Cell-mediated Low-density Lipoprotein Oxidation Research Articles

Paraoxonase: The Universal Factor of Antioxidant Defense in Human Body

The paraoxonase (PON) gene family includes three members: PON1, PON2, and PON3 aligned in tandem on chromosome 7 in humans. All PON proteins share considerable structural homology and have the capacity to protect cells from oxidative stress; therefore, they have been implicated in the pathogenesis of several inflammatory diseases, particularly atherosclerosis. Increased production of reactive oxygen species as a result of decreased activities of mitochondrial electron transport chain complexes plays a role in the development of many inflammatory diseases, including atherosclerosis. PON1 and PON3 proteins can be detected in plasma and reside in the high-density lipoprotein fraction and protect against oxidative stress by hydrolyzing certain oxidized lipids in lipoproteins, macrophages, and atherosclerotic lesions. Paraoxonase 2 (PON2) possesses antiatherogenic properties and is associated with lower ROS levels. PON2 is involved in the antioxidative and anti-inflammatory response in intestinal epithelial cells. In contrast to PON1 and PON3, PON2 is cell-associated and is not found in plasma. It is widely expressed in a variety of tissues, including the kidney, and protects against cellular oxidative stress. Overexpression of PON2 reduces oxidative status, prevents apoptosis in vascular endothelial cells, and inhibits cell-mediated low density lipoprotein oxidation. PON2 also inhibits the development of atherosclerosis, via mechanisms involving the reduction of oxidative stress. In this review we explore the physiological roles of PON in disease development and modulation of PONs by infective (bacterial, viral) agents.

Pine bark extract prevents low-density lipoprotein oxidation and regulates monocytic expression of antioxidant enzymes

Polyphenols are widely distributed in leaves, seeds, bark, and flowers and considered to have beneficial effects on cardiovascular health. We hypothesized that the potent antioxidant properties of pine bark extract (PBE) are exerted by its ability to scavenge free radicals and induce antioxidant enzymes. Therefore, we investigated the effects of PBE on low-density lipoprotein (LDL) oxidation and the antioxidant defense system in monocytes. Oxidative susceptibility of LDL was determined by lag time assay in vitro and by using a human umbilical vein endothelial cell–mediated oxidation model. THP-1 monocytic cells were treated with PBE, and the expression of antioxidant enzymes was measured by real-time polymerase chain reaction and Western blot. Pine bark extract showed radical scavenging ability and significantly inhibited free radical-induced and endothelial cell-mediated LDL oxidation in vitro. Pine bark extract treatment resulted in increases in the expressions of antioxidant enzymes, glutathione peroxidase-1, catalase, and heme oxygenase-1 in THP-1 cells. In addition, PBE induced nuclear factor-erythroid-2–related factor 2 activation, which was accompanied by the activation of extracellular signal-regulated kinase and Akt despite a down-regulation of reactive oxygen species. After the monocyte investigations, we further examined the antioxidant effect after the intake of PBE by 10 healthy male volunteers. Pine bark extract significantly prolonged the lag time of LDL oxidation. Based on our findings, it appears that PBE enhances the antioxidant defense capacity of LDL and monocytes and may play a preventive role in atherosclerosis progression.

Coriandrum sativum L. seed extract mitigates lipotoxicity in RAW 264.7 cells and prevents atherogenic changes in rats.

This study was designed to assess the efficacy of Coriandrum sativum L. (CS) in preventing in vitro low density lipoprotein (LDL) oxidation mediated macrophage modification. Further, an in vivo study was also conducted to confirm upon the efficacy of CS seed extract in alleviating pathophysiological alterations of high cholesterol diet induced atherosclerosis in rats. Copper mediated cell free oxidation of LDL accounted for elevated indices of malondialdehyde (MDA), lipid hydroperoxide (LHP)and protein carbonyl (PC) and a progressive increment in conjugate diene (CD) levels whereas, reverse set of changes were recorded in presence of CS extract. Cell mediated LDL oxidation (using RAW 264.7 cells) accounted for lowered MDA production and oxidized LDL (Ox-LDL) mediated cell death in presence of CS extract and the same was attributed to its potent antioxidant and free radical scavenging potentials. High cholesterol fed atherogenic rats showed elevated lipid indices, evidences of LDL oxidation, plaque formation in thoracic aorta. The same was further validated with immunostaining of cell adhesion molecules and hematoxylin and eosin (HXE) staining. However, co-supplementation of CS to atherogenic rats recorded significant lowering of the above mentioned parameters further strengthening the claim that CS extract is instrumental in preventing onset and progression of atherosclerosis.

