the extract enhanced transcription of the glutathione peroxidase 3 (GPX3), α-synuclein (SNCA) and endothelial nitrogen-oxide synthase (eNOS) genes. Furthermore, atherosclerotic lesions in rabbits were reduced by intake of A. blazei powder. Therefore, A. blazei may be useful for preventing atherosclerosis via dual roles in cell signaling, suppression of macrophage development and the recovery of endothelial cells from vascular damage.

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.

Sakiyama Y, Kato R, Inoue S, Suzuki K, Itabe H, Yamamoto M. Detection of oxidized low-density lipoproteins in gingival crevicular fluid from dental patients. J Periodont Res 2009; doi: 10.1111/j.1600-0765.2009.01226.x. © 2009 John Wiley & Sons A/S Background and Objective: Oxidative modification of low-density lipoprotein (LDL) occurs in various diseased tissues and sites of local inflammation. For example, an increased plasma oxidized low-density lipoprotein (OxLDL) level is a well-known risk marker for cardiovascular diseases. Gingival crevicular fluid, the exudate from gingival tissues into the sulci, can be easily collected in a non-invasive manner. However, the possible presence of OxLDL in gingival crevicular fluid has not been studied. In this study, we established a procedure to measure OxLDL in human gingival crevicular fluid. Material and Methods: Human gingival crevicular fluid was sampled with paper points or paper strips. The gingival crevicular fluid samples from healthy gingival sulci (pocket depth

A TLR4/MyD88 pathway was activated via oxidized phosphatidylcholine caused by Vpr of HIV-1. Vpr, a HIV-1 accessory protein, was believed to be present in the plasma of HIV-1-positive patients, and our previous work demonstrated the presence of plasma Vpr in 20 out of 52 patients. Interestingly, our data revealed that patients’ viral titer was correlated with the level of Vpr detected in their plasma. Here, we first show that rVpr, when incubated with human monocytes or MDMs, caused viral production from latently infected cells, and IL-6 was identified as a responsible factor. The induction of IL-6 by rVpr was dependent on signaling through TLR4 and its adaptor molecule, MyD88. We next provide evidence that rVpr induced the formation of OxPC and that a mAb against OxPC blocked rVpr-induced IL-6 production with the concomitant attenuation of MAPK activation. Moreover, the addition of NAC, a scavenger of ROS, abrogated the rVpr-induced formation of OxPC, the phosphorylation of C/EBP-β, a substrate of MAPK, and IL-6 production. As rIL-6 reactivated viral replication in latently infected cells, our data indicate that rVpr-induced oxidative stress triggers cell-based innate immune responses and reactivates viral production in latently infected cells via IL-6 production. Our results suggest that Vpr should be monitored based on the viral titer, and they provide the rationale for the development of novel, anti-AIDS therapeutics targeting Vpr.

STAT1 mediates Interferon (IFN)-dependent positive and negative regulation of inflammatory gene expression in lung. In this study, we examined the effect of IFN-gamma on the expression of SCGB3A1 which is thought to play crucial roles in inflammation and epithelial cell differentiation in lung. We found that expression of SCGB3A1 was down-regulated by IFN-gamma in a time- and dose-dependent manner in the murine transformed Clara Cells (mtCC) line. IFN-gamma induced the phosphorylation of STAT1, which binds to a STAT-binding element (SBE) in the SCGB3A1 gene promoter, leading to decreased transcriptional activation of this gene.

Background— Plasma level of oxidized low-density lipoprotein (OxLDL) is a risk marker for cardiovascular diseases. The behavior of plasma OxLDL before disease progression has not been studied previously. Methods and Results— In this study, we developed a sensitive ELISA procedure for detecting mouse circulating OxLDL using a monoclonal antibody that recognizes oxidized phosphatidylcholine and a rabbit antimouse apolipoprotein B-48 polyclonal antibody. Apolipoprotein E knockout mice were fed on a chow diet for 40 weeks. Oil red O–positive lesions developed gradually by 20 weeks, and the percentage area covered by the lesions increased dramatically after 28 weeks; it covers 33.4% of the surface area by 40 weeks. The OxLDL level, measured after LDL fraction was isolated from each mouse, at 10 weeks was 0.015 ng/μg LDL. It increased 3-fold at 20 weeks of age and then decreased to the basal level by 40 weeks of age, suggesting that OxLDL appears before the development of atherosclerotic lesions. The occurrence of lipid peroxidation products, acrolein and oxidized phosphatidylcholines, in aortic tissue were revealed by immunohistochemical staining as early as 10 weeks. Conclusion— These results suggest that OxLDL might be involved in the early stages of progression of atherosclerotic lesions.

