Lysosomal oxidation of LDL alters lysosomal pH, induces senescence, and increases secretion of pro-inflammatory cytokines in human macrophages.

We have shown that aggregated LDL is internalized by macrophages and oxidized in lysosomes by redox-active iron. We have now investigated to determine whether the lysosomal oxidation of LDL impairs lysosomal function and whether a lysosomotropic antioxidant can prevent these alterations. LDL aggregated by SMase (SMase-LDL) caused increased lysosomal lipid peroxidation in human monocyte-derived macrophages or THP-1 macrophage-like cells, as shown by a fluorescent probe, Foam-LPO. The pH of the lysosomes was increased considerably by lysosomal LDL oxidation as shown by LysoSensor Yellow/Blue and LysoTracker Red. SMase-LDL induced senescence-like properties in the cells as shown by ß-galactosidase staining and levels of p53 and p21. Inflammation plays a key role in atherosclerosis. SMase-LDL treatment increased the lipopolysaccharide-induced secretion of TNF-α, IL-6, and MCP-1. The lysosomotropic antioxidant, cysteamine, inhibited all of the above changes. Targeting lysosomes with antioxidants, such as cysteamine, to prevent the intralysosomal oxidation of LDL might be a novel therapy for atherosclerosis.

Oxidation of low-density lipoprotein by iron at lysosomal pH: implications for atherosclerosis.

Low-density lipoprotein (LDL) has recently been shown to be oxidized by iron within the lysosomes of macrophages, and this is a novel potential mechanism for LDL oxidation in atherosclerosis. Our aim was to characterize the chemical and physical changes induced in LDL by iron at lysosomal pH and to investigate the effects of iron chelators and α-tocopherol on this process. LDL was oxidized by iron at pH 4.5 and 37 °C and its oxidation monitored by spectrophotometry and high-performance liquid chromatography. LDL was oxidized effectively by FeSO(4) (5-50 µM) and became highly aggregated at pH 4.5, but not at pH 7.4. The level of cholesteryl esters decreased, and after a pronounced lag, the level of 7-ketocholesterol increased greatly. The total level of hydroperoxides (measured by the triiodide assay) increased up to 24 h and then decreased only slowly. The lipid composition after 12 h at pH 4.5 and 37 °C was similar to that of LDL oxidized by copper at pH 7.4 and 4 °C, i.e., rich in hydroperoxides but low in oxysterols. Previously oxidized LDL aggregated rapidly and spontaneously at pH 4.5, but not at pH 7.4. Ferrous iron was much more effective than ferric iron at oxidizing LDL when added after the oxidation was already underway. The iron chelators diethylenetriaminepentaacetic acid and, to a lesser extent, desferrioxamine inhibited LDL oxidation when added during its initial stages but were unable to prevent aggregation of LDL after it had been partially oxidized. Surprisingly, desferrioxamine increased the rate of LDL modification when added late in the oxidation process. α-Tocopherol enrichment of LDL initially increased the rate of oxidation of LDL but decreased it later. The presence of oxidized and highly aggregated lipid within lysosomes has the potential to perturb the function of these organelles and to promote atherosclerosis.

Effects of lysosomal low-density lipoprotein oxidation by ferritin on macrophage function.

We have previously demonstrated that low-density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Some of the iron content of lysosomes might be delivered through autophagy of ferritin (the main iron-storage protein in the body). We have now investigated the effects of ferritin-mediated LDL oxidation on macrophage function. The addition of ferritin to human THP-1 cells and human monocyte-derived macrophages increased lysosomal lipid peroxidation, as shown by LPO-Foam, a fluorescent probe targeted to lysosomes. Incubating THP-1 cells with ferritin and native LDL or LDL aggregated by sphingomyelinase, to allow their endocytosis and delivery to lysosomes, led to the formation of lysosomal ceroid (an advanced lipid oxidation product), indicative of lysosomal LDL oxidation. Incubating THP-1 cells with ferritin and LDL caused metabolic activation of the cells, as shown by increased extracellular acidification and oxygen consumption measured by a Seahorse analyzer. LDL oxidized by ferritin in lysosomes might be released from macrophages when the cells die and lyse and affect neighboring cells in atherosclerotic lesions. Adding LDL oxidized by ferritin at lysosomal pH (pH 4.5) to macrophages increased their intracellular reactive oxygen species formation, shown using dihydroethidium, and increased apoptosis. Ferritin might therefore contribute to LDL oxidation in the lysosomes of macrophages and have atherogenic effects.