Antiatherogenic effect of guava leaf extracts inhibiting leucocyte-type 12-lipoxygenase activity

Oxidative modification of low density lipoprotein (LDL) is one of the critical steps for the development of atherosclerosis and leucocyte-type 12-lipoxygenase, highly expressed in macrophages, has been suggested to play an essential role in this process. In the present study, we show that guava leaf extracts inhibited, not only the leucocyte-type 12-lipoxygenase activity, but also LDL oxidation, mediated by the enzyme-overexpressing macrophage-like J774A.1 cells. Oral administration of guava leaf extracts to apoE-knockout mice at 100mg of dry extracts/kg of body weight, once a day for 16weeks, significantly reduced the area of atherogenic lesions developed in the aorta and aortic sinus. The major components inhibiting leucocyte-type 12-lipoxygenase contained in guava leaf extracts were identified as ethyl gallate and quercetin. The inhibitory effects of guava leaf extracts on the leucocyte-type 12-lipoxygenase activity, as well as on cell-mediated LDL oxidation, might be involved in the antiatherogenic effect of the extracts.

In Vitro Evidence for the Protective role of Sida rhomboidea. Roxb Extract Against LDL Oxidation and Oxidized LDL-Induced Apoptosis in Human Monocyte–Derived Macrophages

The present study was undertaken to evaluate protective role of S. rhomboidea. Roxb (SR) leaf extract against in vitro low-density lipoprotein (LDL) oxidation and oxidized LDL (Ox-LDL) induced macrophage apoptosis. Copper and cell-mediated LDL oxidation, Ox-LDL-induced peroxyl radical generation, mitochondrial activity, and apoptosis in human monocyte-derived macrophages (HMDMs) were assessed in presence of SR extract. Results clearly indicated that SR was capable of reducing LDL oxidation and formation of intermediary oxidation products. Also, SR successfully attenuated peroxyl radical formation, mitochondrial dysfunction, nuclear condensation, and apoptosis in Ox-LDL-exposed HMDMs. This scientific report is the first detailed investigation that establishes anti-atherosclerotic potential of SR extract.

Bioactive constituents and medicinal importance of genus Alnus

The genus Alnus has been reviewed for its chemical constituents and biological activities including traditional importance of some common species. The plants of this genus contain terpenoids, flavonoids, diarylheptanoids, phenols, steroids, and tannins. Diarylheptanoids are the dominant constituents within the genus Alnus, few of them exhibited antioxidant effects and inhibitory activity against nuclear factor kappaB activation, nitric oxide and tumor necrosis factor-α production, human umbilical vein endothelial cells, farnesyl protein transferase, cell-mediated low-density lipoprotein oxidation, HIF-1 in AGS cells, and the HIV-1-induced cytopathic effect in MT-4 cells. Some ellagitannines showed hepatoprotective activity even in a dose of 1 mg/kg which is ten-fold smaller compared with the dose of traditional flavonoid-based drugs. The members of genus Alnus are well known for their traditional uses in the treatment of various diseases like cancer, hepatitis, inflammation of uterus, uterine cancer, rheumatism, dysentery, stomachache, diarrhea, fever, etc. The aim of the present review is to summarize the various researches related to the chemistry and pharmacology of genus Alnus.