Among the many mammalian secreted phospholipase A2 (sPLA2) enzymes, PLA2G3 (group III secreted phospholipase A2) is unique in that it possesses unusual N- and C-terminal domains and in that its central sPLA2 domain is homologous to bee venom PLA2 rather than to other mammalian sPLA2s. To elucidate the in vivo actions of this atypical sPLA2, we generated transgenic (Tg) mice overexpressing human PLA2G3. Despite marked increases in PLA2 activity and mature 18-kDa PLA2G3 protein in the circulation and tissues, PLA2G3 Tg mice displayed no apparent abnormality up to 9 months of age. However, alterations in plasma lipoproteins were observed in PLA2G3 Tg mice compared with control mice. In vitro incubation of low density (LDL) and high density (HDL) lipoproteins with several sPLA2s showed that phosphatidylcholine was efficiently converted to lysophosphatidylcholine by PLA2G3 as well as by PLA2G5 and PLA2G10, to a lesser extent by PLA2G2F, and only minimally by PLA2G2A and PLA2G2E. PLA2G3-modified LDL, like PLA2G5- or PLA2G10-treated LDL, facilitated the formation of foam cells from macrophages ex vivo. Accumulation of PLA2G3 was detected in the atherosclerotic lesions of humans and apoE-deficient mice. Furthermore, following an atherogenic diet, aortic atherosclerotic lesions were more severe in PLA2G3 Tg mice than in control mice on the apoE-null background, in combination with elevated plasma lysophosphatidylcholine and thromboxane A2 levels. These results collectively suggest a potential functional link between PLA2G3 and atherosclerosis, as has recently been proposed for PLA2G5 and PLA2G10.

Objective Our recent studies have indicated that urotensin II, the most potent vasoconstrictor peptide identified to date, potentiates human macrophage foam cell formation and vascular smooth muscle cell proliferation, and its levels are increased in the plasma of hypertensive patients with carotid atherosclerotic plaques. In the present study, we investigated the enhancing effect of urotensin II on atherosclerosis in apolipoprotein E-knockout mice and its suppression by 4-aminoquinoline, an urotensin II receptor-selective antagonist. Methods Urotensin II, urotensin II + 4-aminoquinoline, or vehicle was infused for 4 weeks through an osmotic mini-pump into 9-week-old apolipoprotein E-knockout mice on a high-fat diet. Aortic atherosclerosis and foam cell formation in exudate peritoneal macrophages were examined. Results Atherosclerotic lesions as well as plasma levels of urotensin II, reactive oxygen species, and oxidized low-density lipoprotein and oxidized low-density lipoprotein-induced foam cell formation were significantly greater in urotensin II-infused mice than vehicle-infused controls. Western blotting analysis showed increased expression of scavenger receptors (CD36 and scavenger receptor class A) and acyl-CoA:cholesterol acyltransferase-1 in these macrophages. Increases in these parameters were significantly reduced by addition of 4-aminoquinoline. In apolipoprotein E-knockout mice even without urotensin II infusion, the treatment with 4-aminoquinoline for 8 weeks significantly prevented the development of atherosclerotic lesions. Conclusion Our results provide the first evidence that increased plasma urotensin II level stimulates oxidized low-density lipoprotein and reactive oxygen species production and macrophage foam cell formation via increased expression of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1, contributing to the development of atherosclerosis in apolipoprotein E-deficient mice. Urotensin II receptor antagonism may be a promising therapeutic strategy against atherosclerosis.

It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma. However, the detailed mechanisms of Ox-LDL-induced PPARalpha and PPARgamma activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARalpha and PPARgamma activation in macrophages. Ox-LDL, but not LDL, induced PPARalpha and PPARgamma activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARalpha and PPARgamma activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARalpha and PPARgamma activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ(2) induced both PPARalpha and PPARgamma activation. Furthermore, COX-2 and 15d-PGJ(2) expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARalpha and PPARgamma on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARalpha and PPARgamma suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ(2) level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARalpha and PPARgamma activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.

We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K(2) (VK(2); menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK(2).Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment.We found that PA-1 cells were the most sensitive to VK(2) (IC(50) = 5.0 +/- 0.7 microM), while SK-OV-3 cells were resistant to VK(2). Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 microM VK(2). In the VK(2)-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK(2)-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK(2) and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

The role of plasma levels of oxidized low density lipoprotein (OxLDL) in the development of coronary heart disease (CHD) has not been fully elucidated. We examined the relationship among plasma levels of OxLDL, measured by an enzyme immunoassay using an antibody against OxLDL (FOH1a/DLH3) and apolipoprotein B, CHD, and modalities at the onset of acute coronary syndrome (ACS). A total of 115 individuals who underwent coronary angiography were studied. Of these, 21 patients complicated with extracoronary cardiovascular diseases were excluded. Consequently, 94 patients (63 men) (ACS: 23, stable angina pectoris (SAP): 46, and normal coronary artery (NCA):25) were eligible for inclusion in the study. Elevated plasma levels of OxLDL were associated with CHD, especially with ACS. In patients with NCA, hypertension was associated with plasma OxLDL. Plasma levels of OxLDL were significantly higher in patients with new-onset type ACS than in those with worsening type ACS (2.98 versus 1.53 mg/dL, P = 0.002). In conclusion, plasma levels of OxLDL are associated with CHD and significantly higher in patients with new-onset ACS. The findings of the present study suggest that plasma OxLDL can be a marker of the development of CHD and modalities of ACS.

