Bianca Fuhrman

Reduced Progression of Atherosclerosis in Apolipoprotein E–Deficient Mice Following Consumption of Red Wine, or Its Polyphenols Quercetin or Catechin, Is Associated With Reduced Susceptibility of LDL to Oxidation and Aggregation

The effect of consuming red wine, or its major polyphenol constituents catechin or quercetin, on the development of atherosclerotic lesions, in relation to the susceptibility of plasma LDL to oxidation and to aggregation, was studied in atherosclerotic apolipoprotein E deficient (E degree) mice. Forty E degree mice at the age of 4 weeks were divided into four groups, 10 mice in each group, and were supplemented for up to 6 weeks in their drinking water with placebo (1.1% alcohol); catechin or quercetin (50 micrograms/d per mouse), or red wine (0.5 mL/d per mouse). Consumption of catechin, quercetin, or red wine had no effect on plasma LDL or HDL cholesterol levels. The atherosclerotic lesion area was smaller in the treated mice by 39%, 46%, and 48%, respectively, in comparison with E degree mice that were treated with placebo. In accordance with these findings, cellular uptake of LDL derived after catechin, quercetin, or red wine consumption was found to be reduced by 31%, 40%, and 52%, respectively. These results were associated with reduced susceptibility to oxidation (induced by different modes such as copper ions, free radical generator, or macrophages) of LDL isolated after red wine or quercetin and, to a lesser extent after catechin consumption, in comparison with LDL isolated from the placebo group. Similar results were obtained when LDL was preincubated in vitro with red wine or with the polyphenols prior to its oxidation. Even in the basal oxidative state (not induced oxidation), LDL isolated from E degree mice that consumed catechin, quercetin, or red wine for 2 weeks was found to be less oxidized in comparison with LDL isolated from E degree mice that received placebo, as evidenced by 39%, 48%, and 49% reduced content of LDL-associated lipid peroxides, respectively. This effect could be related to enhanced serum paraoxonase activity in the polyphenol-treated mice. LDL oxidation was previously shown to lead to its aggregation. The present study demonstrated that the susceptibility of LDL to aggregation was reduced in comparison with placebo-treated mice, by 63%, 48%, or 50% by catechin, quercetin, and red wine consumption, respectively, and this effect could be shown also in vitro. The inhibition of LDL oxidation by polyphenols could be related, at least in part, to a direct effect of the polyphenols on the LDL, since both quercetin and catechin were found to bind to the LDL particle via the formation of an ether bond. We thus conclude that dietary consumption by E degree mice of red wine or its polyphenolic flavonoids quercetin and, to a lesser extent, catechin leads to attenuation in the development of the atherosclerotic lesion, and this effect is associated with reduced susceptibility of their LDL to oxidation and aggregation.

Flavonoids protect Ldl from oxidation and attenuate atherosclerosis

Consumption of some plant-derived flavonoids results in their absorption and appearance in plasma and tissues. The inverse relationship between dietary flavonoids consumption and cardiovascular diseases may be associated with the ability of flavonoids to attenuate LDL oxidation, macrophage foam cell formation and atherosclerosis. The effect of flavonoids on arterial cell-mediated oxidation of LDL is determined by their accumulation in the lipoprotein and in arterial cells, such as macrophages. Flavonoids can reduce LDL lipid peroxidation by scavenging reactive oxygen/nitrogen species, chelation of transition metal ions and sparing of LDL-associated antioxidants. They can also reduce macrophage oxidative stress by inhibition of cellular oxygenases [such as nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase] or by activating cellular antioxidants (such as the glutathione system). Thus, plant flavonoids, as potent natural antioxidants that protect against lipid peroxidation in arterial cells and lipoproteins, significantly attenuate the development of atherosclerosis.

Lycopene synergistically inhibits LDL oxidation in combination with vitamin E, glabridin, rosmarinic acid, carnosic acid, or garlic.

