Low density lipoprotein undergoes oxidation within lysosomes in cells

The oxidised 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) nonoxidised LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidised 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 internalised 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 oxidised 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.

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.

Antioxidant and anti-atherogenic activities of olive oil phenolics

The aim of the current study was to investigate the antioxidant and cellular activity of the olive oil phenolics oleuropein, tyrosol, hydroxytyrosol, and homovanillic alcohol (which is also a major metabolite of hydroxytyrosol). Well-characterized chemical and biochemical assays were used to assess the antioxidant potential of the compounds. Further experiments investigated their influence in cell culture on cytotoxic effects of hydrogen peroxide and oxidized low-density lipoprotein (LDL), nitric oxide production by activated macrophages, and secretion of chemoattractant and cell adhesion molecules by the endothelium. Inhibitory influences on in vitro platelet aggregation were also measured. The antioxidant assays indicated that homovanillic alcohol was a significantly more potent antioxidant than the other phenolics, both in chemical assays and in prolonging the lag phase of LDL oxidation. Cell culture experiments suggested that the olive oil phenolics induce a significant reduction in the secretion of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (and a trend towards a reduced secretion of monocyte chemoattractant protein-1), and protect against cytotoxic effects of hydrogen peroxide and oxidized LDL. However, no influence on nitric oxide production or platelet aggregation was evident. The data show that olive oil phenolics have biochemical and cellular actions, which, if also apparent in vivo, could exert cardioprotective effects.

Antioxidant and anti-atherogenic activities of olive oil phenolics

The aim of the current study was to investigate the antioxidant and cellular activity of the olive oil phenolics oleuropein, tyrosol, hydroxytyrosol, and homovanillic alcohol (which is also a major metabolite of hydroxytyrosol). Well-characterized chemical and biochemical assays were used to assess the antioxidant potential of the compounds. Further experiments investigated their influence in cell culture on cytotoxic effects of hydrogen peroxide and oxidized low-density lipoprotein (LDL), nitric oxide production by activated macrophages, and secretion of chemoattractant and cell adhesion molecules by the endothelium. Inhibitory influences on in vitro platelet aggregation were also measured. The antioxidant assays indicated that homovanillic alcohol was a significantly more potent antioxidant than the other phenolics, both in chemical assays and in prolonging the lag phase of LDL oxidation. Cell culture experiments suggested that the olive oil phenolics induce a significant reduction in the secretion of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 (and a trend towards a reduced secretion of monocyte chemoattractant protein-1), and protect against cytotoxic effects of hydrogen peroxide and oxidized LDL. However, no influence on nitric oxide production or platelet aggregation was evident. The data show that olive oil phenolics have biochemical and cellular actions, which, if also apparent in vivo, could exert cardioprotective effects.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subtractions and the relative atherogenicity of these subtractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation (S-f) rates: chylomicron (CM, S-f > 400), very low-density lipoprotein (VLDL)-1 (S-f 60-400) and VLDL-2 (S-f 20-60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-I caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Triglyceride-rich lipoproteins inhibit cholesterol efflux to apolipoprotein (apo) A1 from human macrophage foam cells

High circulating levels of triglyceride-rich lipoproteins (TGRL) represent an independent risk factor for coronary artery disease. Here, we show that TGRL inhibit the efflux of cholesterol from 'foam cell' macrophages to lipid-poor apolipoprotein (apo) A1, and may thereby inhibit arterial reverse cholesterol transport and promote the formation of atherosclerotic lesions. Human (THP-1) monocyte-derived macrophages were pre-incubated (48h) with acetylated low-density lipoprotein (AcLDL) to provide a foam cell model of cholesterol efflux to apoA1. Pre-incubation of macrophage 'foam cells' with TGRL (0-200 mug/ml, 0-24 h) inhibited the efflux of exogenously radiolabelled ([H-3]), endogenously synthesised ([C-14]) and cellular cholesterol mass to lipid-poor apoA1, but not control medium, during a (subsequent) efflux period. This inhibition is dependent upon the length of prior exposure to, and concentration of, TGRL employed, but is independent of changes in intracellular triglyceride accumulation or turnover of the cholesteryl ester pool. Despite the negative impact of TGRL on cholesterol efflux, major proteins involved in this process-namely apoE, ABCA1, SR-B1 and caveolin-1-were unaffected by TGRL pre-incubation, suggesting that exposure to these lipoproteins inhibits an alternate, and possibly novel, anti-atherogenic pathway. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Dietary vitamin C down-regulates inflammatory gene expression in apoE4 smokers