Second generation antipsychotics are useful for the treatment of schizophrenia, but concerns have been raised about the side effects of diabetes mellitus and obesity. Olanzapine, especially, is associated with more weight gain than the others. It has been reported that olanzapine promotes adipocyte-differentiation in rodents both in vivo and in vitro. In this study the effects of antipsychotics on human adipocytes were investigated by using human mesenchymal stem cells (hMSCs). When hMSCs were differentiated and treated with various antipsychotics, olanzapine and clozapine increased intracellular lipids. Olanzapine induced lipid accumulation in a dose-dependent manner. Proteomic analysis revealed that PLIN4 and several enzymes for lipid metabolism were increased in the hMSCs after olanzapine treatment. During adipocyte differentiation, olanzapine increased the protein expression of PLIN1, PLIN2 and PLIN4. These proteins are known to be associated with the initial stage of lipid droplet formation. Immunocytochemistry showed that olanzapine increased and enlarged the lipid droplets coated with PLIN1 and PLIN2 while PLIN4 was largely distributed in the cytosol. mRNA expression of PLIN2, but not PLIN1 or PLIN4, was increased by olanzapine. On the other hand, olanzapine did not alter the mRNA level of transcription regulators involved in adipocyte-differentiation or adipokines. The present study shows that olanzapine induced transient PLIN2 expression in hMSCs that could result in an accumulation of lipid droplets and overexpression of PLIN1 and PLIN4, providing information of possible interest for olanzapine-induced weight gain.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical glycolytic enzyme that is involved in cellular energy production and has important housekeeping functions. We used the natural prooxidant and proatherogenic molecule oxidized low-density lipoprotein (OxLDL) to determine a potential link between OxLDL-promoted oxidative stress, GAPDH expression, and smooth muscle cell energy metabolism. OxLDL but not native LDL (nLDL) produced a 60% to 100% dose- and time-dependent reduction of GAPDH protein. OxLDL increased reactive oxygen species (ROS) formation, including rapid elevation of H 2 O 2 levels. OxLDL decreased intracellular catalase expression, likely contributing to the increase in H 2 O 2 . Antioxidants, anti-CD36 receptor antibody, NADPH oxidase, or lipoxygenase blockers decreased OxLDL-specific ROS and prevented GAPDH downregulation. 12/15-Lipoxygenase or p47phox deficiency resulted in attenuation of GAPDH downregulation, but 5-lipoxygenase suppression had no effect. OxLDL or exogenous H 2 O 2 oxidized GAPDH thiols, decreasing GAPDH protein half-life and increasing GAPDH sensitivity to proteasome-mediated protein degradation in vitro. OxLDL- or small interfering RNA–specific downregulation of GAPDH resulted in 65% reduction in glycolysis rate and 82% decrease in ATP levels. In conclusion, our data demonstrate that OxLDL downregulated GAPDH via a H 2 O 2 -dependent decrease in protein stability. GAPDH protein damage resulted in marked depletion of cellular ATP levels. Our data have important implications for understanding the metabolic effect of OxLDL on the vessel wall and mechanism of atherogenesis.

Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 microg/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescence staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

Nonalcoholic steatohepatitis/nonalcoholic fatty liver disease is considered to be a hepatic manifestation of various metabolic disorders. However, its precise pathogenic mechanism is obscure. Oxidative stress and consequent lipid peroxidation seem to play a pivotal role in disease progression. In this study, we analyzed the localization of oxidized phosphatidylcholine (oxPC), a lipid peroxide that serves as a ligand for scavenger receptors, in livers of patients with this steatotic disorder. Specimens of non-alcoholic fatty liver disease (15 autopsy livers with simple steatosis and 32 biopsy livers with steatohepatitis) were examined via immunohistochemistry and immunoelectron microscopy using a specific antibody against oxPC. In addition, scavenger receptor expression, hepatocyte apoptosis, iron deposition, and inflammatory cell infiltration in the diseased livers were also assessed. Oxidized phosphatidylcholine was mainly localized to steatotic hepatocytes and some macrophages/Kupffer cells. A few degenerative or apoptotic hepatocytes were also positive for oxPC. Immunoelectron microscopy showed oxPC localized to cytoplasmic/intracytoplasmic membranes including lipid droplets. Steatotic livers showed enhanced expression of scavenger receptors. The number of oxPC cells was correlated with disease severity and the number of myeloperoxidase-positive neutrophils, but not with the degree of iron deposition. In conclusion, distinct localization of oxPC in liver tissues suggest that neutrophil myeloperoxidase-derived oxidative stress may be crucial in the formation of oxPC and the progression of steatotic liver disease.