Several lines of evidence suggest that oxidatively modified low-density lipoprotein (LDL) is atherogenic, and that atherosclerosis can be attenuated by natural antioxidants, which inhibit LDL oxidation. This study was conducted to determine the effect of tomato lycopene alone, or in combination with other natural antioxidants, on LDL oxidation. LDL (100 microg of protein/ml) was incubated with increasing concentrations of lycopene or of tomato oleoresin (lipid extract of tomatoes containing 6% lycopene, 0.1% beta-carotene, 1% vitamin E, and polyphenols), after which it was oxidized by the addition of 5 micromol/liter of CuSO4. Tomato oleoresin exhibited superior capacity to inhibit LDL oxidation in comparison to pure lycopene, by up to five-fold [97% vs. 22% inhibition of thiobarbituric acid reactive substances (TBARS) formation, and 93% vs. 27% inhibition of lipid peroxides formation, respectively]. Because tomato oleoresin also contains, in addition to lycopene, vitamin E, flavonoids, and phenolics, a possible cooperative interaction between lycopene and such natural antioxidants was studied. A combination of lycopene (5 micromol/liter) with vitamin E (alpha-tocopherol) in the concentration range of 1-10 micromol/liter resulted in an inhibition of copper ion-induced LDL oxidation that was significantly greater than the expected additive individual inhibitions. The synergistic antioxidative effect of lycopene with vitamin E was not shared by gamma-to-cotrienol. The polyphenols glabridin (derived from licorice), rosmarinic acid or carnosic acid (derived from rosemary), as well as garlic (which contains a mixture of natural antioxidants) inhibited LDL oxidation in a dose-dependent manner. When lycopene (5 micromol/liter) was added to LDL in combination with glabridin, rosmarinic acid, carnosic acid, or garlic, synergistic antioxidative effects were obtained against LDL oxidation induced either by copper ions or by the radical generator AAPH. Similar interactive effects seen with lycopene were also observed with beta-carotene, but, however, to a lesser extent of synergism. Because natural antioxidants exist in nature in combination, the in vivo relevance of lycopene in combination with other natural antioxidants was studied. Four healthy subjects were administered a fatty meal containing 30 mg of lycopene in the form of tomato oleoresin. The lycopene concentration in postprandial plasma was elevated by 70% in comparison to plasma obtained before meal consumption. Postprandial LDL isolated 5 hr after meal consumption exhibited a significant (p < 0.01) reduced susceptibility to oxidation by 21%. We conclude that lycopene acts synergistically, as an effective antioxidant against LDL oxidation, with several natural antioxidants such as vitamin E, the flavonoid glabridin, the phenolics rosmarinic acid and carnosic acid, and garlic. These observations suggest a superior antiatherogenic characteristic to a combination of different natural antioxidants over that of an individual one.

Wine Flavonoids Protect against LDL Oxidation and Atherosclerosis

Abstract: We have previously shown that consumption of red wine, but not of white wine, by healthy volunteers, resulted in the enrichment of their plasma LDL with flavonoid antioxidants such as quercetin, the potent free radicals scavenger flavanol, which binds to the LDL via a glycosidic ether bond. This phenomenon was associated with a significant three‐fold reduction in copper ion‐induced LDL oxidation. The ineffectiveness of flavonoid‐poor white wine could be overcome by grapes skin contact for 18 hours in the presence of alcohol, which extracts grapes skin flavonoids. Recently, we observed that the high antioxidant potency of Israeli red wine could be related to an increased content of flavonols, which are very potent antioxidants and their biosynthesis is stimulated by sunlight exposure. To find out the effect (and mechanisms) of red wine consumption on atherosclerosis, we used the apo E deficient (E0) mice. In these mice, red wine consumption for two months resulted in a 40% decrement in basal LDL oxidation, a similar decrement in LDL oxidizability and aggregation, a 35% reduction in lesion size, and a marked attenuation in the number and morphology of lesions macrophage foam cells. Red wine consumption resulted in accumulation of flavonoids in the mouse macrophages and these cells oxidized LDL and took up LDL about 40% less than macrophages from placebo‐treated mice. Finally, the activity of serum paraoxonase (which can hydrolyze specific lipid peroxides in oxidized LDL and in atherosclerotic lesions) was significantly increased following consumption of red wine by E0 mice. Red wine consumption thus acts against the accumulation of oxidized LDL in lesions as a first line of defense (by a direct inhibition of LDL oxidation), and as a second line of defense (by paraoxonase elevation and removal of atherogenic lesions and lipoproteins oxidized lipids).

Polyphenolic flavonoids inhibit macrophage-mediated oxidation of LDL and attenuate atherogenesis.

Macrophage-mediated oxidation of LDL, a hallmark in early atherosclerosis, depends on the oxidative state of the LDL, and that of the macrophages. The LDL oxidative state is determined by the balance between the LDL polyunsaturated fatty acids and cholesterol, which are prone to oxidation, and the LDL associated antioxidants. Dietary consumption of nutrients rich in polyphenols, such as red wine or liquorice results in LDL enrichment with these polyphenolic flavonoids, and hence, subsequent LDL oxidation is reduced. In addition, enrichment of LDL with polyphenols results in a marked decrease in the susceptibility of the lipoprotein to aggregation (another lipoprotein atherogenic modification). The oxidative status of the macrophages depends on the balance between cellular oxygenases and antioxidants. Macrophage enrichment with polyphenolic flavonoids in vitro or in vivo also reduce macrophage oxidative state, and subsequently cell-mediated oxidation of LDL. The present review article summarizes our current data on these aspects of the antiatherogenic potential of polyphenolic flavonoids.

