Nina Volkova

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.

Pomegranate Phenolics from the Peels, Arils, and Flowers Are Antiatherogenic: Studies in Vivo in Atherosclerotic Apolipoprotein E-Deficient (E0) Mice and in Vitro in Cultured Macrophages and Lipoproteins

We have analyzed in vivo and in vitro the antiatherogenic properties and mechanisms of action of all pomegranate fruit parts: peels (POMxl, POMxp), arils (POMa), seeds (POMo), and flowers (POMf), in comparison to whole fruit juice (PJ). Atherosclerotic E 0 mice consumed POM extracts [200 microg of gallic acid equivalents (GAE)/mouse/day] for 3 months. Blood samples, peritoneal macrophages (MPM), and aortas were then collected. All POM extracts possess antioxidative properties in vitro. After consumption of PJ, POMxl, POMxp, POMa, or POMf by E (0) mice, the atherosclerotic lesion area was significantly decreased by 44, 38, 39, 6, or 70%, respectively, as compared to placebo-treated group, while POMo had no effect. POMf consumption reduced serum lipids, and glucose levels by 18-25%. PJ, POMxl, POMxp, POMf, or POMa consumption resulted in a significant decrement, by 53, 42, 35, 27, or 13%, respectively, in MPM total peroxides content, and increased cellular paraoxonase 2 (PON2) activity, as compared to placebo-treated mice. The uptake rates of oxidized-LDL by E (0)-MPM were significantly reduced by approximately 15% after consumption of PJ, POMxl, or POMxp. Similar results were obtained on using J774A.1 macrophage cell line. Finally, pomegranate phenolics (punicalagin, punicalin, gallic acid, and ellagic acid), as well as pomegranate unique complexed sugars, could mimic the antiatherogenic effects of pomegranate extracts. We conclude that attenuation of atherosclerosis development by some of the POM extracts and, in particular, POMf, could be related to the combined beneficial effects on serum lipids levels and on macrophage atherogenic properties.

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.

Pomegranate juice polyphenols increase recombinant paraoxonase-1 binding to high-density lipoprotein: Studies in vitro and in diabetic patients

OBJECTIVE The high-density lipoprotein (HDL)-associated paraoxonase-1 (PON1)/free PON1 ratio is lower in diabetic patients in comparison with healthy controls. Because diabetes is associated with increased oxidative stress, we hypothesized that a labeled recombinant PON1 (rePON1) would detect differences in HDL capacity to bind PON1 under specific experimental conditions, such as oxidation, addition of polyphenols, or in vivo dosing of diabetic patients with polyphenols. METHODS In the present study we determined labeled rePON1 binding to HDL under various oxidative conditions by using polyacrylamide gel electrophoresis for the separation of free labeled rePON1 from HDL-bound labeled rePON1. RESULTS The HDL-rePON1/free rePON1 ratio gradually decreased as the extent of HDL oxidation increased, and the antioxidants vitamin E or pomegranate juice (PJ) inhibited the redistribution of rePON1. PJ or its purified polyphenols, punicalagin, gallic acid, or ellagic acid, increased rePON1 binding also to non-oxidized HDL. Further, rePON1 associated more efficiently with HDLs isolated from diabetic patients after PJ consumption versus HDLs isolated before PJ consumption. CONCLUSIONS We conclude that 1) oxidative stress impairs binding of fluorescein isothiocyanate-labeled rePON1 to HDL and 2) PJ polyphenols directly increase the HDL-rePON1 association beyond their antioxidative effect.

Amphiphilic/Bipolar Metallocorroles That Catalyze the Decomposition of Reactive Oxygen and Nitrogen Species, Rescue Lipoproteins from Oxidative Damage, and Attenuate Atherosclerosis in Mice