Reactive species of oxygen and nitrogen mediate the oxidative modification of low-density lipoprotein (LDL). Oxidation of LDL is inhibited by endogenous radical scavenging enzymes such as manganese superoxide dismutase (SOD) and Cu-ZnSOD that catalyze dismutation of oxygen to H2O2. Low-molecular antioxidants such as uric acid regulate the inactivation that appears to be linked to an increase in peroxynitrite resulting in oxidized LDL (OxLDL) elevation. The authors evaluated whether a focal imbalance between pro- and antioxidant systems induces plaque vulnerability in patients with carotid artery (CA) stenosis.Carotid artery plaques obtained in 35 patients who had undergone carotid endarterectomy were classified as vulnerable or stable based on histopathological findings. In vulnerable plaques, OxLDL, measured using enzyme-linked immunosorbent assay, was significantly higher (p0.01) and SOD activity significantly lower than in stable plaques (p0.05). The plaque and plasma OxLDL levels were inversely correlated with plaque SOD activity (p0.01). The physiological uric acid level in all plaques was one fourth to one eighth of that in plasma and appeared to be unable to protect Cu-ZnSOD from degradation by H2O2. Immunohistochemical analysis showed increased peroxynitrite and OxLDL in vulnerable plaques. There was a significant correlation between plaque and plasma OxLDL levels (p0.01).Analysis of the results suggests that a focal imbalance between pro- and antioxidant defense systems in patients with CA plaques induces an increase in plaque OxLDL levels and consequent plaque instability, contributing to high levels of plasma OxLDL.

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.

Oxidized low-density lipoprotein (OxLDL) present in circulating plasma and in human atherosclerotic lesions has been measured using a monoclonal antibody recognizing oxidized phosphatidylcholine (OxPC), DLH3. OxLDL levels in patients with acute myocardial and cerebral infarctions were significantly higher than those of controls. Thus, the plasma OxLDL level could be a marker for vascular diseases. Immunohistochemical studies demonstrated the foam cell-associated accumulation of OxLDL in human atherosclerotic lesions. To investigate the intracellular fate of OxLDL in foam cells, OxLDL taken up by J774 macrophages was chased. We found that partially degraded OxLDL particles accumulated in secondary lysosomes. Similarly, apolipoprotein B (apoB) recovered from human carotid plaques was partially degraded, suggesting that OxLDL actually contribute to foam cell formation in atherosclerotic lesions. Minimally modified LDL (MM-LDL) was prepared by dialyzing LDL in the presence of a low concentration of FeSO4. Macrophages did not bind to the MM-LDL effectively, but the DLH3 antibody did bind to MM-LDL as well as with CuSO4-induced OxLDL. Furthermore, MM-LDL appeared to be enriched with aldehyde-containing OxPC. Metabolic dynamics of OxLDL is discussed.

Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction.

Oxidized low-density lipoprotein (OxLDL) is thought to be involved in the early development of atherosclerosis through foam cell formation. However, the actual nature of OxLDL found in vivo remained obscure until recently. We have established a sensitive method of measuring OxLDL present in human plasma by utilizing an anti-OxLDL monoclonal antibody, DLH3, together with an anti-apoB antibody. OxLDL levels appeared to increase in patients with acute myocardial infarction, cerebral infarction and several other pathological conditions. Evidence is accumulating which supports the close relation between vascular diseases and plasma OxLDL levels. In this article, a major concern is the source of the OxLDL. Our recent studies on the OxLDL present in both atherosclerotic lesions and in plasma, together with observations of minimally modified LDL, provide some further clues to the understanding of some of the features and behavior of OxLDL in the circulation. A possible scenario detailing the origin and dynamics of OxLDL in vivo is discussed.

We previously demonstrated that the deficiency of class A macrophage scavenger receptor type I/II was involved in the delayed phagocytosis of degraded myelin by macrophages in class A macrophage scavenger receptor type I/II knockout mice after crush injury of the sciatic nerve [Naba et al. (2000) Exp. Neurol., 166, 83-89]. In order to elucidate the role of CD36, one of the scavenger receptors, here we inflicted crush injury to the sciatic nerves of CD36 knockout mice and investigated the remyelination after crush injury in comparison with that of class A macrophage scavenger receptor type I/II knockout mice. Although we previously reported a lot of onion-bulbs in class A macrophage scavenger receptor type I/II knockout mice at 3 weeks, the number of onion-bulbs was limited both in CD36 knockout mice and wild-type mice. In the morphometry, the remyelination was seriously delayed, and the infiltrating macrophages into the nerve fascicles were quite frequent in CD36 knockout mice compared with wild-type mice at 3 and 6 weeks postinjury. The immunohistochemistry with the monoclonal antibody reacted with oxidized phosphatidylcholine and oil red O staining were positive in wild-type mice, but were negative in CD36 knockout mice, suggesting that the oxidation of phosphatidylcholine and the generation of neutral lipids in macrophages were disturbed in CD36 knockout mice. We hypothesize that the delayed phagocytosis by macrophages and the defect in reuse of lipids from degraded myelin are related to seriously delayed remyelination and a small number of onion-bulbs in CD36 knockout mice.