Sweet potato (Ipomoea batatas L.) leaves suppressed oxidation of low density lipoprotein (LDL) in vitro and in human subjects

Sweet potato (Ipomoea batatas L.) leaves are consumed as vegetables around the world, especially in Southeast Asia. The aim of this study was to investigate the inhibitory effect of sweet potato leaves on low-density lipoprotein oxidation in vitro and in human subjects. We compared the antioxidant activity of 8 kinds of sweet potato leaves. Every sweet potato leaf had high radical scavenging activity and prolonged a lag time for starting low-density lipoprotein oxidation in vitro. We found that sweet potato leaves contained abundant polyphenol compounds and the radical scavenging activity and prolongation rate of lag time were highly correlated with total polyphenol content. We also confirmed that thiobarbituric acid reactive substances production was increased in endothelial cell-mediated low-density lipoprotein oxidation, which was decreased by treatment with sweet potato leaves. We further measured the low-density lipoprotein oxidizability in 13 healthy volunteers after their intake of 18 g of “Suioh”, raw sweet potato leaves. “Suioh” prolonged a lag time for starting low-density lipoprotein oxidation and decreased low-density lipoprotein mobility. These results suggest that sweet potato leaves have antioxidant activity leading to the suppression of low-density lipoprotein oxidation.

Synthesis and antioxidant activity evaluation of a syringic hydrazones family

A novel series of hydrazones derived from syringaldehyde and their antioxidant properties have been explored. Several employed methods such as scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS • +) radical cation expressed as Trolox equivalent antioxidant capacity (TEAC), inhibition of superoxide anion (O 2 •−) generation and of human cell-mediated low-density lipoprotein oxidation (monitored by the formation of TBARS) exhibited their potent antioxidant properties. The carbonyl scavenger efficacy was also evaluated by measuring the ability to decrease the protein carbonyl content in cells challenged with oxidized LDL. In this report, we discuss about the synthesis of hydrazones and their dual biological role, antioxidant and carbonyl scavenger for further application in atherosclerosis.

Oxidized LDL impair adipocyte response to insulin by activating serine/threonine kinases

Oxidized LDL (oxLDL) increase in patients affected by type-2 diabetes, obesity, and metabolic syndrome. Likewise, insulin resistance, an impaired responsiveness of target tissues to insulin, is associated with those pathological conditions. To investigate a possible causal relationship between oxLDL and the onset of insulin resistance, we evaluated the response to insulin of 3T3-L1 adipocytes treated with oxLDL. We observed that oxLDL inhibited glucose uptake (-40%) through reduced glucose transporter 4 (GLUT4) recruitment to the plasma membrane (-70%), without affecting GLUT4 gene expression. These findings were associated to the impairment of insulin signaling. Specifically, in oxLDL-treated cells insulin receptor (IR) substrate-1 (IRS-1) was highly degraded likely because of the enhanced Ser(307)phosphorylation. This process was largely mediated by the activation of the inhibitor of kappaB-kinase beta (IKKbeta) and the c-Jun NH(2)-terminal kinase (JNK). Moreover, the activation of IKKbeta positively regulated the nuclear content of nuclear factor kappaB (NF-kappaB), by inactivating the inhibitor of NF-kappaB (IkappaBalpha). The activated NF-kappaB further impaired per se GLUT4 functionality. Specific inhibitors of IKKbeta, JNK, and NF-kappaB restored insulin sensitivity in adipocytes treated with oxLDL. These data provide the first evidence that oxLDL, by activating serine/threonine kinases, impaired adipocyte response to insulin affecting pathways involved in the recruitment of GLUT4 to plasma membranes (PM). This suggests that oxLDL might participate in the development of insulin resistance.

Development of Novel Antiatherogenic Biaryls: Design, Synthesis, and Reactivity

On the basis of the 5,5'-bisvanillin scaffold, a series of compounds has been synthesized presenting symmetric or dissymmetric frames on each phenolic moiety. These frames are alpha,beta-unsaturated (fluoro)phosphonate and/or alpha,beta-unsaturated hydrazone(s) formed by coupling aldehydic with isoniazid or hydralazine. All compounds were tested for their ability to inhibit cell-mediated low-density lipoprotein oxidation. Oxidized low-density lipoprotein induced cytotoxicity was also evaluated along with the carbonyl scavenger properties of selected compounds. The most efficient agents were found to be those possessing at least one hydralazinone frame, with the most potent being the symmetrical compound: 4,4'-dihydroxy-3,3'-dimethoxy-5,5'-biphenyl-1,1'-(diphthalazin-1-yl)methylhydrazone hydrochloride.