Monocytic Cell Adhesion to Oxidised Ligands: Relevance to Cardiovascular Disease.

Atherosclerosis, the major cause of vascular disease, is an inflammatory process driven by entry of blood monocytes into the arterial wall. LDL normally enters the wall, and stimulates monocyte adhesion by forming oxidation products such as oxidised phospholipids (oxPLs) and malondialdehyde. Adhesion molecules that bind monocytes to the wall permit traffic of these cells. CD14 is a monocyte surface receptor, a cofactor with TLR4 forming a complex that binds oxidised phospholipids and induces inflammatory changes in the cells, but data have been limited for monocyte adhesion. Here, we show that under static conditions, CD14 and TLR4 are implicated in adhesion of monocytes to solid phase oxidised LDL (oxLDL), and also that oxPL and malondialdehyde (MDA) adducts are involved in adhesion to oxLDL. Similarly, monocytes bound to heat shock protein 60 (HSP60), but this could be through contaminating lipopolysaccharide. Immunohistochemistry on atherosclerotic human arteries demonstrated increased endothelial MDA adducts and HSP60, but endothelial oxPL was not detected. We propose that monocytes could bind to MDA in endothelial cells, inducing atherosclerosis. Monocytes and platelets synergized in binding to oxLDL, forming aggregates; if this occurs at the arterial surface, they could precipitate thrombosis. These interactions could be targeted by cyclodextrins and oxidised phospholipid analogues for therapy.

Vitamins E and C do not effectively inhibit low density lipoprotein oxidation by ferritin at lysosomal pH.

Low density lipoprotein (LDL) might be oxidized by iron in the lysosomes of macrophages in atherosclerotic lesions. We have shown previously that the iron-storage proteinferritin can oxidize LDL at lysosomal pH. We have now investigated the roles of the most important antioxidant contained in LDL, α-tocopherol (the main form of vitamin E) and of ascorbate (vitamin C), a major water-soluble antioxidant, on LDL oxidation by ferritin at lysosomal pH (pH 4.5). We incubated LDL with ferritin at pH 4.5 and 37 °C and measured its oxidation by monitoring the formation of conjugated dienes at 234 n min a spectrophotometer. α-Tocopherol is well known to inhibit LDL oxidation at pH 7.4, but enrichment of LDL with α-tocopherol was unable to inhibit LDL oxidation by ferritin at pH 4.5. Ascorbate had a complex effect on LDL oxidation by ferritin at lysosomal pH and exhibited both antioxidant and pro-oxidant effects. It had no antioxidant effect on partially oxidized LDL, only a pro-oxidant effect. Ascorbate completely inhibited LDL oxidation by copper at pH 7.4 for a long period, but in marked contrast did not inhibit LDL oxidation by copper at lysosomal pH. Dehydroascorbate, the oxidation product of ascorbate, had a pronounced pro-oxidant effect on LDL incubated with ferritin at pH 4.5. The inability of α-tocopherol and ascorbate to effectively inhibit LDL oxidation by ferritin at lysosomal pH might help to explain why the large clinical trials with these vitamins failed to show protection against cardiovascular diseases.

Cysteamine Decreases Low-Density Lipoprotein Oxidation, Causes Regression of Atherosclerosis, and Improves Liver and Muscle Function in Low-Density Lipoprotein Receptor-Deficient Mice.

Background We have shown previously that low-density lipoprotein (LDL) can be oxidized in the lysosomes of macrophages, that this oxidation can be inhibited by cysteamine, an antioxidant that accumulates in lysosomes, and that this drug decreases atherosclerosis in LDL receptor-deficient mice fed a high-fat diet. We have now performed a regression study with cysteamine, which is of more relevance to the treatment of human disease. Methods and Results LDL receptor-deficient mice were fed a high-fat diet to induce atherosclerotic lesions. They were then reared on chow diet and drinking water containing cysteamine or plain drinking water. Aortic atherosclerosis was assessed, and samples of liver and skeletal muscle were analyzed. There was no regression of atherosclerosis in the control mice, but cysteamine caused regression of between 32% and 56% compared with the control group, depending on the site of the lesions. Cysteamine substantially increased markers of lesion stability, decreased ceroid, and greatly decreased oxidized phospholipids in the lesions. The liver lipid levels and expression of cluster of differentiation 68, acetyl-coenzyme A acetyltransferase 2, cytochromes P450 (CYP)27, and proinflammatory cytokines and chemokines were decreased by cysteamine. Skeletal muscle function and oxidative fibers were increased by cysteamine. There were no changes in the plasma total cholesterol, LDL cholesterol, high-density lipoprotein cholesterol, or triacylglycerol concentrations attributable to cysteamine. Conclusions Inhibiting the lysosomal oxidation of LDL in atherosclerotic lesions by antioxidants targeted at lysosomes causes the regression of atherosclerosis and improves liver and muscle characteristics in mice and might be a promising novel therapy for atherosclerosis in patients.