Oxidized low-density lipoprotein (OxLDL) has previously been thought to promote atherogenesis through foam cell formation. However, the actual nature of OxLDL present in vivo remained obscure until recently. We have produced a monoclonal antibody, DLH3, which specifically binds to OxLDL but not to native LDL. The presence of OxLDL in the LDL fraction of human plasma was demonstrated by introducing a sandwich ELISA procedure using DLH3 together with an anti-apoB antibody. Furthermore, OxLDL levels appeared to increase in certain pathological conditions including acute myocardial infarction and carotid artery atherosclerosis. Accumulation of OxLDL in atherosclerotic lesions has also been demonstrated by immunohistochemical and biochemical studies using the DLH3 antibody. This antibody recognizes oxidized phosphatidylcholines (OxPC) generated during oxidative modification of LDL, and OxPC-apoB adducts formed in OxLDL are the presumed antigens. Measuring OxLDL in plasma would be a useful diagnostic tool for cardiovascular diseases. However, there still remain some major questions related to OxLDL, the answers to which are crucial for understanding the mechanisms of atherogenesis.

Objective— Oxidation of LDL plays a significant pathogenic role in atherosclerosis. In this study, we attempted to clarify the correlation between the morphology of human atherosclerotic plaques and the oxidized LDL (OxLDL) levels in plasma and carotid plaques. Methods and Results— OxLDL levels (ng/μg apolipoprotein B) in plasma and carotid plaques from 44 patients undergoing carotid endarterectomy and OxLDL levels in 17 control plasma and 9 normal intima samples were determined by a sandwich ELISA by using specific antibodies against OxLDL (DLH3) and apolipoprotein B. The plaques were immunohistochemically classified as macrophage (Mφ)-rich and Mφ-poor. In paired samples from individual patients, plaque OxLDL was nearly 70 times higher than plasma OxLDL (mean±SEM, 11.9±1.7 vs 0.18±0.01 ng/μg apoB, P P P =0.02). Conclusions— Our results suggest that LDL undergoes further oxidation in plaques, and that high plasma and plaque levels of OxLDL are correlated with the vulnerability to rupture of atherosclerotic lesions.

OBJECTIVE: Lowering the blood concentration of low-density lipoprotein (LDL) cholesterol is the primary strategy employed in treating atherosclerotic disorders; however, most commonly prescribed statins prevent cardiovascular events in just 30% to 40% of treated patients. Therefore, additional treatment is required for patients in whom statins have been ineffective. In this study of atherosclerosis in rabbits, we examined the effect of probucol, a lipid-lowering drug with potent antioxidative effects, added to treatment with atorvastatin. METHODS AND RESULTS: Atherosclerosis was induced by feeding rabbits chow containing 0.5% cholesterol for 8 weeks. Probucol 0.1%, atorvastatin 0.001%, and atorvastatin 0.003% were administered solely or in combination for 6 weeks, beginning 2 weeks after the start of atherosclerosis induction. Atorvastatin decreased the plasma concentration of non-high-density lipoprotein cholesterol (non-HDLC) dose-dependently; atorvastatin 0.003% decreased the plasma concentration of non-HDLC by 25% and the area of atherosclerotic lesions by 21%. Probucol decreased the plasma concentration of non-HDLC to the same extent as atorvastatin (i.e., by 22%) and the area of atherosclerotic lesions by 41%. Probucol with 0.003% atorvastatin decreased the plasma concentration of non-HDLC by 38% and the area of atherosclerotic lesions by 61%. Co-administration of probucol with atorvastatin did not affect the antioxidative effects of probucol, which were not evident on treatment with atorvastatin alone, such as prevention of in vitro LDL-oxidation, increase in paraoxonase-1 activity of HDL, and decreases in plasma and plaque levels of oxidized-LDL in vivo. CONCLUSIONS: Probucol has significant add-on anti-atherosclerotic effects when combined with atorvastatin treatment; suggesting that this combination might be beneficial for treatment of atherosclerosis.

We examined the effects of reduction of sphingomyelin level on cholesterol behavior in cells using 2 types of Chinese hamster ovary cell mutants deficient in sphingomyelin synthesis: LY-A strain defective in intracellular trafficking of ceramide for sphingomyelin synthesis, and LY-B strain defective in the enzyme catalyzing the initial step of sphingolipid biosynthesis. Although the sphingomyelin content in LY-A and LY-B cells was approximately 40 and approximately 15%, respectively, of the wild-type level without accumulation of ceramide, these mutant cells were almost identical in cholesterol content and also in plasma membrane cholesterol level to the wild-type cells. However, density gradient fractionation analysis of Triton X-100-treated lysates of cells prelabeled with [(3)H]cholesterol showed that the [(3)H]cholesterol level in the low-density floating fraction was lower in sphingomyelin-deficient cells than in wild-type cells. When cells were exposed to methyl-beta-cyclodextrin, cholesterol was more efficiently fluxed from sphingomyelin-deficient cells than wild-type cells. These results suggest that the steady state level of cholesterol at the plasma membrane is little affected by the sphingomyelin levels in Chinese hamster ovary cells, but that sphingomyelin levels play an important role in the retention of cholesterol in the plasma membrane against efflux to extracellular cholesterol-acceptors, due to interaction between sphingomyelin and cholesterol in detergent-resistant membrane domains.