Accumulating evidence points towards the involvement of reactive oxygen and nitrogen species (ROS and RNS, respectively) in a very wide variety of diseases. The relationship between atherosclerosis and oxidative/nitrosative stress—the imbalance between the desired production of ROS/RNS and the capability of biological systems to detoxify excess amounts thereof—is often analyzed in terms of damage to the cholesterol-transporting highand low-density lipoproteins (HDL and LDL, respectively). Oxidized LDL are taken up at an enhanced rate by arterial macrophages in a noncontrolled fashion and modified HDL are less effective in performing cellular cholesterol efflux, thus leading to the formation of cholesterol-loaded foam cells, the hallmark of early atherosclerosis. Although the atherosclerosis-preventing effect of certain natural antioxidants is in harmony with this hypothesis, it is seriously challenged by the fact that many potent in vitro antioxidants fail to display beneficial in vivo effects. Recent focus on peroxynitrite (HOONO) revealed that this molecule is involved (directly and through COH and CNO2 derived from it) in the damage of a wide variety of molecules, including those that are of vital importance for proper cardiovascular function. The problem unique to peroxynitrite is that, in contrast to other ROS and RNS and their precursors, there is no known biological defense system against it, and most natural antioxidants are very poor peroxynitrite scavengers. This calls for the development of synthetic reagents that could act on and neutralize peroxynitrite by one or more of the following ways: 1) interfere with its formation by eliminating its precursors (superoxide anion and nitric oxide); 2) decompose it to biologically benign products; and 3) repair the damage caused by it. The motivation for the current investigations was the fact that the particular iron(III) and manganese(III) corroles shown in Scheme 1A were disclosed as potent catalysts for decomposition of peroxynitrite in purely chemical systems, with the former acting very fast and the latter operating through the novel mechanism shown in Scheme 1B. The

Pomegranate juice (PJ) consumption antioxidative properties on mouse macrophages, but not PJ beneficial effects on macrophage cholesterol and triglyceride metabolism, are mediated via PJ-induced stimulation of macrophage PON2

OBJECTIVE To examine whether the beneficial effects of PJ consumption by mice on their macrophages are mediated via PJ-induced increment in serum paraoxonase 1 (PON1) activity and/or in macrophage PON2 expression. METHODS AND RESULTS We performed studies in peritoneal macrophages (MPM) from C57BL/6 control mice, or from PON1KO mice, or from PON2KO mice that consumed PJ (200 microg of gallic acid equivalents/mouse/day, for 1 month period). PJ consumption by C57BL/6 mice resulted in a significant increment, by 36% in serum PON1 catalytic activities, and upregulated MPM PON2 expression. In MPM from C57BL/6 or from PON1KO mice that consumed PJ, the extent of cell-mediated LDL oxidation was decreased by 22%, and that of cellular superoxide release by 20-26%. In contrast, PJ consumption by PON2KO mice resulted in a minimal inhibitory effect on macrophage oxidative stress by only 4-9%. Unlike PJ antioxidative effects in MPM, PJ anti-atherogenic effects on MPM cholesterol and triglyceride metabolism were similar in all mice groups that consumed PJ. After PJ consumption, cellular cholesterol content was decreased by 14-19%, and this could be attributed to a significant inhibition in MPM cholesterol biosynthesis rate by 20-32%, and/or to stimulation of HDL-mediated cholesterol efflux from the cells by 22-37%. Similarly, MPM triglyceride content and triglyceride biosynthesis rate were both significantly decreased after PJ consumption, by 16-27% and by 22-28%, respectively. CONCLUSION PJ consumption antioxidative properties on mouse macrophages, but not PJ beneficial effects on macrophage cholesterol and triglyceride metabolism, are mediated via PJ-induced stimulation of macrophage PON2 expression. Serum PON1 stimulation by PJ consumption, however, was not involved in PJ-induced effects on macrophages.

Urokinase activates macrophage PON2 gene transcription via the PI3K/ROS/MEK/SREBP-2 signalling cascade mediated by the PDGFR-β

AIMS We have recently shown that urokinase plasminogen activator (uPA) increases oxidative stress (OS), cholesterol biosynthesis, and paraoxonase 2 (PON2) expression in macrophages via binding to its receptor, the uPAR. Since PON2 is regulated by both OS and cholesterol content, we hypothesized that uPA elicits a cascade of signal transduction events shared by NADPH oxidase and cholesterol biosynthesis that culminates in PON2 gene expression. Here, we investigated the signalling pathway that leads to the expression of PON2 in macrophages in response to uPA. METHODS AND RESULTS The increase in macrophage PON2 mRNA levels in response to uPA was shown to depend on PON2 gene promoter activation and mRNA transcription. LDL abolished these effects, suggesting a possible role for a transcription factor involved in cellular cholesterogenesis. Indeed, uPA upregulated PON2 expression in a sterol regulatory binding protein-2 (SREBP-2)-dependent manner, since blocking SREBP-2 maturation by 4-(2-aminoethyl)-benzenesulfonyl fluoride abolished uPA-stimulation of PON2, whereas inhibition of SREBP-2 catabolism by N-acetyl-leucyl-norleucinal had an opposite effect. The upstream signalling mechanisms include uPA activation of extracellular signal-regulated kinases (ERK1/2), which was dependent on NADPH oxidase and phosphatidylinositol 3-kinase activation, and these latter effects were mediated by the tyrosine kinase activity of the platelet-derived growth factor receptor-beta. CONCLUSION These findings provide a framework linking interactions among cellular signalling pathways associated with reactive oxygen species production, macrophage cholesterol biosynthesis, and cellular PON2 expression in vascular pathophysiology.