Macrophage mediated protein hydroperoxide formation and lipid oxidation in low density lipoprotein are inhibited by the inflammation marker 7,8-dihydroneopterin

The formation of oxidised low density lipoprotein (LDL) within the atherosclerotic plaque appears to be a factor in the development of advanced atherosclerotic plaques. LDL oxidation is dependent on the balance of oxidants and antioxidants within the intima. In addition to producing various oxidants, human macrophages release 7,8-dihydroneopterin which in vivo is oxidised to the inflammation marker neopterin. Using macrophage-like THP-1 cells and human monocyte-derived macrophages, we demonstrate that 7,8-dihydroneopterin is a potent inhibitor of cell-mediated LDL oxidation. 7,8-Dihydroneopterin scavenges the chain propagating lipid peroxyl radical, inhibiting both lipid and protein hydroperoxide formation. A significant amount of the hydroperoxide formed during cell-mediated LDL oxidation was protein hydroperoxide. 7,8-Dihydroneopterin oxidation to 7,8-dihydroxanthopterin was only observed in the presence of both cells and LDL, showing that 7,8-dihydroneopterin had no effect on initiating oxidant generation by the cells. 7,8-Dihydroneopterin did not regenerate α-tocopherol but competed with it for the lipid peroxyl radical. Although stimulation of both cell types with γ-interferon failed to produce sufficient 7,8-dihydroneopterin to inhibit LDL oxidation in tissue culture, analysis of advanced atherosclerotic plaque removed from patients showed that total neopterin levels could reach low micromolar concentrations. This suggests that 7,8-dihydroneopterin synthesis by macrophages could play a significant role in the development of atherosclerotic plaques.

Redistribution of metal ions to control low density lipoprotein oxidation in Ham's F10 medium

The study of cell-mediated low density lipoprotein (LDL) oxidation has traditionally been undertaken using Ham's F10 media due to its high metal content and low levels of antioxidants. Although there has been no acknowledged change to this media in recent years by the suppliers, Ham's F10 medium has been found to be extremely inconsistent in its promotion of LDL oxidation in the absence of cells. This variability contrasts with the relatively consistent rates of THP-1 cell-mediated LDL oxidation. This study has now shown that the variability in cell-free LDL oxidation is medium-dependent and not an artefact of experimental protocol. It presents evidence that suggests the variable rates of cell-free LDL oxidation are caused by iron auto-oxidation during storage of the Ham's F10 medium. The medium can be standardized by removal of all transition metals, by treatment with Chelex, before the addition of known amounts of iron or copper. This treatment generates a cell culture medium that only allows very slow LDL oxidation in the absence of cells.

Cyclic diarylheptanoids inhibit cell-mediated low-density lipoprotein oxidation

Two cyclic diarylheptanoids, garugamblin-3 (1) and acerogenin L (2), isolated from the MeOH extract of the fruits of Alnus japonica Steud., inhibited human low-density lipoprotein (LDL) oxidation in the thiobarbituric acid-reactive substance assay with IC50 values of 2.9 and 1.7 μM, respectively, and they also inhibited cell-mediated LDL oxidation more than five times more strongly than that of a well-known antioxidant, probucol, at a concentration of 10 μM. 1 had no effect on the anti-atherogenesis in low-density lipoprotein receptor- deficient mice.