Abstract —Recent studies have established oxidative modification of low density lipoprotein (LDL) as an important atherogenic factor. We examined the clinical relevance of circulating oxidized LDL (OxLDL) levels in atherosclerotic disease by an enzyme immunoassay with use of specific antibodies against OxLDL (FOH1a/DLH3) and apolipoprotein B. Plasma OxLDL levels were significantly higher in patients with coronary heart disease (n=65) than in control subjects (n=181; 201.3±11.2 versus 112.4±3.3 U/dL, respectively; P

Although there have been suggestions that the glycation and oxidation of low density lipoprotein (LDL) might increase its atherogenic potential, little is known about the presence of glycoxidative LDL in human atherosclerotic lesions. We developed specific antibodies against different immunological epitopes of AGE structures, including N o -(carboxymethyl)lysine-protein adduct (CML), a glycoxidation product, and structure(s) other than CML (nonCML), and a monoclonal antibody against oxidized phosphatidylcholine (oxPC), as an epitope of oxidized LDL. Immunohistochemical analysis demonstrated that the CML- and oxPC-epitopes were accumulated mainly in macrophage-derived foam cells in atherosclerotic lesions, including fatty streaks and atherosclerotic plaques. On the other hand, the nonCML-epitope and apolipoprotein B were localized mainly in extracellular matrices of atherosclerotic lesions. The CML- and oxPC-epitopes were characterized by a model antigen-generating system using the copper ion-induced peroxidation and:or glucose-induced glycation of LDL. The glycoxidation of LDL caused the formation of CML-epitope with increasing concentrations of copper ion and glucose. It was also formed to some extent in LDL incubated with high concentrations (500 mM) of glucose. However, no CML-epitope was observed in oxidized LDL induced by copper ion alone. On the other hand, the formation of oxPC-epitope in LDL was dependent on copper ion-induced peroxidation, but independent of glucose-induced glycation. The addition of chelators, ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid, reduced the increase in electrophoretic mobility and TBARS caused by the peroxidation and glycoxidation of LDL, but had no effects on the formation of fructosamine caused by the glycation and glycoxidation of LDL. Chelators as well as aminoguanidine protected the formation of CML-epitope in glycated or glycoxidative LDL. Although the formation of oxPC-epitope was completely inhibited by the addition of chelators, it was partially protected by aminoguanidine. These in vitro results suggest that the glycoxidative modification of LDL may occur in the arterial intima, and may contribute to the development of human atherosclerotic lesions. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

To develop a simple and practical enzyme immunoassay (EIA) for oxidatively modified low density lipoprotein (Ox-LDL) in human blood, a biological marker of atherogenesis.A sandwich EIA suitable for the measurement of human Ox-LDL was developed using the mouse monoclonal antibody FOH1a/DLH3. This antibody, specific for oxidized phosphatidylcholine, was used as the capture antibody, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein-B (Apo-B) IgG was used for detection. Copper-oxidized human LDL, prepared under controlled conditions, was used as a standard and the results of the EIA were expressed in arbitrary units (U/mL).This EIA meets all the requirements for use in routine clinical assays in terms of sensitivity (detection limit: 1 U/mL), reproducibility (total CV: 2.3-7.7%), accuracy (recovery: 90.6-103.8%), simplicity and rapidity (4 h). Clinical performance of the assay was assessed by measurement of the Ox-LDL in the plasma of normal subjects (10.8 +/- 2.8 U/mL, mean +/- SD) and patients with coronary heart disease (CHD) (19.7 +/- 10.2). The present EIA had a sensitivity of 79% and a specificity of 75% for CHD.We have developed a new assay, suitable for the measurement of Ox-LDL in human blood, which meets the requirements for routine clinical assay.

Stimulated phospholipid degradation was observed in liver homogenates harvested from rats who had received an injection of carbon tetrachloride (CCl4). When the liver homogenates obtained from CCl4-treated rats were incubated for 1 h at 37°C, approximately half of the phosphatidylethanolamine (PE) was hydrolyzed, producing a stoichiometrical amount of lysophosphatidylethanolamine (LysoPE). The homogenates obtained from control rats showed only poor hydrolytic activity toward endogenous PE. The fatty acid composition of lysoPE produced was essentially identical to that at the sn-1 position of PE, suggesting that A2 type of phospholipase (PLase A2) was involved in the degradation during incubation. The hydrolysis of endogenous PE was dependent on calcium ion. The hydrolytic activity was almost exclusively associated with the membrane fraction which was precipitated by centrifugation at 10000 × g, and did not require the cytosol fraction. Para-bromophenacylbromide, rabbit antibody against rat 14 kDa type II PLase A2, and thielocin A1, a specific inhibitor of type II PLase A2, suppressed the hydrolysis almost completely. These results indicate that the accelerated degradation of endogenous PE observed in liver homogenates of CCl4-treated rat may be mainly due to the activity of the enzyme closely related to type II PLase A2.