The deleterious impact of cigarette smoking on cardiovascular health may be in part attributable to a free radical mediated proinflammatory response in circulating monocytes. In the current investigation, the impact of vitamin C supplementation on monocyte gene expression was determined in apoE4 smokers versus non-smokers. A total of 10 smokers and 11 non-smokers consumed 60 mg/day of vitamin C for four weeks and a fasting blood sample was taken at baseline and post-intervention for the determination of plasma vitamin C and monocyte gene expression profiles using cDNA array and real time PCR. In apoE4 smokers, supplementation resulted in a 43% increase in plasma vitamin C concentrations. Furthermore, a number of genes were differentially expressed more than 2-fold in response to treatment, including a downregulation of the proinflammatory mediators tumor necrosis factor (TNF) beta, TNF receptor, neurotrophin-3 growth factor receptor, and monocyte chemoattractant protein I receptor. The study has identified a number of molecular mechanisms underlying the benefit of vitamin C supplementation in smokers. (c) 2005 Elsevier Inc. All rights reserved.

Effects of oils rich in eicosapentaenoic and docosahexaenoic acids on immune cell composition and function in healthy humans

Background: Supplementation of the diet with fish oil, which is rich in the long-chain n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is reported to decrease several markers of immune function. However, whether EPA, DHA, or a combination of the 2 exerts these immunomodulatory effects is unclear. Objective: The objective of the study was to determine the effects of supplementation with an EPA-rich or DHA-rich oil on a range of immune outcomes representing key functions of human neutrophils, monocytes, and lymphocytes in healthy humans. Design: In a placebo-controlled, double-blind, parallel study, 42 healthy subjects were randomly allocated to receive supplementation with either placebo (olive oil), EPA (4.7 g/d), or DHA (4.9 g/d) for 4 wk. Blood samples were taken before and after supplementation. Results: The fatty acid composition of plasma phospholipids and neutrophils was dramatically altered by supplementation with EPA or DHA, and the effects of EPA differed notably from those of DHA. DHA supplementation decreased T lymphocyte activation, as assessed by expression of CD69, whereas EPA supplementation had no significant effect. Neither the EPA-rich oil nor the DHA-rich oil had any significant effect on monocyte or neutrophil phagocytosis or on cytokine production or adhesion molecule expression by peripheral blood mononuclear cells. Conclusions: Supplementation with DHA, but not with EPA, suppresses T lymphocyte activation, as assessed by expression of CD69. EPA alone does not, therefore, influence CD69 expression. No other marker of immune function assessed in this study was significantly affected by either EPA or - DHA.

Relation between the fatty acid composition of peripheral blood mononuclear cells and measures of immune cell function in health free-living subjects aged 25-72 y

Background: There is little information about the relation between the fatty acid composition of human immune cells and the function of those cells over the habitual range of fatty acid intakes. Objective: The objective of the study was to determine the relation between the fatty acid composition of human peripheral blood mononuclear cell (PBMC) phospholipids and the functions of human immune cells. Design: One hundred fifty healthy adult subjects provided a fasting blood sample. The phagocytic and oxidative burst activities of monocytes and neutrophils were measured in whole blood. PBMCs were isolated and used to measure lymphocyte proliferation in response to the T cell mitogen concanavalin A and the production of cytokines in response to concanavalin A or bacterial lipopolysaccharide. The fatty acid composition of plasma and PBMC phospholipids was determined. Results: Wide variations in fatty acid composition of PBMC phospholipids and immune cell functions were identified among the subjects. The proportions of total Polyunsaturated fatty acids (PUFAs), of total n-6 and n-3 PUFAs, and of several individual PUFAs in PBMC phospholipids were positively correlated with phagocytosis by neutrophils and monocytes, neutrophil oxidative burst, lymphocyte proliferation, and interferon gamma production. The ratios of saturated fatty acids to PUFAs and of n-6 to n-3 PUFAs were negatively correlated with these same immune functions. The relation of PBMC fatty acid composition to monocyte oxidative burst was the reverse of its relation to monocyte phagocytosis and neutrophil oxidative burst. Conclusion: Variations in the fatty acid composition of PBMC phospholipids account for some of the variability in immune cell functions among healthy adults.

