Julie M. Proudfoot

Specific dietary polyphenols attenuate atherosclerosis in apolipoprotein E-knockout mice by alleviating inflammation and endothelial dysfunction.

Objective—Animal and clinical studies have suggested that polyphenols in fruits, red wine, and tea may delay the development of atherosclerosis through their antioxidant and anti-inflammatory properties. We investigated whether individual dietary polyphenols representing different polyphenolic classes, namely quercetin (flavonol), (−)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan), or chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the apolipoprotein E (ApoE)−/− gene–knockout mouse. Methods and Results—Quercetin and theaflavin (64-mg/kg body mass daily) significantly attenuated atherosclerotic lesion size in the aortic sinus and thoracic aorta (P<0.05 versus ApoE−/− control mice). Quercetin significantly reduced aortic F2-isoprostane, vascular superoxide, vascular leukotriene B4, and plasma-sP-selectin concentrations; and augmented vascular endothelial NO synthase activity, heme oxygenase-1 protein, and urinary nitrate excretion (P<0.05 versus control ApoE−/− mice). Theaflavin showed similar, although less extensive, significant effects. Although (−)-epicatechin significantly reduced F2-isoprostane, superoxide, and endothelin-1 production (P<0.05 versus control ApoE−/− mice), it had no significant effect on lesion size. Sesamin and chlorogenic acid treatments exerted no significant effects. Quercetin, but not (−)-epicatechin, significantly increased the expression of heme oxygenase-1 protein in lesions versus ApoE−/− controls. Conclusion—Specific dietary polyphenols, in particular quercetin and theaflavin, may attenuate atherosclerosis in ApoE−/− gene–knockout mice by alleviating inflammation, improving NO bioavailability, and inducing heme oxygenase-1. These data suggest that the cardiovascular protection associated with diets rich in fruits, vegetables, and some beverages may in part be the result of flavonoids, such as quercetin.

Mangostin Inhibits the Oxidative Modification of Human Low Density Lipoprotein

The oxidation of low density lipoprotein (LDL) may play an important role in atherosclerosis. We investigated the possible antioxidant effects of mangostin, isolated from Garcinia mangostana, on metal ion dependent (Cu2+) and independent (aqueous peroxyl radicals) oxidation of human LDL. Mangostin prolonged the lagtime to both metal ion dependent and independent oxidation of LDL in a dose dependent manner over 5 to 50 microM as monitored by the formation of conjugated dienes at 234 nm (P < 0.001). There was no significant effect of mangostin on the rate at which conjugated dienes were formed in the uninhibited phase of oxidation. Levels of thiobarbituric reactive substances (TBARS) generated in LDL were measured 4 and 24 hours after oxidation with 5 microM Cu2+ in the presence or absence of 50 microM or 100 microM mangostin. We observed an inhibition of TBARS formation with 100 microM mangostin at 4 hours (P = 0.027) but not at 24 hours (P = 0.163). Similar results were observed in the presence of 50 microM mangostin. Mangostin, at 100 microM, retarded the relative electrophoretic mobility of LDL at both 4 and 24 hours after Cu2+ induced oxidation. Mangostin (100 microM) significantly inhibited the consumption of alpha-tocopherol in the LDL during Cu2+ initiated oxidation over a 75 minute period (P < 0.001). From these results, we conclude that mangostin is acting as a free radical scavenger to protect the LDL from oxidative damage in this in vitro system.

Inhibition of lipoprotein oxidation by prenylated xanthones derived from mangostin

Oxidative damage is thought to play a critical role in cardiovascular and other chronic diseases. This has led to considerable interest in the antioxidant activity of dietary compounds. Flavonoids have received the most attention and much is known about the structural requirements for antioxidant activity. However, little is known about the antioxidant activity of other plant derived phenolic compounds such as the xanthones. We have previously shown that the prenylated xanthone, mangostin, can inhibit the oxidation of low density lipoprotein. In order to examine the effects of structure modification on antioxidant activity of this class of compound we have prepared a number of derivatives of mangostin and tested antioxidant activity in an isolated LDL and plasma assay. The results of this study show that structural modification of mangostin can have a profound effect on antioxidant activity. Derivatisation of the C-3 and C-6 hydroxyl groups with either methyl, acetate, propane diol or nitrile substantially reduces antioxidant activity. In contrast, derivatisation of C-3 and C-6 with aminoethyl derivatives enhanced antioxidant activity, which may be related to changes in solubility. Cyclisation of the prenyl chains had little influence on antioxidant activity.