Effect of vitamin E on low density lipoprotein oxidation at lysosomal pH.

Many cholesterol-laden foam cells in atherosclerotic lesions are macrophages and much of their cholesterol is present in their lysosomes and derived from low density lipoprotein (LDL). LDL oxidation has been proposed to be involved in the pathogenesis of atherosclerosis. We have shown previously that LDL can be oxidised in the lysosomes of macrophages. α-Tocopherol has been shown to inhibit LDL oxidation in vitro, but did not protect against cardiovascular disease in large clinical trials. We have therefore investigated the effect of α-tocopherol on LDL oxidation at lysosomal pH (about pH 4.5). LDL was enriched with α-tocopherol by incubating human plasma with α-tocopherol followed by LDL isolation by ultracentrifugation. The α-tocopherol content of LDL was increased from 14.4 ± 0.2 to 24.3 ± 0.3 nmol/mg protein. LDL oxidation was assessed by measuring the formation of conjugated dienes at 234 nm and oxidised lipids (cholesteryl linoleate hydroperoxide and 7-ketocholesterol) by HPLC. As expected, LDL enriched with α-tocopherol was oxidised more slowly than control LDL by Cu2+ at pH 7.4, but was not protected against oxidation by Cu2+ or Fe3+ or a low concentration of Fe2+ at pH 4.5 (it was sometimes oxidised faster by α-tocopherol with Cu2+ or Fe3+ at pH 4.5). α-Tocopherol-enriched LDL reduced Cu2+ and Fe3+ into the more pro-oxidant Cu+ and Fe2+ faster than did control LDL at pH 4.5. These findings might help to explain why the large clinical trials of α-tocopherol did not protect against cardiovascular disease.

Low density lipoprotein undergoes oxidation within lysosomes in cells.

The oxidized low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidized LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidized intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalized rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. The intracellular levels of oxysterols, measured by HPLC, increased during the chase incubation period, showing that LDL must have been oxidized inside the cells. Furthermore, we found that this oxidative modification was inhibited by lipid-soluble antioxidants, an iron chelator taken up by fluid-phase pinocytosis and the lysosomotropic drug chloroquine, which increases the pH of lysosomes. The results indicate that LDL oxidation can occur intracellularly, most probably within lysosomes.

Cysteamine inhibits lysosomal oxidation of low density lipoprotein in human macrophages and reduces atherosclerosis in mice.

BACKGROUND AND AIMS: We have shown previously that low density lipoprotein (LDL) aggregated by vortexing is internalised by macrophages and oxidised by iron in lysosomes to form the advanced lipid/protein oxidation product ceroid. We have now used sphingomyelinase-aggregated LDL, a more pathophysiological form of aggregated LDL, to study lysosomal oxidation of LDL and its inhibition by antioxidants, including cysteamine (2-aminoethanethiol), which concentrates in lysosomes by several orders of magnitude. We have also investigated the effect of cysteamine on atherosclerosis in mice. METHODS: LDL was incubated with sphingomyelinase, which increased its average particle diameter from 26 to 170 nm, and was then incubated for up to 7 days with human monocyte-derived macrophages. LDL receptor-deficient mice were fed a Western diet (19-22 per group) and some given cysteamine in their drinking water at a dose equivalent to that used in cystinosis patients. The extent of atherosclerosis in the aortic root and the rest of the aorta was measured. RESULTS: Confocal microscopy revealed lipid accumulation in lysosomes in the cultured macrophages. Large amounts of ceroid were produced, which colocalised with the lysosomal marker LAMP2. The antioxidants cysteamine, butylated hydroxytoluene, amifostine and its active metabolite WR-1065, inhibited the production of ceroid. Cysteamine at concentrations well below those expected to be present in lysosomes inhibited the oxidation of LDL by iron ions at lysosomal pH (pH 4.5) for prolonged periods. Finally, we showed that the extent of atherosclerotic lesions in the aortic root and arch of mice was significantly reduced by cysteamine. CONCLUSIONS: These results support our hypothesis that lysosomal oxidation of LDL is important in atherosclerosis and hence antioxidant drugs that concentrate in lysosomes might provide a novel therapy for this disease.