Urokinase Plasminogen Activator Upregulates Paraoxonase 2 Expression in Macrophages via an NADPH Oxidase-Dependent Mechanism

Objective—Macrophage foam cells are characterized by increased oxidative stress. Macrophage urokinase plasminogen activator (uPA) was shown to contribute to atherosclerosis progression. We hypothesized that uPA atherogenicity is related to its ability to increase macrophage oxidative stress. Increased macrophage oxidative stress in turn was shown to enhance PON2 expression. In the present study we investigated the effect of uPA on macrophage PON2 expression in relation to cellular oxidative stress. Methods and Results—uPA increased PON2 expression in THP-1 macrophages in a dose-dependent manner. This effect required uPA/uPAR interaction and was abolished by cell treatment with antioxidants. uPA increased macrophage oxidative stress, measured by increased lipid peroxides, reactive oxygen species formation, superoxide anion release, and cell-mediated LDL oxidation. These effects were related to uPA-mediated activation of NADPH oxidase, and could not be reproduced in mouse peritoneal macrophages (MPM) harvested from p47phox−/− mice, suggesting a causal relationship between NADPH oxidase activation and the effects of uPA on macrophage oxidative stress and PON2 expression. Finally, MPM from PON2−/− mice were more susceptible to uPA-induced cellular oxidative stress than wild-type MPM, suggesting that PON2 protects against uPA-stimulated macrophage oxidative stress. Conclusions—Upregulation of macrophage PON2 may provide a compensatory protective mechanism against uPA-stimulation of macrophage oxidative stress during atherogenesis.

Paraoxonase 1 (PON1) inhibits monocyte-to-macrophage differentiation

OBJECTIVE To analyze paraoxonase 1 (PON1) effect on monocyte-to-macrophage differentiation. METHODS AND RESULTS THP-1 monocytic cell-line and mouse peritoneal macrophages (MPM) were studied. Markers for monocytes differentiation included: morphological changes, CD11b and CD36 expression, and cellular oxidative stress. PON1KO MPM were more differentiated than control C57BL/6 MPM. Intraperitoneal injection of recombinant PON1 (rePON1) to C57BL/6 or to PON1KO mice significantly increased serum, MPM, and tissues PON1 activities. These effects were associated with a significant decrease in CD11b in C57BL/6 and PON1KO MPM (by 21% and 35%, respectively), in CD36 (by 35% and 38%, respectively), and in cellular total peroxides content (by 18% and 20%, respectively). rePON1 also significantly inhibited CD11b and CD36 expression, and cellular total peroxides during PMA-induced THP-1 monocytes differentiation, by 68%, 56% and 53%, respectively. Similar effects were observed upon using reconstituted HDL (rHDL) +rePON1, or human HDL +rePON1, in comparison to rHDL or to human HDL, as well as, HDL from C57BL/6 vs. PON1KO mice. Inhibition of monocyte-to-macrophage differentiation was demonstrated also by several dietary antioxidants such as vitamin E, gallic acid, or punicalagin (the major polyphenol in pomegranate). Whereas NADPH oxidase was not involved in PON1 anti-differentiation effect, mitochondrial complex I could be involved, as rotenone (complex I inhibitor) significantly decreased (by 77%) the expression of CD11b during THP-1 differentiation. Finally, blocking PON1 sulfhydryl group with N-ethylmalemide significantly attenuated PON1 inhibitory effect on THP-I monocyte-to-macrophage differentiation. CONCLUSION HDL-associated PON1 inhibits monocyte-to-macrophage differentiation, and this effect could be related to PON1 peroxidase-like activity which involves its free sulfhydryl group.