Oxidative stress increases the expression of the CD36 scavenger receptor and the cellular uptake of oxidized low-density lipoprotein in macrophages from atherosclerotic mice: protective role of antioxidants and of paraoxonase

Little is known about the effects of oxidative stress on macrophage lipid peroxidation and on their atherogenic consequences. Therefore, we questioned the causal relationship between cellular lipid peroxides content and macrophage uptake of oxidized low-density lipoprotein (Ox-LDL). Lipid peroxide content in mouse peritoneal macrophages (MPMs) from E-deficient (E(0)) mice increased progressively by up to 4.6 fold during mice aging, and this was accompanied by an age-dependent increase in the cellular uptake of Ox-LDL (90%), and in the expression of the scavenger receptor CD36 mRNA (41%). Inhibition or stimulation of cellular oxidative stress by administration of dietary potent antioxidants (vitamin E or glabridin) or by inducing cellular glutathione depletion (by using buthionine sulfoximine), respectively, resulted in a significant increment or inhibition of macrophage uptake of Ox-LDL and in cellular CD36 mRNA expression, respectively. Intraperitoneal injection of human serum paraoxonase (PON1) into E(0) mice, resulted in a 40-65% decrement in the lipid peroxide content in MPM harvested from E(0) mice at 2-5 months of age, which subsequently resulted in a similar reduced uptake of Ox-LDL and expression of CD36 mRNA (by 30-40%). In conclusion, our results are the first to demonstrate that macrophage lipid peroxidation stimulates CD36 mRNA expression and enhances the cellular uptake of Ox-LDL.

Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes

Atherosclerosis is characterized by macrophage foam cells formation, which originate from differentiating blood monocytes that have taken up oxidized LDL (Ox-LDL) at enhanced rate. Statin therapy exhibit pleiotropic effects on many components of atherosclerosis. We have studied the effect of atorvastatin therapy in hypercholesterolemic patients, on the cellular uptake of Ox-LDL by their monocytes during differentiation into macrophages. Eleven hypercholesterolemic men were treated with 20 mg/day of atorvastatin for a period of 1 month. Peripheral blood monocytes harvested from control subjects and from patients before and after atorvastatin therapy were allowed to differentiate in culture for up to 9 days in the presence of 20% autologous serum. In control monocytes/macrophages the cellular uptake of Ox-LDL and the scavenger receptors CD36, SRA-I and SRA-II mRNA expression were upregulated during differentiation, and this upregulation was significantly enhanced in cells from hypercholesterolemic patients. Atorvastatin therapy suppressed the upregulation in Ox-LDL degradation and scavenger receptors expression in differentiating monocytes. These effects could be related at least in part to antioxidant characteristics of atorvastatin. Reduced susceptibility of plasma to free radical-induced lipid peroxidation (by 35%), increased plasma total antioxidant status (TAS; by 30%), and increased serum paraoxonase activity (by 53%), were noted following drug therapy. We conclude that atorvastatin therapy in hypercholesterolemic patients reduces the enhanced cellular uptake of Ox-LDL during ex-vivo differentiation of monocytes into macrophages, and decreases cellular scavenger receptors gene expression. These effects may account for the attenuation of atherogenesis in hypercholesterolemic patients following atorvastatin treatment.

Iron induces lipid peroxidation in cultured macrophages, increases their ability to oxidatively modify LDL, and affects their secretory properties