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When (123)I-labeled ASH1a/256C antibody is injected intravenously into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purified by high performance liquid chromatography, was shown to be phosphatidylcholine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing (1)H and (13)C nuclear magnetic resonance, Fourier transfer-infrared spectrum, and mass spectrometry. The antibody did not react with other classes of phospholipids or neutral lipids when tested using an enzyme-linked immunosorbent assay. When PC was mixed with either cholesterol, cholesteryl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal and WHHL rabbits were fractionated using sucrose density gradient ultracentrifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions from WHHL rabbits was twice as high as that of normal rabbits, and there was an enormous difference in the antigenic activity in these fractions. The content of cholesterol in the cellular membrane fraction of WHHL rabbits was approximately 50 times higher than that of normal rabbits. Addition of neutral lipids to the cellular membrane fraction of normal rabbit markedly increased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-neutral lipid complex domains are formed in atherosclerotic lesions.

To better understand the role of advanced glycation end products (AGEs) in atherogenesis, we developed specific antibodies against different immunological epitopes of AGE structures, including N e -(carboxymethyl)lysine-protein adduct (CML) and a structure(s) other than CML (nonCML), and demonstrated the immunohistochemical localization of CML- and nonCML-epitopes in atherosclerotic lesions of human aorta, which were obtained at autopsy from 20 nondiabetic patients (12 males and eight females; mean age, 60.8±16.7 years). Monoclonal anti-CML antibody (6D12) recognized not only AGE-modified proteins, but also CML-modified proteins. On the other hand, polyclonal anti-nonCML antibody reacted to AGE-modified proteins, but not to CML-modified proteins. Both antibodies were unreactive to the early-stage products of glycation, including fructose-modified butyloxycarbonyl-lysine and fructose- e -aminocaproic acid. Atherosclerotic lesions included diffuse intimal thickening (DIT), fatty streaks (FS), atherosclerotic plaques (AP) and complicated lesions. An immunohistochemical analysis showed both CML- and nonCML-epitopes to be found along the collagen fibers in DIT in subjects more than 40 years old, but not in subjects less than 40 years old. CML-epitopes accumulated mainly in the cytoplasm of macrophage/foam cells, while nonCML-epitopes accumulated exclusively in the extracellular spaces in FS. APs showed the CML-epitope stored macrophage/foam cells, and the accumulation of both CML- and nonCML-epitopes in the lipid-rich fibrous area. An immunohistochemical analysis with a monoclonal antibody against oxidized low density lipoprotein (FOH1a/DLH3) showed the presence of this antigen within the cytoplasm of the macrophage/foam cells in atherosclerotic lesions, which were also positive for the CML-epitopes. These findings thus suggest that the heterogeneous localization of AGEs in atherosclerotic lesions depends on their different epitopes, and that a close link, therefore, exists between the peroxidation of LDL and the formation of AGEs in atherosclerotic lesions.

A new monoclonal antibody against malondialdehyde (MDA)-treated low density lipoprotein (LDL) was raised using homogenate of human atheroma as immunogen. This antibody, DLH2, was obtained by selecting the clones which did not react to native LDL but did react to copper-induced oxidized LDL (OxLDL). DLH2 showed a greater reactivity to MDA-LDL than to OxLDL. When LDL was treated with various aldehyde containing reagents, treatment of LDL with glutaraldehyde or MDA greatly increased the reactivity to the antibody, while LDL treated with 2,4-hexadienal or 4-hydroxynonenal was not reactive. Among many proteins tested, high density lipoprotein, bovine serum albumin and hemoglobin showed significant reactivity to DLH2 after they were treated with MDA or glutaraldehyde. When low density and high density lipoproteins treated with MDA were subjected to immunoblot analysis, newly formed products larger than the original apolipoproteins were detected with the antibody, suggesting that this antibody recognizes aggregated proteins with divalent short chain cross linkers. The antigenic materials were shown by immunohistochemical analysis to be present in foamy macrophages in human atheromatous lesions. DLH2 antigen did not colocalize either with apolipoprotein B. Furthermore, we found a massive accumulation of the antigenic material in Kupffer cells in the liver of rats treated with alcohol and carbonyl iron, a model of hepatic fibrosis due to oxidative stress. These results suggest the presence of cross linked proteins in damaged tissues.

Oxidatively modified low density lipoprotein (OxLDL) is known to be involved in atherogenesis. We have previously developed a murine monoclonal antibody, FOH1a/DLH3, which recognized oxidatively modified lipoproteins as well as foam cells in human atherosclerotic lesions (Itabe, H., Takeshima, E., Iwasaki, H., Kimura, J., Yoshida, Y., Imanaka, T., and Takano, T. (1994) J. Biol. Chem. 269, 15274-15279). The antigen of this monoclonal antibody was formed by peroxidation of phosphatidylcholine (PC), and the antigenic oxidized PC (OxPC) derivatives are thought to form complexes with polypeptides including apolipoproteins. OxLDL was measured by a sensitive sandwich enzyme-linked immunosorbent assay using the monoclonal antibody and anti-human apolipoprotein B antibody, in which antigenic OxPC competed with OxLDL. When antigenic activities of PC analogs were tested by the competition assay, 1-palmitoyl-2-(9-oxononanoyl) PC (9-CHO PC) and the hydroperoxide of egg PC potently inhibited the detection of OxLDL. 1-Palmitoyl-2-linoleoyl PC was oxidized with ferrous ion and ascorbic acid, and the antigenic products were purified from the OxPC extracts on high pressure liquid chromatography columns and subsequently analyzed by laser desorption mass spectrometry. Molecular weight determination and retention times of high pressure liquid chromatography suggest that one of these products was 9-CHO PC. Other products are thought to be 8-carbon aldehyde, dihydroxy, and ketohydroxy derivatives of PC. When a C-terminal 16-mer synthetic peptide of the 70-kDa peroxisomal membrane protein was simply incubated with 9-CHO PC, it was found to be reactive in a sandwich enzyme-linked immunosorbent assay using FOH1a/DLH3 and an anti-peptide antiserum. These results suggest that the anti-OxLDL monoclonal antibody FOH1a/DLH3 reacts with several oxidized products of PC including aldehyde derivatives of PC, which covalently modify polypeptides.