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity.

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.

Low density lipoprotein oxidation by ferritin at lysosomal pH.

Oxidation of low density lipoprotein (LDL) has been proposed to be involved in the pathogenesis of atherosclerosis. We have previously shown that LDL can be oxidised by iron in lysosomes. As the iron-storage protein ferritin might enter lysosomes by autophagy, we have investigated the ability of ferritin to catalyse LDL oxidation at lysosomal pH. LDL was incubated with ferritin at 37 °C and pH 4.5 and its oxidation monitored spectrophotometrically at 234 nm by the formation of conjugated dienes and by measuring oxidised lipids by HPLC or a tri-iodide assay. Iron released from ferritin was measured using the ferrous iron chelator bathophenanthroline and by ultrafiltration followed by atomic absorption spectroscopy. LDL was oxidised effectively by ferritin (0.05-0.2 µM). The oxidation at lysosomal pH (pH 4.5) was much faster than at pH 7.4. Ferritin increased cholesteryl linoleate hydroperoxide, total lipid hydroperoxides and 7-ketocholesterol. Iron was released from ferritin at acidic pH. The iron chelators, diethylenetriaminepentaacetate and EDTA, and antioxidant N,N׳-diphenyl-p-phenylenediamine inhibited the oxidation considerably, but not entirely. The antioxidant tempol did not inhibit the initial oxidation of LDL, but inhibited its later oxidation. Cysteamine, a lysosomotropic antioxidant, inhibited the initial oxidation of LDL in a concentration-dependent manner, however, the lower concentrations exhibited a pro-oxidant effect at later times, which was diminished and then abolished as the concentration increased. These results suggest that ferritin might play a role in lysosomal LDL oxidation and that antioxidants that accumulate in lysosomes might be a novel therapy for atherosclerosis.

Antioxidants inhibit low density lipoprotein oxidation less at lysosomal pH: A possible explanation as to why the clinical trials of antioxidants might have failed.

Oxidised low density lipoprotein (LDL) was considered to be important in the pathogenesis of atherosclerosis, but the large clinical trials of antioxidants, including the first one using probucol (the PQRST Trial), failed to show benefit and have cast doubt on the importance of oxidised LDL. We have shown previously that LDL oxidation can be catalysed by iron in the lysosomes of macrophages. The aim of this study was therefore to investigate the effectiveness of antioxidants in preventing LDL oxidation at lysosomal pH and also establish the possible mechanism of oxidation. Probucol did not effectively inhibit the oxidation of LDL at lysosomal pH, as measured by conjugated dienes or oxidised cholesteryl esters or tryptophan residues in isolated LDL or by ceroid formation in the lysosomes of macrophage-like cells, in marked contrast to its highly effective inhibition of LDL oxidation at pH 7.4. LDL oxidation at lysosomal pH was inhibited very effectively for long periods by N,N'-diphenyl-1,4-phenylenediamine, which is more hydrophobic than probucol and has been shown by others to inhibit atherosclerosis in rabbits, and by cysteamine, which is a hydrophilic antioxidant that accumulates in lysosomes. Iron-induced LDL oxidation might be due to the formation of the superoxide radical, which protonates at lysosomal pH to form the much more reactive, hydrophobic hydroperoxyl radical, which can enter LDL and reach its core. Probucol resides mainly in the surface monolayer of LDL and would not effectively scavenge hydroperoxyl radicals in the core of LDL. This might explain why probucol failed to protect against atherosclerosis in various clinical trials. The oxidised LDL hypothesis of atherosclerosis now needs to be re-evaluated using different and more effective antioxidants that protect against the lysosomal oxidation of LDL.

The synthetic glycolipid-based TLR4 antagonist FP7 negatively regulates in vitro and in vivo haematopoietic and non-haematopoietic vascular TLR4 signalling.