HDL is the major lipoprotein carrier of plasma F2-isoprostanes

Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and animal models. F2-isoprostanes are formed in vivo via free radical peroxidation of arachidonic acid, and their quantification has allowed assessment of oxidative stress in vivo. F2-isoprostanes associate with lipids, although their distribution in human plasma lipoproteins is unknown. Our aim was to determine the distribution and levels of F2-isoprostanes in lipoproteins isolated from human plasma by ultracentrifugation and fast protein liquid chromatography (FPLC). F2-isoprostanes were significantly higher in HDL compared with LDL or VLDL after isolation by ultracentrifugation or FPLC. Furthermore, HDL3 particles contained elevated levels of F2-isoprostanes compared with HDL2. Platelet activating factor acetylhydrolase (PAF-AH), which hydrolyses esterified F2-isoprostanes from phospholipids, was predominantly associated with LDL. Reduced F2-isoprostanes in LDL may be related to higher PAF-AH activity in LDL. Paraoxonase 1 (PON-1) activity was associated with HDL2 and may be a contributing factor to the lower F2-isoprostanes in HDL2 compared with HDL3. Further studies are required to establish the implications of these findings on HDL function.

A Metabolite Profiling Approach to Identify Biomarkers of Flavonoid Intake in Humans

Flavonoids are phytochemicals that are widespread in the human diet. Despite limitations in their bioavailability, experimental and epidemiological data suggest health benefits of flavonoid consumption. Valid biomarkers of flavonoid intake may be useful for estimating exposure in a range of settings. However, to date, few useful flavonoid biomarkers have been identified. In this study, we used a metabolite profiling approach to examine the aromatic and phenolic profile of plasma and urine of healthy men after oral consumption of 200 mg of the pure flavonoids, quercetin, (-)-epicatechin, and epigallocatechin gallate, which represent major flavonoid constituents in the diet. Following enzymatic hydrolysis, 71 aromatic compounds were quantified in plasma and urine at 2 and 5 h, respectively, after flavonoid ingestion. Plasma concentrations of different aromatic compounds ranged widely, from 0.01 to 10 micromol/L, with variation among volunteers. None of the aromatic compounds was significantly elevated in plasma 2 h after consumption of either flavonoid compared with water placebo. This indicates that flavonoid-derived aromatic compounds are not responsible for the acute physiological effects reported within 2 h in previous human intervention studies involving flavonoids or flavonoid-rich food consumption. These effects are more likely due to absorption of the intact flavonoid. Our urine analysis suggested that urinary 4-ethylphenol, benzoic acid, and 4-ethylbenzoic acid may be potential biomarkers of quercetin intake and 1,3,5-trimethoxybenzene, 4-O-methylgallic acid, 3-O-methylgallic acid, and gallic acid may be potential markers of epigallocatechin gallate intake. Potential biomarkers of (-)-epicatechin were not identified. These urinary biomarkers may provide an accurate indication of flavonoid exposure.

Comparison of the effects of black and green tea on in vitro lipoprotein oxidation in human serum

Lipoprotein oxidation is a process thought to be involved in atherogenesis. Dietary antioxidants that prevent or inhibit oxidative damage to lipoproteins may help to prevent atherosclerosis. Both black and green teas can be major dietary sources of flavonoids and other phenolics with antioxidant activity. Results of previous studies suggest that green tea may have a greater antioxidant potential than black tea. The aim of this study was to assess and compare the effects of black and green tea on in vitro lipoprotein oxidation. The tea extracts were prepared using a method similar to that used to prepare infusions of tea for drinking. Antioxidant activities of seven black teas and four green teas were assessed using an in vitro assay that measures Cu2+ -induced oxidation of lipoproteins in human serum. All tea extracts inhibited in vitro lipoprotein oxidation in human serum to a similar extent. No significant difference in antioxidant activity was found between black and green tea. Caffeine prepared to a comparable concentration to that found in tea had no effect on lipoprotein oxidation. Further studies are required to determine the importance of these findings in relation to possible protective effects of black and green tea consumption against atherogenesis and cardiovascular disease. © 1999 Society of Chemical Industry

The Role of Copper Reduction by α-Tocopherol in Low-Density Lipoprotein Oxidation

Abstract The oxidation of lipoproteins is thought to be an important early step in atherogenesis. The measurement of lipid peroxidation in low-density lipoprotein (LDL) challenged with Cu2+ has become a widespread test to determine the “susceptibility” of LDL to oxidation. The determination of lag time to oxidation is thought to be a measure of the total antioxidant capacity of the LDL. However, we and others have failed to observe any correlation between lag time and the LDL content of its major lipid antioxidant, α-tocopherol. In fact, several studies now suggest a pro-oxidant role for tocopherol under some conditions of LDL oxidation. In the present study we sought to determine if there was a relationship between Cu2+ reduction by LDL and kinetic parameters of LDL oxidation. LDL (0.3 mmol/l cholesterol, ∼0.1 mg protein/ml) was incubated at 30°C with 2 μM Cu2+ and the formation of conjugated dienes measured over a 4-h period. Using neocuproine, an indicator molecule that specifically complexes Cu+ but not Cu2+, the reduction of Cu2+ by LDL was monitored. The final Cu concentration in these assays was 100 μM and neocuproine 750 μM. Cu+ formation was measured by absorbance at 454 nm. A strong negative correlation was observed between copper reduction by LDL and lag time to oxidation (r = −0.66, p

Comparative reactivity of the myeloperoxidase-derived oxidants HOCl and HOSCN with low-density lipoprotein (LDL): Implications for foam cell formation in atherosclerosis

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), results in extensive lipid accumulation by macrophages. However, the reactivity of the other major MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and F2-isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature of LDL oxidation in vivo, and has implications for the progression of atherosclerosis.