The present study demonstrates for the first time that iron ions can induce lipid peroxidation in intact macrophages without causing cell death. Macrophage lipid peroxidation increases cell-mediated oxidation of LDL, enhances the release of interleukin 1 and inhibits the release of apolipoprotein E from the macrophages. When cultured macrophages were exposed to ferrous ions (50 microM FeSO4) for 4 h at 37 degrees C, cellular lipid peroxidation (measured by analyses of malondialdehyde (MDA), conjugated dienes (CD), and lipid peroxides (PD)) increased 2-4-fold in comparison with non-treated cells. This process was iron-dose dependent, reached its maximum after 4 h of incubation, and was accompanied by 68% and 53% reductions in the content of the cellular linoleic (18:2), and arachidonic acid (20:4), respectively, and by 29% and 36% reductions of cellular vitamin E and vitamin A, respectively. Cell viability (measured by trypan blue exclusion, by [3H]thymidine incorporation into DNA, by analysis of the release of lactate dehydrogenase (LDH) or [3H]adenine), and cell morphology (studied by scanning electron microscopy) were not significantly affected by the iron-induced oxidative stress. Manitol and dimethylthiourea (DMTU), but not catalase or superoxide dismutase (SOD), significantly inhibited iron-induced cellular lipid peroxide formation, suggesting that hydroxyl radical, but not superoxides or hydrogen peroxides, mediated the iron-induced cellular lipid peroxidation. Incubation of LDL (0.2 mg of protein/ml) with oxidized macrophages resulted in LDL lipids peroxidation, as evidenced by an 8-fold increase in the LDL associated MDA in comparison with LDL that was incubated under similar conditions with non-oxidized macrophages. Furthermore, oxidation of LDL by oxidized macrophages in the presence of copper ions (10 microM CuSO4) was 2-fold higher in comparison with oxidation of LDL by non-oxidized macrophages. The release of apolipoprotein E from oxidized macrophages decreased by 50%, whereas macrophage release of beta-glucuronidase and of interleukin-1 beta increased by 83% and by a factor of 6, respectively. This study demonstrates for the first time that iron ions induce oxidation of the cellular polyunsaturated fatty acids in intact macrophages and that this cellular lipid peroxidation can subsequently induce LDL oxidation.

LDL oxidation by arterial wall macrophages depends on the oxidative status in the lipoprotein and in the cells: Role of prooxidants vs. antioxidants

Oxidized LDL is highly atherogenic as it stimulates macrophage cholesterol accumulation and foam cell formation, it is cytotoxic to cells of the arterial wall and it stimulates inflammatory and thrombotic processes. LDL oxidation can lead to its subsequent aggregation, which further increases cellular cholesterol accumulation.All major cells in the arterial wall including endothelial cells, smooth muscle cells and monocyte derived macrophages can oxidize LDL. Macrophage-mediated oxidation of LDL is probably a hallmark in early atherosclerosis, and it depends on the oxidative state of the LDL and that of the macrophages. The LDL oxidative state is elevated by increased ratio of poly/mono unsaturated fatty acids, and it is reduced by elevation of LDL-associated antioxidants such as vitamin E, β-carotene, lycopene, and polyphenolic flavonoids.The macrophage oxidative state depends on the balance between cellular NADPH -oxidase and the glutathione system. LDL-associated polyphenolic flavonoids which inhibit its oxidation, can also reduce macrophage oxidative state, and subsequently the cell-mediated oxidation of LDL. Oxidation of the macrophage lipids, which occurs under oxidative stress, can lead to cell-mediated oxidation of LDL even in the absence of transition metal ions ,and may be operable in vivo.Finally, elimination of Ox-LDL from extracellular spaces, after it was formed under excessive oxidative stress, can possibly be achieved by the hydrolytic action of HDL-associated paraoxonase on lipoproteins lipid peroxides. The present review article summarizes the above issues with an emphasis on our own data.

Paraoxonase 1 (PON1) expression in hepatocytes is upregulated by pomegranate polyphenols: A role for PPAR-γ pathway

OBJECTIVE Serum paraoxonase-1 (PON1) expression is regulated by polyphenols, shown to activate the peroxisome proliferator-activated receptor gamma (PPARgamma). Pomegranate juice (PJ) is a polyphenol-rich fruit. Because promoter sequence of PON1 gene indicates that it could be regulated by nuclear receptors, we investigated the effect of PJ polyphenols on PON1 gene expression in HuH7 hepatocytes. METHODS AND RESULTS PON1 protein or mRNA expression, determined by immunocytochemistry, or quantitative PCR, respectively, as well as PON1 gene promoter activation, was significantly increased in hepatocytes incubated with PJ or with its major polyphenols punicalagin, or gallic acid (GA). Ellagic acid (EA) elicited only modest stimulatory effect. Accordingly, PJ, punicalagin, GA, and less so EA, dose-dependently increased cell-associated and hepatocyte-secreted PON1 arylesterase activity. Functionally, the secreted PON1 exhibited biological activity by protecting LDL and HDL from oxidation. Finally, PJ polyphenols upregulated the hepatocyte PON1 expression, at least in part, via the intracellular signaling cascade PPARgamma-PKA-cAMP. CONCLUSIONS This study shows for the first time that PJ polyphenols have a specific transcriptional role in hepatocyte PON1 expression upregulation, and its secretion to the medium. We conclude that the anti-atherogenic characteristics of PJ polyphenols are modulated, at least in part, via hepatocyte PON1 upregulation and its subsequent release to the medium.