TLRs, including TLR4, have been shown to play a crucial role in cardiovascular inflammatory-based diseases. The main goal of this study was to determine the potential of FP7, a synthetic glycolipid active as a TLR4 antagonist, to modulate haematopoietic and non-haematopoietic vascular TLR4 pro-inflammatory signalling. HUVEC, human THP-1 monocytes, THP-1-derived macrophages, mouse RAW-264.7 macrophages and Angiotensin II-infused apolipoprotein E-deficient mice were in vitro and in vivo models, respectively. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 on TLR4 functional activity in response to bacterial LPS ( in vitro) and endogenous ligands of sterile inflammation ( in vitro and in vivo). Following activation of TLR4, in vitro and in vivo data revealed that FP7 inhibited p38 MAPK and p65 NF-kB phosphorylation associated with down-regulation of a number of TLR4-dependent pro-inflammatory proteins. In addition to inhibition of LPS-induced TLR4 signalling, FP7 negatively regulated TLR4 activation in response to ligands of sterile inflammation (hydroperoxide-rich oxidised LDL, in vitro and Angiotensin II infusion, in vivo). These results demonstrate the ability of FP7 to negatively regulate in vitro and in vivo haematopoietic and non-haematopoietic vascular TLR4 signalling both in humans and mice, suggesting the potential therapeutic use of this TLR4 antagonist for pharmacological intervention of vascular inflammatory diseases.

Common variants of apolipoprotein A-IV differ in their ability to inhibit low density lipoprotein oxidation.

Apolipoprotein A-IV (apoA-IV) inhibits lipid peroxidation, thus demonstrating potential anti-atherogenic properties. The aim of this study was to investigate how the inhibition of low density lipoprotein (LDL) oxidation was influenced by common apoA-IV isoforms. Recombinant wild type apoA-IV (100 microg/ml) significantly inhibited the oxidation of LDL (50 microg protein/ml) by 5 microM CuSO(4) (P<0.005), but not by 100 microM CuSO(4), suggesting that it may act by binding copper ions. ApoA-IV also inhibited the oxidation of LDL by the water-soluble free-radical generator 2,2'-azobis(amidinopropane) dihydrochloride (AAPH; 1 mM), as shown by the two-fold increase in the time for half maximal conjugated diene formation (T(1/2); P<0.05) suggesting it can also scavenge free radicals in the aqueous phase. Compared to wild type apoA-IV, apoA-IV-S347 decreased T(1/2) by 15% (P=0.036) and apoA-IV-H360 increased T(1/2) by 18% (P=0.046). All apoA-IV isoforms increased the relative electrophoretic mobility of native LDL, suggesting apoA-IV can bind to LDL and acts as a site-specific antioxidant. The reduced inhibition of LDL oxidation by apoA-IV-S347 compared to wild type apoA-IV may account for the previous association of the APOA4 S347 variant with increased CHD risk and oxidative stress.

In vitro antioxidant activity of coffee compounds and their metabolites.

In this paper we report the antioxidant activity of different compounds which are present in coffee or are produced as a result of the metabolism of this beverage. In vitro methods such as the ABTS*+ [ABTS = 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] decolorization assay and the oxygen radical absorbance capacity assay (ORAC) were used to assess the capacity of coffee compounds to scavenge free radicals. The importance of caffeine metabolites and colonic metabolites in the overall antioxidant activity associated with coffee consumption is shown. Colonic metabolites such as m-coumaric acid and dihydroferulic acid showed high antioxidant activity. The ability of these compounds to protect human low-density lipoprotein (LDL) oxidation by copper and 2,2'-azobis(2-amidinopropane) dihydrochloride was also explored. 1-Methyluric acid was particularly effective at inhibiting LDL oxidative modification. Different experiments showed that this caffeine metabolite is not incorporated into LDL particles. However, at physiologically relevant concentrations, it was able to delay for more than 13 h LDL oxidation by copper.

Effects of dairy products naturally enriched with cis-9,trans-11 conjugated linoleic acid on the blood lipid profile in healthy middle-aged men.