F2-Isoprostanes in HDL are bound to neutral lipids and phospholipids

Abstract Low HDL cholesterol (HDL-C) is a risk factor for coronary artery disease (CAD). However, interventions that raise HDL-C have failed to reduce cardiovascular events. We previously reported that HDL is the main carrier of plasma F2-isoprostanes (F2-IsoPs) that are markers of oxidative stress formed upon oxidation of arachidonic acid. F2-IsoPs are predominantly associated with phospholipids. However, there is evidence that F2-IsoPs in the liver of rats treated with carbon tetrachloride associate with the neutral lipids. To date it is not known whether F2-IsoPs are found in the neutral lipids in HDL in humans. Possible candidate neutral lipids include cholesteryl esters, triglycerides, diglycerides, and monoglycerides. This study aimed to identify the lipid classes within native and oxidized HDL that contain F2-IsoPs. We showed that F2-IsoPs in HDL are bound to neutral lipids as well as phospholipids. HDL-3 contained the highest concentration of F2-IsoPs in all lipid classes before and after in vitro oxidation. Using targeted LC/MS and high resolution MS, we were unable to provide conclusive evidence for the presence of the synthesized standards 15(R)-15-F2t-isoP cholesterol and 1-ent-15(RS)-15-F2t-isoprostanoyl-sn-glycerol in the neutral lipids of HDL. Our findings show that oxidized lipids such as F2-IsoPs are found in the core and surface of HDL. However, the exact molecular species remain to be definitively characterized. Future studies are required to determine whether the presence of F2-IsoPs in neutral lipids alters HDL function.

F2-isoprostanes affect macrophage migration and CSF-1 signalling

Abstract F2‐isoprostanes (F2‐IsoP) are formed in vivo via free radical peroxidation of arachidonic acid. Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and F2‐IsoP have been detected in atherosclerotic plaque. Colony stimulating factor‐1 (CSF‐1) is essential to macrophage survival, proliferation and differentiation and has been detected in human atherosclerotic plaques. Accumulation of macrophages within the vascular wall is an important component of atherosclerosis but little is known about the effect of F2‐IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid‐bound 15‐F2t‐isoprostane (15‐F2t‐IsoP) on macrophage migration and investigate the signalling pathways involved. Mouse macrophages (cell line BAC1.2F5) were pre‐incubated with 15‐F2t‐IsoP (free, bound to cholesterol or monoacylglycerol or within oxidized phospholipid) and cell migration was assessed using chemotaxis towards CSF‐1 in Boyden chambers. Migration was also measured using the wound healing assay with primary mouse bone marrow derived macrophages. We showed that 15‐F2t‐IsoP dose‐dependently inhibited BAC1.2F5 macrophage spreading and adhesion but stimulated their migration towards CSF‐1, with maximum effect at 10 &mgr;M. Analysis of CSF‐1 stimulated signalling pathways in BAC1.2F5 macrophages showed that phosphorylation of Akt, a key mediator of cell migration, and one of its regulators, the mTORC2 component, Rictor, was significantly decreased. In contrast, phosphorylation of the adhesion kinases, FAK and Pyk2, and the adhesion scaffold protein, paxillin, was enhanced after treatment with 15‐F2t‐IsoP. Mouse bone marrow macrophages were transfected with FAK or Pyk2 small interfering RNA (siRNA) to examine the role of FAK and Pyk2 in 15‐F2t‐IsoP signalling. Pyk2 silencing inhibited 15‐F2t‐IsoP‐induced reduction in cell area and phospho‐paxillin adhesion numbers. The size distribution of adhesions in the presence of 15‐F2t‐IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15‐F2t‐IsoP‐stimulated macrophage migration. These results demonstrate that 15‐F2t‐IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis. Graphical abstract Figure. No caption available. HighlightsOxidized fatty acid 15‐F2t‐isoprostane decreased macrophage adhesions and increased motility15‐F2t‐isoprostane reduced phosphorylation of Akt and Rictor, but enhanced phosphorylation of FAK, Pyk2 and paxillin.Pyk2 siRNA inhibited effect of 15‐F2t‐isoprostane on focal adhesions and migration in primary mouse bone marrow macrophages.15‐F2t‐isoprostane signalling showed cross‐talk with key migration factor CSF‐1.Stimulation of macrophage migration is an additional bioactivity for F2‐isoprostanes.