Troglitazone Inhibits Long-Term Glycation and Oxidation of Low-Density Lipoprotein

Troglitazone (T) is a member of a new class of antidiabetic drugs termed thiazolidinediones (TZDs), which has previously been used as an anti-diabetic agent. In this study we investigated the influence of T, a ligand for PPAR-gamma receptor, on copper-catalyzed or cell-mediated oxidation of native, glycated, and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition of copper-mediated low-density lipoprotein-oxidation, as monitored by the formation of oxidation-specific fluorescence, was observed for both native and glycated low-density lipoprotein. At the concentration of 20 microg/mL the inhibition amounted from 14.7% to 64.7% by all low-density lipoprotein forms. For glycated low-density lipoprotein we obtained the highest oxidation rate, but the most pronounced inhibition by T was found for glycoxidated low-density lipoprotein (goLDL). Inhibitory effects of T were also investigated by measurement of relative electrophoretic mobility (REM) in the concentration range of 0 to 20 microg/mL. The inhibition of 4h oxidation of native low-density lipoprotein was found in the entire concentration range, but significance was seen at 10 microg/mL. The long-term glycation and glycoxidation of low-density lipoprotein as measured by 5-hydroxymethyl-2-furaldehyde (5-HMF) formation and binding of fructosamine was found to be inhibited by T. In endothelial cell-mediated oxidation of low-density lipoprotein cytotoxicity of T in the concentration range of 0 to 160 microg/mL during 2 to 24 h oxidation was investigated. In the non-cytotoxic concentration range of 5 to 20 microg/mL, a significantly reduced liberation of isoprostane 8-epi-PGF2alpha during 24 h cell-mediated oxidation of low-density lipoprotein and its modifications was found. This inhibitory action of T was most significant in the case of goLDL and amounted to approximately 20% to 60% inhibition at 5 to 20 microg/mL T, respectively. In the concentration range of 40 to 160 microg/mL, however, T showed an increasing cytotoxic action, as evidenced by loss of cell adhesion, loss of cellular protein, morphological changes, and cell disintegration as well as by strongly enhanced troglitazone-mediated isoprostane 8-IP liberation (fivefold to sixfold). T may be used as a model to explore the thiazolidinediones' mechanism on oxidation in a more general aspect for treatment for T2DM, because T is not clinically available.

Edaravone, a novel radical scavenger, inhibits oxidative modification of low-density lipoprotein (LDL) and reverses oxidized LDL-mediated reduction in the expression of endothelial nitric oxide synthase

Edaravone, a newly synthesized synthetic radical scavenger, has been identified and adopted as an anti-stroke agent. However, its mechanism and the effect of edaravone on lipoprotein oxidation are not fully understood. Therefore, whether edaravone could suppress oxidation of low-density lipoprotein (LDL) and be involved in the expression of endothelial nitric oxide synthase (eNOS) in relation to anti-atherogenesis by improving and conserving vascular circulation was investigated. We investigated the in vitro effects of edaravone on copper- and endothelial cell-mediated LDL oxidation, and the expression of eNOS in human umbilical vein endothelial cells (HUVEC) modulated by oxidized LDL. The in vivo effect of edaravone on antioxidative effect was also studied in male rats intravenously administered with edaravone. Edaravone apparently inhibited copper- and HUVEC-mediated LDL oxidation at the concentration equivalent to serum concentrations in clinical use. The intravenous administration of edaravone also enhanced serum radical-scavenging property in rats. We tested the effect of edaravone on protein and mRNA expression of eNOS in HUVEC. Edaravone enhanced eNOS expression in HUVEC, presumably because of increased stability of eNOS mRNA, and reversed eNOS expression reduced by oxidized LDL nearly to the control levels. The present study demonstrates for the first time that edaravone increases eNOS expression with the inhibition of LDL oxidation, and that edaravone can reverse oxidized LDL-mediated reduction in eNOS expression in endothelial cells. The preventive action of edaravone from ischemic disease consequence may be attributed to these eNOS up-regulation with decreased oxidation.