Effects of apoE genotype on macrophage inflammation and heme oxygenase-1 expression

In order to gain a more comprehensive understanding of the aetiology of apolipoprotein E4 genotype-cardiovascular disease (CVD) associations, the impact of the apoE genotype on the macrophage inflammatory response was examined. The murine monocyte-macrophage cell line (RAW 264.7) stably transfected to produce equal amounts of human apoE3 or apoE4 was used. Following LPS stimulation, apoE4-macrophages showed higher and lower concentrations of tumour necrosis factor alpha (pro-inflammatory) and interleukin 10 (anti-inflammatory), respectively, both at mRNA and protein levels. In addition, increased expression of heme oxygenase-1 (a stress-induced anti-inflammatory protein) was observed in the apoE4-cells. Furthermore, in apoE4-macrophages, an enhanced transactivation of the key redox sensitive transcription factor NF-kappa B was shown. Current data indicate that apoE4 macrophages have an altered inflammatory response, which may contribute to the higher CVD risk observed in apoE4 carriers. (c) 2007 Elsevier Inc. All rights reserved.

Soy-isoflavone-enriched foods and inflammatory biomarkers of cardiovascular disease risk in postmenopausal women: interactions with genotype and equol production

Background: Dietary isoflavones are thought to be cardioprotective because of their structural similarity to estrogen. The reduction of concentrations of circulating inflammatory markers by estrogen may be one of the mechanisms by which premenopausal women are protected against cardiovascular disease. Objective: Our aim was to investigate the effects of isolated soy isoflavones on inflammatory biomarkers [von Willebrand factor, intracellular adhesion molecule 1, vascular cell adhesion molecule 1 (VCAM-1), E-selectin, monocyte chemoattractant protein 1, C-reactive protein (CRP), and endothelin 1 concentrations]. Differences with respect to single-nucleotide polymorphisms in selected genes [estrogen receptor alpha (XbaI and PvuII), estrogen receptor beta [ER beta (AluI) and ER beta[cx] (Tsp5091), endothelial nitric oxide synthase (Glu298Asp), apolipoprotein E (Apo E2, E3, and E4), and cholesteryl ester transfer protein (TaqIB)] and equol production were investigated. Design: One hundred seventeen healthy European postmenopausal women participated in this randomized, double-blind, placebo-controlled, crossover dietary intervention trial. Isoflavone-enriched (genistein-to-daidzein ratio of 2:1;50 mg/d) or placebo cereal bars were consumed for 8 wk, with a washout period of 8 wk between the crossover. Plasma inflammatory factors were measured at 0 and 8 wk of each study arm. Results: Isoflavones improved CRP concentrations [odds ratio (95% Cl) for CRP values >1 mg/L for isoflavone compared with placebo: 0.43 (0.27, 0.69)]; no significant effects of isoflavone treatment on other plasma inflammatory markers were observed. No significant differences in the response to isoflavones were observed according to subgroups of equol production. Differences in the VCAM-1 response to isoflavones and to placebo were found with ER beta AluI genotypes. Conclusion: Isoflavones have beneficial effects on CRP concentrations, but not on other inflammatory biomarkers of cardiovascular disease risk in postmenopausal women, and may improve VCAM-1 in an ER beta gene polymorphic subgroup.