BACKGROUND: Interest in the development of dairy products naturally enriched in conjugated linoleic acid (CLA) exists. However, feeding regimens that enhance the CLA content of milk also increase concentrations of trans-18:1 fatty acids. The implications for human health are not yet known. OBJECTIVE: This study investigated the effects of consuming dairy products naturally enriched in cis-9,trans-11 CLA (and trans-11 18:1) on the blood lipid profile, the atherogenicity of LDL, and markers of inflammation and insulin resistance in healthy middle-aged men. DESIGN: Healthy middle-aged men (n = 32) consumed ultra-heat-treated milk, butter, and cheese that provided 0.151 g/d (control) or 1.421 g/d (modified) cis-9,trans-11 CLA for 6 wk. This was followed by a 7-wk washout and a crossover to the other treatment. RESULTS: Consumption of dairy products enriched with cis-9,trans-11 CLA and trans-11 18:1 did not significantly affect body weight, inflammatory markers, insulin, glucose, triacylglycerols, or total, LDL, and HDL cholesterol but resulted in a small increase in the ratio of LDL to HDL cholesterol. The modified dairy products changed LDL fatty acid composition but had no significant effect on LDL particle size or the susceptibility of LDL to oxidation. Overall, increased consumption of full-fat dairy products and naturally derived trans fatty acids did not cause significant changes in cardiovascular disease risk variables, as may be expected on the basis of current health recommendations. CONCLUSION: Dairy products naturally enriched with cis-9,trans-11 CLA and trans-11 18:1 do not appear to have a significant effect on the blood lipid profile.

Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: activation by oxidatively modified LDL and 4-hydroxynonenal.

CD36 is an important scavenger receptor mediating uptake of oxidized low-density lipoproteins (oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase-1 (HO-1), and peroxiredoxin I (Prx I). 4-Hydroxy-2-nonenal (HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2-deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO-1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO-1, and Prx I protein levels. Because PPAR-gamma can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.

Oxidation affects the flow-induced aggregation of low density lipoprotein and its inhibition by albumin.

We investigated whether oxidation alters the self-aggregation of low density lipoprotein (LDL) and the inhibition of such aggregation by albumin. Incubation with copper for different durations produced mildly, moderately, and highly oxidised LDL (having, respectively, ca. 60, 300 and 160 nM lipid hydroperoxides/mg protein, and electrophoretic mobilities 1.2, 2.6 and 4.4 times that of native LDL). The rate of flow-induced aggregation was the same for native, mildly oxidised and moderately oxidised LDL, but decreased for highly oxidised LDL. The inhibitory effect of albumin (40 mg/ml) on aggregation was reduced by mild oxidation and further reduced by moderate or severe oxidation. The net result of the two effects was that in the presence of albumin, moderately oxidised LDL had the highest rate of aggregation and native the lowest. The reduction in the anti-aggregatory effect of albumin provides a new mechanism by which LDL oxidation might enhance net aggregation in vivo.

Induction of heme oxygenase 1 by moderately oxidized low-density lipoproteins in human vascular smooth muscle cells: role of mitogen-activated protein kinases and Nrf2.

Oxidized low-density lipoproteins (LDL) play a central role in atherogenesis and induce expression of the antioxidant stress protein heme oxygenase 1 (HO-1). In the present study we investigated induction of HO-1 and adaptive increases in reduced glutathione (GSH) in human aortic smooth muscle cells (SMC) in response to moderately oxidized LDL (moxLDL, 100 microg protein/ml, 24 h), a species containing high levels of lipid hydroperoxides. Expression and activity of HO-1 and GSH levels were elevated to a greater extent by moxLDL than highly oxidized LDL but unaffected by native or acetylated LDL. Inhibitors of protein kinase C (PKC) or mitogen-activated protein kinases (MAPK) p38(MAPK) and MEK or c-jun-NH2-terminal kinase (JNK) significantly attenuated induction of HO-1. Phosphorylation of p38(MAPK), extracellular signal-regulated kinase (ERK1/2), or JNK and nuclear translocation of the transcription factor Nrf2 were enhanced following acute exposure of SMC to moxLDL (100 microg protein/ml, 1-2 h). Pretreatment of SMC with the antioxidant vitamin C (100 microM, 24 h) attenuated the induction of HO-1 by moxLDL. Native and oxidized LDL did not alter basal levels of intracellular ATP, mitochondrial dehydrogenase activity, or expression of the lectin-like oxidized LDL receptor (LOX-1) in SMC. These findings demonstrate for the first time that activation of PKC, p38(MAPK), JNK, ERK1/2, and Nrf2 by oxidized LDL in human SMC leads to HO-1 induction, constituting an adaptive response against oxidative injury that can be ameliorated by vitamin C.

Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?

OBJECTIVE: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously. METHODS: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO(4), and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. RESULTS: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis. CONCLUSIONS: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering-activation-apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.