Flavonoids-rich nutrients with potent antioxidant activity prevent atherosclerosis development: the licorice example

Background: Macrophage-mediated oxidation of low-density lipoprotein (LDL) has a major role in early atherogenesis, and, thus, intervention means to inhibit this process by flavonoids-rich nutrients (pomegranate, red wine, licorice) are being extensively studied. In the present study, we questioned the ability of the isoflavan glabridin from licorice root to accumulate in macrophages and to affect cell-mediated oxidation of LDL and atherogenesis. Results: Macrophages accumulated glabridin, and this process was time and glabridin dose dependent. In parallel, macrophage-mediated oxidation of LDL was inhibited by up to 80%. Glabridin inhibited superoxides release from MPM by up to 60%, and in glabridin-enriched macrophages, reduced protein kinase C activity by 70% was noted, in comparison to control cells. Translocation of P-47phox, the cytosolic component of NADPH oxidase, to the plasma membrane was substantially inhibited by glabridin. Macrophages (MPM) isolated from the atherosclerotic apolipoprotein E-deficient (E o) mice that consumed glabridin (20 μg/mouse/day) for 6 weeks demonstrated reduced capability to oxidize LDL by 80%, in comparison to placebo-treated mice. This latter phenomenon was associated with a substantial 50% reduction in the mice aortic lesion size. Conclusions: Glabridin accumulation in macrophages affects cell-signaling processes, which are associated with activation of the cellular NADPH oxidase system. These phenomena are responsible for the inhibition of cell-mediated oxidation of LDL and the attenuation of atherosclerosis developments by dietary glabridin.

Inhibition of THP-1 cell-mediated low-density lipoprotein oxidation by the macrophage-synthesised pterin, 7,8-dihydroneopterin

Human macrophages release the pterin, 7,8-dihydroneopterin when exposed to the immune stimulant γ-interferon (IFN-γ). Previous in vitro studies have shown 7,8-dihydroneopterin is a potent antioxidant, which inhibits copper- and peroxyl-radical mediated low-density lipoprotein (LDL) oxidation. Using THP-1 cells, a human derived monocyte-like cell line, we have found that low micromolar concentrations of 7,8-dihydroneopterin inhibit cell mediated oxidation of LDL, as measured by electrophoretic mobility, α-tocopherol loss, and lipid oxidation. Stimulation of the THP-1 cells with IFN-γ caused a significant reduction in the cells' ability to oxidise LDL. The extracellular pterin concentration increased from 0 to 16 nM with IFN-γ stimulation, while the intracellular concentration increased from 0.21 to 1.69 nmol/mg cell protein.

Protein Hydroperoxides are a Major Product of Low Density Lipoprotein Oxidation During Copper, Peroxyl Radical and Macrophage-mediated Oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2′-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble α-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.

ATP-binding cassette transporter 1 participates in LDL oxidation by artery wall cells.

We have previously reported that products of the lipoxygenase pathway, hydroperoxyoctadecadienoic acid and hydroperoxyeicosatetraenoic acid, as well as cholesterol linoleate hydroperoxides, collectively termed seeding molecules, are removed by apolipoprotein A-I (apoA-I) from the artery wall cells and render low density lipoprotein (LDL) resistant to oxidation by human artery wall cells. The mechanisms by which oxidized lipids are transported and/or transferred to lipoproteins and the pathways by which apoA-I facilitates their removal remain unclear. ATP-binding cassette transporter 1 (ABCA1) is known to facilitate the release of cellular phospholipids and cholesterol from the plasma membrane to apoA-I and high density lipoprotein. Therefore, we evaluated whether ABCA1 participates in LDL oxidation. In this report, we show that (1) chemical inhibitors of ABCA1 function, glyburide and DIDS, block artery wall cell-mediated oxidative modification of LDL, (2) inhibition of ABCA1 with the use of antisense (but not sense) oligonucleotides prevents LDL-induced lipid hydroperoxide formation and LDL-induced monocyte chemotactic activity by the artery wall cells, and (3) oxysterols that induce ABCA1 expression, such as 22(R)hydroxycholesterol, enhance cell-mediated LDL oxidation. Furthermore, we also show that 22(R)hydroxycholesterol induces the production of reactive oxygen species in the artery wall cells, which can be removed by incubating the artery wall cells with apoA-I. Our data suggest that ABCA1 plays an important role in artery wall cell-mediated modification/oxidation of LDL by modulating the release of reactive oxygen species from artery wall cells that are necessary for LDL oxidation.

Low Density Lipoprotein Receptor-related Protein Is Required for Macrophage-mediated Oxidation of Low Density Lipoprotein by 12/15-Lipoxygenase

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.