BACKGROUND & AIMS: Lipid islands are a common finding in the gastric mucosa, but their pathogenesis has not yet been established. The aim of this study was to investigate the morphology and immunophenotype of the various cells in lipid islands and to consider the possible mechanisms involved in the pathogenesis of these lesions. METHODS: Morphological and immunohistochemical investigations using antibodies against macrophages, smooth muscle cells, and lymphocytes were performed. Unfixed tissue was available for immunostaining for low-density lipoprotein (LDL) and oxidized LDL in one case. RESULTS: The lipid islands were composed of KP1-, KiM1p-, and cathepsin D-positive foam cells that were only weakly reactive for lysozyme. In cryostat sections, the foam cells were found to contain LDL and oxidized LDL. A few smooth muscle cells, plasma cells, lymphocytes, pericytes, fibroblasts, and Schwann cells that contained lipid droplets were also found. CONCLUSIONS: In gastric lipid islands, the presence of oxidized LDL, which is taken up by macrophages and smooth muscle cells via scavenger receptors, suggests that oxidized LDL is of key significance in the development and persistence of these lesions. Because the metabolism of LDL to oxidized LDL may occur by various mechanisms, various different initial conditions, including gastritis, may precede the development of lipid islands. Thus, anti-inflammatory treatment may be appropriate. (Gastroenterology 1996 Feb;110(2):369-74)

We have established a new method capable of measuring the very low concentrations of oxidized low density lipoprotein (OxLDL). In our previous study, we obtained a novel murine monoclonal antibody against oxidized lipoproteins (Itabe, H. et al. 1994. J. Biol. Chem. 269: 15274-15279). The epitope of this antibody resides in oxidized products of phosphatidylcholine that can form complexes with polypeptides, including apolipoprotein B. When the monoclonal antibody was precoated onto microtiter wells prior to carrying out a sandwich ELISA using an anti-human apolipoprotein B antibody, it was possible to detect 0.5 ng protein of copper-induced OxLDL. The detection of OxLDL was dependent on the presence of monoclonal antibody and was blocked by oxidized phosphatidylcholine (OxPC). Under the same sandwich ELISA condition, native LDL showed a dose-dependent increase of absorbance that was inhibited by complex of OxPC with BSA. These results suggest the possible occurrence of oxidative modification of human plasma LDL, which is recognized by the antibody against OxPC. The level of LDL oxidation of normal human subjects was found to be 0.52 +/- 0.35 units per 5 mu g protein of LDL, where one unit was defined as the reactivity corresponding to 1 ng of copper-induced OxLDL by this assay. Furthermore, we found that the LDL oxidation level in patients who had been receiving hemodialysis treatment was increased more than eightfold over that of normal subjects. We suggest that LDL in human plasma is oxidatively modified under certain conditions and this method for measurement of OxLDL could be used to study the relationship between in vivo oxidation reaction and various pathological conditions.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. Although pathogenic factors, such as oxidative stress, inflammation and genetics are thought to contribute to the development of AMD, little is known about the relationships and priorities between these factors. Here, we show that chronic photo-oxidative stress is an environmental factor involved in AMD pathogenesis. We first demonstrated that exposure to light induced phospholipid oxidation in the mouse retina, which was more prominent in aged animals. The induced oxidized phospholipids led to an increase in the expression of monocyte chemoattractant protein-1, which then resulted in macrophage accumulation, an inflammatory process. Antioxidant treatment prevented light-induced phospholipid oxidation and the subsequent increase of monocyte chemoattractant protein-1 (also known as C-C motif chemokine 2; CCL2), which are the beginnings of the light-induced changes. Subretinal application of oxidized phospholipids induced choroidal neovascularization, a characteristic feature of wet-type AMD, which was inhibited by blocking monocyte chemoattractant protein-1. These findings strongly suggest that a sequential cascade from photic stress to inflammatory processes through phospholipid oxidation has an important role in AMD pathogenesis. Finally, we succeeded in mimicking human AMD in mice with low-level, long-term photic stress, in which characteristic pathological changes, including choroidal neovascularization formation, were observed. Therefore, we propose a consecutive pathogenic pathway involving photic stress, oxidation of phospholipids and chronic inflammation, leading to angiogenesis. These findings add to the current understanding of AMD pathology and suggest protection from oxidative stress or suppression of the subsequent inflammation as new potential therapeutic targets for AMD.