Antioxidant and cellular activities of anthocyanins and their corresponding vitisins A - studies in platelets, monocytes, and human endothelial cells

During red wine aging, there is a loss of anthocyanins and the formation of various other pigments, so-called vitisins A, which are formed through the chemical interaction of the original anthocyanins with pyruvic acid. The objective of this study was to investigate the antioxidant activities of the most abundant anthocyanins present in red wine (glycosides of delphinidin, petunidin, and malvidin) and their corresponding vitisins A. Anthocyanins exhibited a higher iron reducing as well as 2,2'-azinobis (3-ethyl-benzothiazoline-6-sulfonate) and peroxyl radical scavenging activity than their corresponding vitisins A. Delphinidin showed the highest antioxidant effect of the tested compounds in all of the assays used. Furthermore, we studied the effect of anthocyanins and vitisins A on platelet aggregation and monocyte and endothelial function. Anthocyanins and vitisins did not affect nitric oxide production and tumor necrosis factor-alpha (TNF-alpha) secretion in lipopolysaccharide plus interferon-gamma-activated macrophages. Furthermore, anthocyanins and vitisins did not change collagen-induced platelet aggregation in vitro. However, anthocyanins and to a lesser extent vitisins exhibited protective effects against TNF-alpha-induced monocyte chemoattractant protein production in primary human endothelial cells.

Techniques for quantifying effects of dietary antioxidants on transcription factor translocation and nitric oxide production in cultured cells

Dietary antioxidants can affect cellular processes relevant to chronic inflammatory diseases such as atherosclerosis. We have used non- standard techniques to quantify effects of the antioxidant soy isoflavones genistein and daidzein on translocation of Nuclear Factor-KB (NF-KB) and nitric oxide (NO) production, which are important in these diseases. Translocation was quantified using confocal immunofluoresecence microscopy and ratiometric image analysis. NO was quantified by an electrochemical method after reduction of its oxidation products in cell culture supernatants. Activation of the RAW 264.7 murine monocyte/macrophage cell line increased the ratio of nuclear to cytoplasmic immunostaining for NF-kB. The increase was exacerbated by pre-treatment with genistein or daidzein. To show that decreases could also be detected, pre-treatment with the pine bark extract Pycnogenol (R) r was examined, and found to reduce translocation. NO production was also increased by activation, but was reduced by pre-treatment with genistein or daidzein. In the EA. hy926 human endothelial cell line, constitutive production was detectable and was increased by thrombin. The confocal and electrochemical methods gave data that agreed with results obtained using the established electromobility shift and Griess assays, but were more sensitive, more convenient, gave more detailed information and avoided the use of radioisotopes.

Rapid quality assessment of wheat cultivars registered in Poland using the 2-g mixograph and multivariate statistical analysis

Baking and 2-g mixograph analyses were performed for 55 cultivars (19 spring and 36 winter wheat) from various quality classes from the 2002 harvest in Poland. An instrumented 2-g direct-drive mixograph was used to study the mixing characteristics of the wheat cultivars. A number of parameters were extracted automatically from each mixograph trace and correlated with baking volume and flour quality parameters (protein content and high molecular weight glutenin subunit [HMW-GS] composition by SDS-PAGE) using multiple linear regression statistical analysis. Principal component analysis of the mixograph data discriminated between four flour quality classes, and predictions of baking volume were obtained using several selected mixograph parameters, chosen using a best subsets regression routine, giving R-2 values of 0.862-0.866. In particular, three new spring wheat strains (CHD 502a-c) recently registered in Poland were highly discriminated and predicted to give high baking volume on the basis of two mixograph parameters: peak bandwidth and 10-min bandwidth.

Comparison of predictions of baking volume using large deformation rheological properties

Three large deformation rheological tests, the Kieffer dough extensibility system, the D/R dough inflation system and the 2 g mixograph test, were carried out on doughs made from a large number of winter wheat lines and cultivars grown in Poland. These lines and cultivars represented a broad spread in baking performance in order to assess their suitability as predictors of baking volume. The parameters most closely associated with baking volume were strain hardening index, bubble failure strain, and mixograph bandwidth at 10min. Simple correlations with baking volume indicate that bubble failure strain and strain hardening index give the highest correlations, whilst the use of best subsets regression, which selects the best combination of parameters, gave increased correlations with R-2 = 0.865 for dough inflation parameters, R-2 = 0. 842 for Kieffer parameters and R-2 = 0.760 for mixograph parameters. (c) 2007 Elsevier Ltd. All rights reserved.